Default target region bed file in test run

WGLab / SeqMule

Automated human exome/genome variants detection from FASTQ files

http://seqmule.usc.edu

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Default target region bed file in test run #146

Open rashidma opened 7 years ago

rashidma commented 7 years ago

Hi Yun, I recently run seqmule in quick mode with the following commands to test the installation- seqmule pipeline -a normal_R1.fastq.gz -b normal_R2.fastq.gz -prefix example -N 2 -capture default -threads 10 -e The report I got is matched with http://www.openbioinformatics.org/seqmule/example/example_report/example/example.html There are differences which I assume is due to the target regions from bed file. In my run the bed (by default) is - hg19_exonPlus5bp_UCSCrefGene.bed But on your website the default bed is hg19_exome.bed Can you provide me that hg19_exome.bed in order to reproduce the same result mentioned on your website. Please comment overall. Regards Mamoon

yunfeiguo commented 7 years ago

Hi Mamoon,

Please use inst/hg19_exome.legacy.bed after updating SeqMule with seqmule update --git. Thank you!

Best, Yunfei

rashidma commented 7 years ago

Thanks a lot Yunfei, I successfully ran the test run. Now, I was running a real sample from NIST. Out of 51 steps 48 steps ran well. I am stuck in the last few steps. The seqmule was stopped many times due to system shutdown and couple of times I killed the process. But every time I restarted it with--

nohup seqmule run NIST7035-NIST7086.script &

Please let me know any troubleshooting. I will be thankful to you. Regards MR

On Thu, Aug 11, 2016 at 1:02 AM, Yunfei Guo notifications@github.com wrote:

Hi Mamoon,

Please use inst/hg19_exome.legacy.bed after updating SeqMule with seqmule update --git. Thank you!

Best, Yunfei

— You are receiving this because you authored the thread. Reply to this email directly, view it on GitHub https://github.com/WGLab/SeqMule/issues/146#issuecomment-239018748, or mute the thread https://github.com/notifications/unsubscribe-auth/ATyiOAPu4as9L3bQ8R0fedRCtTQ976qQks5qekpygaJpZM4Jf64z .

Mamoon Rashid, PhD *Post-Doctoral Fellow Marine Microbial Ecology Lab Red Sea Research Center 4700 King Abdullah University of Science and Technology (KAUST) Room 2217-WS03, Ibn Al-Haytham Building (2) 4700 King Abdullah University of Science and Technology* Thuwal 23955-6900, Kingdom of Saudi Arabia Office: +966 (0) 2 808-2671

yunfeiguo commented 7 years ago

Hi Rashid,

Could you please provide the contents of nohup.out file? I need to know where it stopped.

Thanks.

Best, Yunfei

On Mon, Aug 15, 2016 at 12:47 AM, rashidma notifications@github.com wrote:

Thanks a lot Yunfei, I successfully ran the test run. Now, I was running a real sample from NIST. Out of 51 steps 48 steps ran well. I am stuck in the last few steps. The seqmule was stopped many times due to system shutdown and couple of times I killed the process. But every time I restarted it with--

nohup seqmule run NIST7035-NIST7086.script &

Please let me know any troubleshooting. I will be thankful to you. Regards MR

On Thu, Aug 11, 2016 at 1:02 AM, Yunfei Guo notifications@github.com wrote:

Hi Mamoon,

Please use inst/hg19_exome.legacy.bed after updating SeqMule with seqmule update --git. Thank you!

Best, Yunfei

— You are receiving this because you authored the thread. Reply to this email directly, view it on GitHub https://github.com/WGLab/SeqMule/issues/146#issuecomment-239018748, or mute the thread https://github.com/notifications/unsubscribe-auth/ ATyiOAPu4as9L3bQ8R0fedRCtTQ976qQks5qekpygaJpZM4Jf64z .

Mamoon Rashid, PhD *Post-Doctoral Fellow Marine Microbial Ecology Lab Red Sea Research Center 4700 King Abdullah University of Science and Technology (KAUST) Room 2217-WS03, Ibn Al-Haytham Building (2) 4700 King Abdullah University of Science and Technology* Thuwal 23955-6900, Kingdom of Saudi Arabia Office: +966 (0) 2 808-2671

— You are receiving this because you modified the open/close state. Reply to this email directly, view it on GitHub https://github.com/WGLab/SeqMule/issues/146#issuecomment-239746634, or mute the thread https://github.com/notifications/unsubscribe-auth/AHYPzsknYKT-MSQOex9rMD0_O_MHhoqLks5qgBmcgaJpZM4Jf64z .

rashidma commented 7 years ago

Hi Yunfei, I sent you the big nohup file (~8MB) and it failed to reach you. Now I am sending you the last 1000 lines of the nohup file. I can see there are errors at step 42 and 43. I thank you so much for your time. MR

On Mon, Aug 15, 2016 at 4:05 PM, Yunfei Guo notifications@github.com wrote:

Hi Rashid,

Could you please provide the contents of nohup.out file? I need to know where it stopped.

Thanks.

Best, Yunfei

On Mon, Aug 15, 2016 at 12:47 AM, rashidma notifications@github.com wrote:

Thanks a lot Yunfei, I successfully ran the test run. Now, I was running a real sample from NIST. Out of 51 steps 48 steps ran well. I am stuck in the last few steps. The seqmule was stopped many times due to system shutdown and couple of times I killed the process. But every time I restarted it with--

nohup seqmule run NIST7035-NIST7086.script &

Please let me know any troubleshooting. I will be thankful to you. Regards MR

On Thu, Aug 11, 2016 at 1:02 AM, Yunfei Guo notifications@github.com wrote:

Hi Mamoon,

Please use inst/hg19_exome.legacy.bed after updating SeqMule with seqmule update --git. Thank you!

Best, Yunfei

— You are receiving this because you authored the thread. Reply to this email directly, view it on GitHub https://github.com/WGLab/SeqMule/issues/146#issuecomment-239018748, or mute the thread https://github.com/notifications/unsubscribe-auth/ ATyiOAPu4as9L3bQ8R0fedRCtTQ976qQks5qekpygaJpZM4Jf64z .

Mamoon Rashid, PhD *Post-Doctoral Fellow Marine Microbial Ecology Lab Red Sea Research Center 4700 King Abdullah University of Science and Technology (KAUST) Room 2217-WS03, Ibn Al-Haytham Building (2) 4700 King Abdullah University of Science and Technology* Thuwal 23955-6900, Kingdom of Saudi Arabia Office: +966 (0) 2 808-2671

— You are receiving this because you modified the open/close state. Reply to this email directly, view it on GitHub https://github.com/WGLab/SeqMule/issues/146#issuecomment-239746634, or mute the thread https://github.com/notifications/unsubscribe- auth/AHYPzsknYKT-MSQOex9rMD0_O_MHhoqLks5qgBmcgaJpZM4Jf64z .

— You are receiving this because you authored the thread. Reply to this email directly, view it on GitHub https://github.com/WGLab/SeqMule/issues/146#issuecomment-239796794, or mute the thread https://github.com/notifications/unsubscribe-auth/ATyiON2yhwvkb3ikEdlt0uiv9ZGe7k45ks5qgGQpgaJpZM4Jf64z .

NOTICE: unable to check for updates. Results will not be affected. Generating variant stats NOTICE: Calculating coverage for NIST7035_result/NIST7035_bwamem.merge.bam. This takes a while NOTICE: checking contig(chromosome) name consistency in nexterarapidcapture_expandedexome_targetedregions.chrmod.nooverlap.bed and NIST7035_result/NIST7035_bwamem.merge.bam. NOTICE: 52514 extracted variants written to NIST7086_result/NIST7086_bwamem.merge.0_freebayes.multi-call.extract.vcf NOTICE: unable to download '/work/rashidma/software/seqmule_by_admin/SeqMule-master/bin/secondary/../../version' from http://www.openbioinformatics.org/seqmule. Bad Internet connection? NOTICE: unable to check for updates. Results will not be affected. Generating variant stats NOTICE: Calculating coverage for NIST7086_result/NIST7086_bwamem.merge.bam. This takes a while NOTICE: checking contig(chromosome) name consistency in nexterarapidcapture_expandedexome_targetedregions.chrmod.nooverlap.bed and NIST7086_result/NIST7086_bwamem.merge.bam. NOTICE: all chromosome names in BED can be found in BAM header. NOTICE: all chromosome names in BED can be found in BAM header. NOTICE: Calculating coverage ... NOTICE: Start reading from SAMTools depth command ... NOTICE: Calculating coverage ... NOTICE: Start reading from SAMTools depth command ... NOTICE: Output written to NIST7086_result/NIST7086_bwamem.merge.realn.0_gatklite.multi-call_var_stat.txt NOTICE: Output written to NIST7035_result/NIST7035_bwamem.merge.realn.0_gatklite.multi-call_var_stat.txt [ => SeqMule Execution Status: step 35 is finished at Thu Aug 11 21:19:46 AST 2016, Extract variants in custom regions]

[ => SeqMule Execution Status: step 36 is finished at Thu Aug 11 21:19:46 AST 2016, Extract variants in custom regions]

NOTICE: Output written to NIST7086_result/NIST7086_bwamem.merge.realn.0_gatklite.multi-call_var_stat.txt [ => SeqMule Execution Status: step 37 is finished at Thu Aug 11 21:19:47 AST 2016, Extract variants in custom regions]

NOTICE: Output written to NIST7035_result/NIST7035_bwamem.merge.0_samtools.multi-call_var_stat.txt NOTICE: Output written to NIST7086_result/NIST7086_bwamem.merge.0_samtools.multi-call_var_stat.txt [ => SeqMule Execution Status: step 38 is finished at Thu Aug 11 21:19:48 AST 2016, Extract variants in custom regions]

NOTICE: Output written to NIST7035_result/NIST7035_bwamem.merge.0_freebayes.multi-call_var_stat.txt [ => SeqMule Execution Status: step 39 is finished at Thu Aug 11 21:19:49 AST 2016, Extract variants in custom regions]

----------NOTICE---------- [ => SeqMule Execution Status: Running 42 of 51 steps: Extract consensus calls, at Thu Aug 11 21:19:50 AST 2016, Time Elapsed: 4 hr 54 min 55 s] [ => SeqMule Execution Status: step 40 is finished at Thu Aug 11 21:19:50 AST 2016, Extract variants in custom regions]

Current version: 1.2.5 Check for upgrade ... NOTICE: Output written to NIST7086_result/NIST7086_bwamem.merge.0_freebayes.multi-call_var_stat.txt

----------NOTICE---------- [ => SeqMule Execution Status: Running 43 of 51 steps: Extract consensus calls, at Thu Aug 11 21:19:51 AST 2016, Time Elapsed: 4 hr 54 min 56 s] [ => SeqMule Execution Status: step 41 is finished at Thu Aug 11 21:19:51 AST 2016, Extract variants in custom regions]

Current version: 1.2.5 Check for upgrade ...

----------NOTICE---------- [ => SeqMule Execution Status: Running 44 of 51 steps: Generate Venn digram, at Thu Aug 11 21:19:52 AST 2016, Time Elapsed: 4 hr 54 min 57 s] Current version: 1.2.5 Check for upgrade ...

----------NOTICE---------- [ => SeqMule Execution Status: Running 47 of 51 steps: Generate Venn digram, at Thu Aug 11 21:19:53 AST 2016, Time Elapsed: 4 hr 54 min 58 s] Current version: 1.2.5 Check for upgrade ...

----------NOTICE---------- [ => SeqMule Execution Status: Running 50 of 51 steps: Remove intermediate files, at Thu Aug 11 21:19:54 AST 2016, Time Elapsed: 4 hr 54 min 59 s] [ => SeqMule Execution Status: step 50 is finished at Thu Aug 11 21:19:55 AST 2016, Remove intermediate files]

NOTICE: unable to download '/work/rashidma/software/seqmule_by_admin/SeqMule-master/bin/secondary/../../version' from http://www.openbioinformatics.org/seqmule. Bad Internet connection? NOTICE: unable to check for updates. Results will not be affected. NOTICE: Counting non-empty lines in NIST7035_result/NIST7035_bwamem.merge.realn.0_gatklite.multi-call.extract.vcf NOTICE: Counting non-empty lines in /tmp/245851528.01878005132_nofiltered.vcf NOTICE: 2984 records removed because of being filtered. NOTICE: Counting non-empty lines in NIST7035_result/NIST7035_bwamem.merge.0_freebayes.multi-call.extract.vcf NOTICE: Counting non-empty lines in /tmp/245854245.50906468008_nofiltered.vcf NOTICE: 0 records removed because of being filtered. NOTICE: Counting non-empty lines in NIST7035_result/NIST7035_bwamem.merge.0_samtools.multi-call.extract.vcf NOTICE: Counting non-empty lines in /tmp/245852975.78281714735_nofiltered.vcf NOTICE: 0 records removed because of being filtered. ERROR: not all VCFs have the same number of samples! NOTICE: Cleaning... NOTICE: unable to download '/work/rashidma/software/seqmule_by_admin/SeqMule-master/bin/secondary/../../version' from http://www.openbioinformatics.org/seqmule. Bad Internet connection? NOTICE: unable to check for updates. Results will not be affected. NOTICE: Counting non-empty lines in NIST7086_result/NIST7086_bwamem.merge.realn.0_gatklite.multi-call.extract.vcf NOTICE: Counting non-empty lines in /tmp/246146010.863221326_nofiltered.vcf NOTICE: 2984 records removed because of being filtered. NOTICE: Counting non-empty lines in NIST7086_result/NIST7086_bwamem.merge.realn.0_gatklite.multi-call.extract.vcf NOTICE: Counting non-empty lines in /tmp/246149034.30842225667_nofiltered.vcf NOTICE: 2984 records removed because of being filtered. NOTICE: Counting non-empty lines in NIST7086_result/NIST7086_bwamem.merge.0_freebayes.multi-call.extract.vcf NOTICE: Counting non-empty lines in /tmp/2461419845.8495834473_nofiltered.vcf NOTICE: 0 records removed because of being filtered. NOTICE: Counting non-empty lines in NIST7086_result/NIST7086_bwamem.merge.0_samtools.multi-call.extract.vcf NOTICE: Counting non-empty lines in /tmp/2461424584.1620939387_nofiltered.vcf NOTICE: 0 records removed because of being filtered. ERROR: not all VCFs have the same number of samples! NOTICE: Cleaning... NOTICE: unable to download '/work/rashidma/software/seqmule_by_admin/SeqMule-master/bin/secondary/../../version' from http://www.openbioinformatics.org/seqmule. Bad Internet connection? NOTICE: unable to check for updates. Results will not be affected.

----------ERROR---------- [ => SeqMule Execution Status: step 42 FAILED at Thu Aug 11 21:20:07 AST 2016, Extract consensus calls]

----------ERROR---------- [ => SeqMule Execution Status: step 43 FAILED at Thu Aug 11 21:20:07 AST 2016, Extract consensus calls] ERROR: command failed /work/rashidma/software/seqmule_by_admin/SeqMule-master/bin/secondary/../../bin/secondary/worker /work/rashidma/Exome_data/GIAB_NIST_exome_data/MyAnalysis/04_seqmule/seqmule.08112016.9339.logs 42 "/work/rashidma/software/seqmule_by_admin/SeqMule-master/bin/secondary/../../bin/seqmule stats -tmpdir /tmp -ref /work/rashidma/software/seqmule_by_admin/SeqMule-master/bin/secondary/../../database/human_g1k_v37.fasta -jmem 1750m -jexe java -t 2 -c-vcf NIST7035_result/NIST7035_bwamem.merge.realn.0_gatklite.multi-call.extract.vcf,NIST7035_result/NIST7035_bwamem.merge.0_freebayes.multi-call.extract.vcf,NIST7035_result/NIST7035_bwamem.merge.0_samtools.multi-call.extract.vcf -p NIST7035_result/NIST7035.extract " /work/rashidma/software/seqmule_by_admin/SeqMule-master/bin/secondary/../../bin/secondary/worker /work/rashidma/Exome_data/GIAB_NIST_exome_data/MyAnalysis/04_seqmule/seqmule.08112016.9339.logs 43 "/work/rashidma/software/seqmule_by_admin/SeqMule-master/bin/secondary/../../bin/seqmule stats -tmpdir /tmp -ref /work/rashidma/software/seqmule_by_admin/SeqMule-master/bin/secondary/../../database/human_g1k_v37.fasta -jmem 1750m -jexe java -t 2 -c-vcf NIST7086_result/NIST7086_bwamem.merge.realn.0_gatklite.multi-call.extract.vcf,NIST7086_result/NIST7086_bwamem.merge.realn.0_gatklite.multi-call.extract.vcf,NIST7086_result/NIST7086_bwamem.merge.0_freebayes.multi-call.extract.vcf,NIST7086_result/NIST7086_bwamem.merge.0_samtools.multi-call.extract.vcf -p NIST7086_result/NIST7086.extract " !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! After fixing the problem, please execute 'cd /work/rashidma/Exome_data/GIAB_NIST_exome_data/MyAnalysis/04_seqmule' and 'seqmule run NIST7035-NIST7086.script' to resume analysis. !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! NOTICE: normalzing VCF with Vt... NOTICE: unable to download '/work/rashidma/software/seqmule_by_admin/SeqMule-master/bin/secondary/../../version' from http://www.openbioinformatics.org/seqmule. Bad Internet connection? NOTICE: unable to check for updates. Results will not be affected. NOTICE: normalzing VCF with Vt... NOTICE: done normalzing. NOTICE: Converting input file to ANNOVAR format, variants that failed to pass filtering will be ignored. NOTICE: Counting non-empty lines in /tmp/2463813132.9333942714_normalized.vcf NOTICE: Counting non-empty lines in /tmp/2463812796.6749276356_nofiltered.vcf NOTICE: 2983 records removed because of being filtered. NOTICE: output files will be written to /tmp/246388742.43345209784_annovar_raw..avinput NOTICE: Finished reading 54779 lines from VCF file NOTICE: A total of 54651 locus in VCF file passed QC threshold, representing 49556 SNPs (34950 transitions and 14606 transversions) and 5250 indels/substitutions NOTICE: Finished writting 97968 SNPs (69304 transitions and 28664 transversions) and 10375 indels/substitutions for 2 samples NOTICE: Converting input file to ANNOVAR format, variants that failed to pass filtering will be ignored. NOTICE: Counting non-empty lines in /tmp/246385740.36024867303_normalized.vcf NOTICE: Counting non-empty lines in /tmp/2463821915.7641346052_nofiltered.vcf NOTICE: 0 records removed because of being filtered. NOTICE: output files will be written to /tmp/246383165.32093791921_annovar_raw..avinput NOTICE: done normalzing. NOTICE: Converting input file to ANNOVAR format, variants that failed to pass filtering will be ignored. NOTICE: Counting non-empty lines in /tmp/2466215106.8313601309_normalized.vcf NOTICE: Counting non-empty lines in /tmp/2466210124.4926337858_nofiltered.vcf NOTICE: 2983 records removed because of being filtered. NOTICE: output files will be written to /tmp/2466214768.4130827634_annovar_raw..avinput NOTICE: Finished reading 53617 lines from VCF file NOTICE: A total of 53496 locus in VCF file passed QC threshold, representing 48758 SNPs (33513 transitions and 15245 transversions) and 5821 indels/substitutions NOTICE: Finished writting 94602 SNPs (66260 transitions and 28342 transversions) and 9256 indels/substitutions for 2 samples NOTICE: Converting input file to ANNOVAR format, variants that failed to pass filtering will be ignored. NOTICE: Counting non-empty lines in /tmp/246382102.67139302551_normalized.vcf NOTICE: Counting non-empty lines in /tmp/2463813089.1766829906_nofiltered.vcf NOTICE: 0 records removed because of being filtered. NOTICE: output files will be written to /tmp/2463810263.0275990118_annovar_raw..avinput NOTICE: Finished reading 54779 lines from VCF file NOTICE: A total of 54651 locus in VCF file passed QC threshold, representing 49556 SNPs (34950 transitions and 14606 transversions) and 5250 indels/substitutions NOTICE: Finished writting 97968 SNPs (69304 transitions and 28664 transversions) and 10375 indels/substitutions for 2 samples NOTICE: Converting input file to ANNOVAR format, variants that failed to pass filtering will be ignored. NOTICE: Counting non-empty lines in /tmp/2466221970.0864549778_normalized.vcf NOTICE: Counting non-empty lines in /tmp/2466222201.1059853663_nofiltered.vcf NOTICE: 2983 records removed because of being filtered. NOTICE: output files will be written to /tmp/2466223653.0993271246_annovar_raw..avinput NOTICE: Finished reading 52571 lines from VCF file NOTICE: A total of 52514 locus in VCF file passed QC threshold, representing 47737 SNPs (33510 transitions and 14227 transversions) and 5059 indels/substitutions NOTICE: Finished writting 47510 SNPs (33447 transitions and 14063 transversions) and 5050 indels/substitutions for 1 samples NOTICE: Finished reading 54779 lines from VCF file NOTICE: A total of 54651 locus in VCF file passed QC threshold, representing 49556 SNPs (34950 transitions and 14606 transversions) and 5250 indels/substitutions NOTICE: Finished writting 97968 SNPs (69304 transitions and 28664 transversions) and 10375 indels/substitutions for 2 samples NOTICE: Converting input file to ANNOVAR format, variants that failed to pass filtering will be ignored. NOTICE: Counting non-empty lines in /tmp/2466211473.4763577923_normalized.vcf NOTICE: Counting non-empty lines in /tmp/246624736.13069420965_nofiltered.vcf NOTICE: 0 records removed because of being filtered. NOTICE: output files will be written to /tmp/2466282.7828975389612_annovar_raw..avinput NOTICE: Generating Venn diagram... NOTICE: Finished reading 53617 lines from VCF file NOTICE: A total of 53496 locus in VCF file passed QC threshold, representing 48758 SNPs (33513 transitions and 15245 transversions) and 5821 indels/substitutions NOTICE: Finished writting 94602 SNPs (66260 transitions and 28342 transversions) and 9256 indels/substitutions for 2 samples NOTICE: Converting input file to ANNOVAR format, variants that failed to pass filtering will be ignored. NOTICE: Counting non-empty lines in /tmp/246629612.8807128095_normalized.vcf NOTICE: Counting non-empty lines in /tmp/246628231.36922920045_nofiltered.vcf Loading required package: VennDiagram Loading required package: grid NOTICE: 0 records removed because of being filtered. NOTICE: output files will be written to /tmp/2466211203.7308176434_annovar_raw..avinput [1] 1 NOTICE: Finished reading 52571 lines from VCF file NOTICE: A total of 52514 locus in VCF file passed QC threshold, representing 47737 SNPs (33510 transitions and 14227 transversions) and 5059 indels/substitutions NOTICE: Finished writting 47510 SNPs (33447 transitions and 14063 transversions) and 5050 indels/substitutions for 1 samples mv: cannot stat `/tmp/2466211203.7308176434_annovar_raw.NIST7086.avinput': No such file or directory ERROR:Bad file descriptor NOTICE: Cleaning... NOTICE: Venn Digram plotted to NIST7035_result/NIST7035_SNV_venn.tiff and NIST7035_result/NIST7035_SNV_venn.jpg NOTICE: Generating Venn diagram... Loading required package: VennDiagram Loading required package: grid [1] 1 NOTICE: Venn Digram plotted to NIST7035_result/NIST7035_NON-SNV_venn.tiff and NIST7035_result/NIST7035_NON-SNV_venn.jpg NOTICE: Cleaning... NOTICE: nexterarapidcapture_expandedexome_targetedregions.chrmod.nooverlap.bed contains 62085286 base pairs Statistics written to NIST7035_result/NIST7035_cov_stat.txt and NIST7035_result/NIST7035_cov_stat_detail.txt NOTICE: coverage plot <<NIST7035_result/NIST7035_cov.jpg>> generated NOTICE: Extracting primary alignments NOTICE: nexterarapidcapture_expandedexome_targetedregions.chrmod.nooverlap.bed contains 62085286 base pairs Statistics written to NIST7086_result/NIST7086_cov_stat.txt and NIST7086_result/NIST7086_cov_stat_detail.txt NOTICE: coverage plot <<NIST7086_result/NIST7086_cov.jpg>> generated NOTICE: Extracting primary alignments Generating alignment stats Output written to NIST7035_result/NIST7035_aln_stat.txt NOTICE: Cleaning... Generating alignment stats Output written to NIST7086_result/NIST7086_aln_stat.txt NOTICE: Cleaning... Current version: 1.2.5 Check for upgrade ... NOTICE: unable to download '/work/rashidma/software/seqmule_by_admin/SeqMule-master/bin/secondary/../../version' from http://www.openbioinformatics.org/seqmule. Bad Internet connection? NOTICE: unable to check for updates. Results will not be affected. ****EXECUTION BEGINS at Sun Aug 14 09:39:34 AST 2016****

----------NOTICE---------- [ => SeqMule Execution Status: Running 42 of 51 steps: Extract consensus calls, at Sun Aug 14 09:39:35 AST 2016, Time Elapsed: 0 hr 0 min 1 s] [ => SeqMule Execution Status: step 1 is finished at Sun Aug 14 09:39:35 AST 2016, QC assesment on input]

[ => SeqMule Execution Status: step 2 is finished at Sun Aug 14 09:39:35 AST 2016, QC assesment on input]

[ => SeqMule Execution Status: step 3 is finished at Sun Aug 14 09:39:35 AST 2016, QC assesment on input]

[ => SeqMule Execution Status: step 4 is finished at Sun Aug 14 09:39:35 AST 2016, QC assesment on input]

[ => SeqMule Execution Status: step 5 is finished at Sun Aug 14 09:39:35 AST 2016, QC assesment on input]

[ => SeqMule Execution Status: step 6 is finished at Sun Aug 14 09:39:35 AST 2016, QC assesment on input]

[ => SeqMule Execution Status: step 7 is finished at Sun Aug 14 09:39:35 AST 2016, QC assesment on input]

[ => SeqMule Execution Status: step 8 is finished at Sun Aug 14 09:39:35 AST 2016, QC assesment on input]

[ => SeqMule Execution Status: step 9 is finished at Sun Aug 14 09:39:35 AST 2016, Generate QC stat]

[ => SeqMule Execution Status: step 10 is finished at Sun Aug 14 09:39:35 AST 2016, Generate QC stat]

[ => SeqMule Execution Status: step 11 is finished at Sun Aug 14 09:39:35 AST 2016, bwamem alignment]

[ => SeqMule Execution Status: step 12 is finished at Sun Aug 14 09:39:35 AST 2016, bwamem alignment]

[ => SeqMule Execution Status: step 13 is finished at Sun Aug 14 09:39:35 AST 2016, bwamem alignment]

[ => SeqMule Execution Status: step 14 is finished at Sun Aug 14 09:39:35 AST 2016, bwamem alignment]

[ => SeqMule Execution Status: step 15 is finished at Sun Aug 14 09:39:35 AST 2016, Remove duplicates in NIST7035_result/NIST7035.0.0_bwamem.sort.bam]

[ => SeqMule Execution Status: step 16 is finished at Sun Aug 14 09:39:35 AST 2016, Remove duplicates in NIST7035_result/NIST7035.1.0_bwamem.sort.bam]

[ => SeqMule Execution Status: step 17 is finished at Sun Aug 14 09:39:35 AST 2016, Remove duplicates in NIST7086_result/NIST7086.0.0_bwamem.sort.bam]

[ => SeqMule Execution Status: step 18 is finished at Sun Aug 14 09:39:35 AST 2016, Remove duplicates in NIST7086_result/NIST7086.1.0_bwamem.sort.bam]

[ => SeqMule Execution Status: step 19 is finished at Sun Aug 14 09:39:35 AST 2016, Filter BAM file by mapping quality]

[ => SeqMule Execution Status: step 20 is finished at Sun Aug 14 09:39:35 AST 2016, Filter BAM file by mapping quality]

[ => SeqMule Execution Status: step 21 is finished at Sun Aug 14 09:39:35 AST 2016, Filter BAM file by mapping quality]

[ => SeqMule Execution Status: step 22 is finished at Sun Aug 14 09:39:35 AST 2016, Filter BAM file by mapping quality]

[ => SeqMule Execution Status: step 23 is finished at Sun Aug 14 09:39:35 AST 2016, Merge BAM by bwamem and NIST7035]

[ => SeqMule Execution Status: step 24 is finished at Sun Aug 14 09:39:35 AST 2016, Merge BAM by bwamem and NIST7086]

Current version: 1.2.5 [ => SeqMule Execution Status: step 25 is finished at Sun Aug 14 09:39:35 AST 2016, gatklite realn]

[ => SeqMule Execution Status: step 26 is finished at Sun Aug 14 09:39:35 AST 2016, gatklite realn]

[ => SeqMule Execution Status: step 27 is finished at Sun Aug 14 09:39:35 AST 2016, gatklite.multi-call variant calling]

[ => SeqMule Execution Status: step 28 is finished at Sun Aug 14 09:39:35 AST 2016, Copy results to other samples]

[ => SeqMule Execution Status: step 29 is finished at Sun Aug 14 09:39:35 AST 2016, gatklite.multi-call variant filtering]

[ => SeqMule Execution Status: step 30 is finished at Sun Aug 14 09:39:35 AST 2016, Copy results to other samples]

[ => SeqMule Execution Status: step 31 is finished at Sun Aug 14 09:39:35 AST 2016, samtools.multi-call variant calling]

[ => SeqMule Execution Status: step 32 is finished at Sun Aug 14 09:39:35 AST 2016, Copy results to other samples]

[ => SeqMule Execution Status: step 33 is finished at Sun Aug 14 09:39:35 AST 2016, freebayes.multi-call variant calling]

[ => SeqMule Execution Status: step 34 is finished at Sun Aug 14 09:39:35 AST 2016, Copy results to other samples]

[ => SeqMule Execution Status: step 35 is finished at Sun Aug 14 09:39:35 AST 2016, Extract variants in custom regions]

[ => SeqMule Execution Status: step 36 is finished at Sun Aug 14 09:39:35 AST 2016, Extract variants in custom regions]

[ => SeqMule Execution Status: step 37 is finished at Sun Aug 14 09:39:35 AST 2016, Extract variants in custom regions]

[ => SeqMule Execution Status: step 38 is finished at Sun Aug 14 09:39:35 AST 2016, Extract variants in custom regions]

[ => SeqMule Execution Status: step 39 is finished at Sun Aug 14 09:39:35 AST 2016, Extract variants in custom regions]

[ => SeqMule Execution Status: step 40 is finished at Sun Aug 14 09:39:35 AST 2016, Extract variants in custom regions]

[ => SeqMule Execution Status: step 41 is finished at Sun Aug 14 09:39:35 AST 2016, Extract variants in custom regions]

Check for upgrade ...

----------NOTICE---------- [ => SeqMule Execution Status: Running 43 of 51 steps: Extract consensus calls, at Sun Aug 14 09:39:36 AST 2016, Time Elapsed: 0 hr 0 min 2 s] Current version: 1.2.5 Check for upgrade ...

----------NOTICE---------- [ => SeqMule Execution Status: Running 44 of 51 steps: Generate Venn digram, at Sun Aug 14 09:39:37 AST 2016, Time Elapsed: 0 hr 0 min 3 s] Current version: 1.2.5 Check for upgrade ...

----------NOTICE---------- [ => SeqMule Execution Status: Running 45 of 51 steps: Generate alignment and coverage stat, at Sun Aug 14 09:39:38 AST 2016, Time Elapsed: 0 hr 0 min 4 s] Current version: 1.2.5 Check for upgrade ...

----------NOTICE---------- [ => SeqMule Execution Status: Running 47 of 51 steps: Generate Venn digram, at Sun Aug 14 09:39:39 AST 2016, Time Elapsed: 0 hr 0 min 5 s] Current version: 1.2.5 Check for upgrade ...

----------NOTICE---------- [ => SeqMule Execution Status: Running 48 of 51 steps: Generate alignment and coverage stat, at Sun Aug 14 09:39:40 AST 2016, Time Elapsed: 0 hr 0 min 6 s] Current version: 1.2.5 Check for upgrade ...

----------NOTICE---------- [ => SeqMule Execution Status: Running 50 of 51 steps: Remove intermediate files, at Sun Aug 14 09:39:41 AST 2016, Time Elapsed: 0 hr 0 min 7 s] [ => SeqMule Execution Status: step 50 is finished at Sun Aug 14 09:39:42 AST 2016, Remove intermediate files]

----------ERROR---------- [ => SeqMule Execution Status: step 42 FAILED at Sun Aug 14 09:39:51 AST 2016, Extract consensus calls] ERROR: command failed /work/rashidma/software/seqmule_by_admin/SeqMule-master/bin/secondary/../../bin/secondary/worker /work/rashidma/Exome_data/GIAB_NIST_exome_data/MyAnalysis/04_seqmule/seqmule.08112016.9339.logs 42 "/work/rashidma/software/seqmule_by_admin/SeqMule-master/bin/secondary/../../bin/seqmule stats -tmpdir /tmp -ref /work/rashidma/software/seqmule_by_admin/SeqMule-master/bin/secondary/../../database/human_g1k_v37.fasta -jmem 1750m -jexe java -t 2 -c-vcf NIST7035_result/NIST7035_bwamem.merge.realn.0_gatklite.multi-call.extract.vcf,NIST7035_result/NIST7035_bwamem.merge.0_freebayes.multi-call.extract.vcf,NIST7035_result/NIST7035_bwamem.merge.0_samtools.multi-call.extract.vcf -p NIST7035_result/NIST7035.extract " !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! After fixing the problem, please execute 'cd /work/rashidma/Exome_data/GIAB_NIST_exome_data/MyAnalysis/04_seqmule' and 'seqmule run NIST7035-NIST7086.script' to resume analysis. !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! NOTICE: unable to download '/work/rashidma/software/seqmule_by_admin/SeqMule-master/bin/secondary/../../version' from http://www.openbioinformatics.org/seqmule. Bad Internet connection? NOTICE: unable to check for updates. Results will not be affected. NOTICE: Counting non-empty lines in NIST7086_result/NIST7086_bwamem.merge.realn.0_gatklite.multi-call.extract.vcf NOTICE: Counting non-empty lines in /tmp/122669157.7039602099_nofiltered.vcf NOTICE: 2984 records removed because of being filtered. NOTICE: Counting non-empty lines in NIST7086_result/NIST7086_bwamem.merge.realn.0_gatklite.multi-call.extract.vcf NOTICE: Counting non-empty lines in /tmp/1226610411.1396007496_nofiltered.vcf NOTICE: 2984 records removed because of being filtered. NOTICE: Counting non-empty lines in NIST7086_result/NIST7086_bwamem.merge.0_freebayes.multi-call.extract.vcf NOTICE: Counting non-empty lines in /tmp/122667252.45206305453_nofiltered.vcf NOTICE: 0 records removed because of being filtered. NOTICE: Counting non-empty lines in NIST7086_result/NIST7086_bwamem.merge.0_samtools.multi-call.extract.vcf NOTICE: Counting non-empty lines in /tmp/122661544.42978128392_nofiltered.vcf NOTICE: 0 records removed because of being filtered. ERROR: not all VCFs have the same number of samples! NOTICE: Cleaning... NOTICE: unable to download '/work/rashidma/software/seqmule_by_admin/SeqMule-master/bin/secondary/../../version' from http://www.openbioinformatics.org/seqmule. Bad Internet connection? NOTICE: unable to check for updates. Results will not be affected. NOTICE: normalzing VCF with Vt... NOTICE: unable to download '/work/rashidma/software/seqmule_by_admin/SeqMule-master/bin/secondary/../../version' from http://www.openbioinformatics.org/seqmule. Bad Internet connection? NOTICE: unable to check for updates. Results will not be affected. NOTICE: Calculating coverage for NIST7035_result/NIST7035_bwamem.merge.bam. This takes a while NOTICE: checking contig(chromosome) name consistency in nexterarapidcapture_expandedexome_targetedregions.chrmod.nooverlap.bed and NIST7035_result/NIST7035_bwamem.merge.bam. NOTICE: unable to download '/work/rashidma/software/seqmule_by_admin/SeqMule-master/bin/secondary/../../version' from http://www.openbioinformatics.org/seqmule. Bad Internet connection? NOTICE: unable to check for updates. Results will not be affected. NOTICE: normalzing VCF with Vt... NOTICE: all chromosome names in BED can be found in BAM header. NOTICE: unable to download '/work/rashidma/software/seqmule_by_admin/SeqMule-master/bin/secondary/../../version' from http://www.openbioinformatics.org/seqmule. Bad Internet connection? NOTICE: unable to check for updates. Results will not be affected. NOTICE: Calculating coverage for NIST7086_result/NIST7086_bwamem.merge.bam. This takes a while NOTICE: checking contig(chromosome) name consistency in nexterarapidcapture_expandedexome_targetedregions.chrmod.nooverlap.bed and NIST7086_result/NIST7086_bwamem.merge.bam. NOTICE: Calculating coverage ... NOTICE: Start reading from SAMTools depth command ... NOTICE: done normalzing. NOTICE: Converting input file to ANNOVAR format, variants that failed to pass filtering will be ignored. NOTICE: all chromosome names in BED can be found in BAM header. NOTICE: Counting non-empty lines in /tmp/122851263.03081712854_normalized.vcf NOTICE: Counting non-empty lines in /tmp/1228511153.2504036918_nofiltered.vcf NOTICE: 2983 records removed because of being filtered. NOTICE: output files will be written to /tmp/122856236.40571601722_annovar_raw..avinput NOTICE: Calculating coverage ... NOTICE: Start reading from SAMTools depth command ... NOTICE: Finished reading 54779 lines from VCF file NOTICE: A total of 54651 locus in VCF file passed QC threshold, representing 49556 SNPs (34950 transitions and 14606 transversions) and 5250 indels/substitutions NOTICE: Finished writting 97968 SNPs (69304 transitions and 28664 transversions) and 10375 indels/substitutions for 2 samples NOTICE: Converting input file to ANNOVAR format, variants that failed to pass filtering will be ignored. NOTICE: Counting non-empty lines in /tmp/122852341.14748387559_normalized.vcf NOTICE: Counting non-empty lines in /tmp/1228511095.591970768_nofiltered.vcf NOTICE: 0 records removed because of being filtered. NOTICE: output files will be written to /tmp/1228510870.9041279158_annovar_raw..avinput NOTICE: Finished reading 53617 lines from VCF file NOTICE: A total of 53496 locus in VCF file passed QC threshold, representing 48758 SNPs (33513 transitions and 15245 transversions) and 5821 indels/substitutions NOTICE: Finished writting 94602 SNPs (66260 transitions and 28342 transversions) and 9256 indels/substitutions for 2 samples NOTICE: Converting input file to ANNOVAR format, variants that failed to pass filtering will be ignored. NOTICE: Counting non-empty lines in /tmp/122856339.23799349625_normalized.vcf NOTICE: Counting non-empty lines in /tmp/1228510481.4337290995_nofiltered.vcf NOTICE: 0 records removed because of being filtered. NOTICE: output files will be written to /tmp/122857037.94419306214_annovar_raw..avinput NOTICE: Finished reading 52571 lines from VCF file NOTICE: A total of 52514 locus in VCF file passed QC threshold, representing 47737 SNPs (33510 transitions and 14227 transversions) and 5059 indels/substitutions NOTICE: Finished writting 47510 SNPs (33447 transitions and 14063 transversions) and 5050 indels/substitutions for 1 samples NOTICE: done normalzing. NOTICE: Converting input file to ANNOVAR format, variants that failed to pass filtering will be ignored. NOTICE: Counting non-empty lines in /tmp/123276567.88189709897_normalized.vcf NOTICE: Counting non-empty lines in /tmp/1232711629.1239015117_nofiltered.vcf NOTICE: 2983 records removed because of being filtered. NOTICE: output files will be written to /tmp/123277320.47123258426_annovar_raw..avinput NOTICE: Finished reading 54779 lines from VCF file NOTICE: A total of 54651 locus in VCF file passed QC threshold, representing 49556 SNPs (34950 transitions and 14606 transversions) and 5250 indels/substitutions NOTICE: Finished writting 97968 SNPs (69304 transitions and 28664 transversions) and 10375 indels/substitutions for 2 samples NOTICE: Converting input file to ANNOVAR format, variants that failed to pass filtering will be ignored. NOTICE: Counting non-empty lines in /tmp/123279980.29479589375_normalized.vcf NOTICE: Generating Venn diagram... NOTICE: Counting non-empty lines in /tmp/123279760.87166573345_nofiltered.vcf NOTICE: 2983 records removed because of being filtered. NOTICE: output files will be written to /tmp/123272050.77081613519_annovar_raw..avinput Loading required package: VennDiagram Loading required package: grid [1] 1 NOTICE: Venn Digram plotted to NIST7035_result/NIST7035_SNV_venn.tiff and NIST7035_result/NIST7035_SNV_venn.jpg NOTICE: Generating Venn diagram... Loading required package: VennDiagram Loading required package: grid NOTICE: Finished reading 54779 lines from VCF file NOTICE: A total of 54651 locus in VCF file passed QC threshold, representing 49556 SNPs (34950 transitions and 14606 transversions) and 5250 indels/substitutions NOTICE: Finished writting 97968 SNPs (69304 transitions and 28664 transversions) and 10375 indels/substitutions for 2 samples NOTICE: Converting input file to ANNOVAR format, variants that failed to pass filtering will be ignored. NOTICE: Counting non-empty lines in /tmp/123274155.04472879646_normalized.vcf NOTICE: Counting non-empty lines in /tmp/123279095.91889599042_nofiltered.vcf NOTICE: 0 records removed because of being filtered. NOTICE: output files will be written to /tmp/123277311.11218015558_annovar_raw..avinput [1] 1 NOTICE: Venn Digram plotted to NIST7035_result/NIST7035_NON-SNV_venn.tiff and NIST7035_result/NIST7035_NON-SNV_venn.jpg NOTICE: Cleaning... NOTICE: Finished reading 53617 lines from VCF file NOTICE: A total of 53496 locus in VCF file passed QC threshold, representing 48758 SNPs (33513 transitions and 15245 transversions) and 5821 indels/substitutions NOTICE: Finished writting 94602 SNPs (66260 transitions and 28342 transversions) and 9256 indels/substitutions for 2 samples NOTICE: Converting input file to ANNOVAR format, variants that failed to pass filtering will be ignored. NOTICE: Counting non-empty lines in /tmp/12327320.042136803374_normalized.vcf NOTICE: Counting non-empty lines in /tmp/1232711378.1579504381_nofiltered.vcf NOTICE: 0 records removed because of being filtered. NOTICE: output files will be written to /tmp/123272550.61668376584_annovar_raw..avinput NOTICE: Finished reading 52571 lines from VCF file NOTICE: A total of 52514 locus in VCF file passed QC threshold, representing 47737 SNPs (33510 transitions and 14227 transversions) and 5059 indels/substitutions NOTICE: Finished writting 47510 SNPs (33447 transitions and 14063 transversions) and 5050 indels/substitutions for 1 samples mv: cannot stat `/tmp/123272550.61668376584_annovar_raw.NIST7086.avinput': No such file or directory ERROR:Bad file descriptor NOTICE: Cleaning... NOTICE: nexterarapidcapture_expandedexome_targetedregions.chrmod.nooverlap.bed contains 62085286 base pairs Statistics written to NIST7035_result/NIST7035_cov_stat.txt and NIST7035_result/NIST7035_cov_stat_detail.txt NOTICE: coverage plot <<NIST7035_result/NIST7035_cov.jpg>> generated NOTICE: Extracting primary alignments NOTICE: nexterarapidcapture_expandedexome_targetedregions.chrmod.nooverlap.bed contains 62085286 base pairs Statistics written to NIST7086_result/NIST7086_cov_stat.txt and NIST7086_result/NIST7086_cov_stat_detail.txt NOTICE: coverage plot <<NIST7086_result/NIST7086_cov.jpg>> generated NOTICE: Extracting primary alignments Generating alignment stats Output written to NIST7035_result/NIST7035_aln_stat.txt NOTICE: Cleaning... Generating alignment stats Output written to NIST7086_result/NIST7086_aln_stat.txt NOTICE: Cleaning... Current version: 1.2.5 Check for upgrade ... NOTICE: unable to download '/work/rashidma/software/seqmule_by_admin/SeqMule-master/bin/secondary/../../version' from http://www.openbioinformatics.org/seqmule. Bad Internet connection? NOTICE: unable to check for updates. Results will not be affected. ****EXECUTION BEGINS at Sun Aug 14 13:17:15 AST 2016****

----------NOTICE---------- [ => SeqMule Execution Status: Running 42 of 51 steps: Extract consensus calls, at Sun Aug 14 13:17:16 AST 2016, Time Elapsed: 0 hr 0 min 1 s] [ => SeqMule Execution Status: step 1 is finished at Sun Aug 14 13:17:16 AST 2016, QC assesment on input]

[ => SeqMule Execution Status: step 2 is finished at Sun Aug 14 13:17:16 AST 2016, QC assesment on input]

[ => SeqMule Execution Status: step 3 is finished at Sun Aug 14 13:17:16 AST 2016, QC assesment on input]

[ => SeqMule Execution Status: step 4 is finished at Sun Aug 14 13:17:16 AST 2016, QC assesment on input]

[ => SeqMule Execution Status: step 5 is finished at Sun Aug 14 13:17:16 AST 2016, QC assesment on input]

[ => SeqMule Execution Status: step 6 is finished at Sun Aug 14 13:17:16 AST 2016, QC assesment on input]

[ => SeqMule Execution Status: step 7 is finished at Sun Aug 14 13:17:16 AST 2016, QC assesment on input]

[ => SeqMule Execution Status: step 8 is finished at Sun Aug 14 13:17:16 AST 2016, QC assesment on input]

[ => SeqMule Execution Status: step 9 is finished at Sun Aug 14 13:17:16 AST 2016, Generate QC stat]

[ => SeqMule Execution Status: step 10 is finished at Sun Aug 14 13:17:16 AST 2016, Generate QC stat]

[ => SeqMule Execution Status: step 11 is finished at Sun Aug 14 13:17:16 AST 2016, bwamem alignment]

[ => SeqMule Execution Status: step 12 is finished at Sun Aug 14 13:17:16 AST 2016, bwamem alignment]

[ => SeqMule Execution Status: step 13 is finished at Sun Aug 14 13:17:16 AST 2016, bwamem alignment]

[ => SeqMule Execution Status: step 14 is finished at Sun Aug 14 13:17:16 AST 2016, bwamem alignment]

[ => SeqMule Execution Status: step 15 is finished at Sun Aug 14 13:17:16 AST 2016, Remove duplicates in NIST7035_result/NIST7035.0.0_bwamem.sort.bam]

[ => SeqMule Execution Status: step 16 is finished at Sun Aug 14 13:17:16 AST 2016, Remove duplicates in NIST7035_result/NIST7035.1.0_bwamem.sort.bam]

[ => SeqMule Execution Status: step 17 is finished at Sun Aug 14 13:17:16 AST 2016, Remove duplicates in NIST7086_result/NIST7086.0.0_bwamem.sort.bam]

[ => SeqMule Execution Status: step 18 is finished at Sun Aug 14 13:17:16 AST 2016, Remove duplicates in NIST7086_result/NIST7086.1.0_bwamem.sort.bam]

[ => SeqMule Execution Status: step 19 is finished at Sun Aug 14 13:17:16 AST 2016, Filter BAM file by mapping quality]

[ => SeqMule Execution Status: step 20 is finished at Sun Aug 14 13:17:16 AST 2016, Filter BAM file by mapping quality]

[ => SeqMule Execution Status: step 21 is finished at Sun Aug 14 13:17:16 AST 2016, Filter BAM file by mapping quality]

[ => SeqMule Execution Status: step 22 is finished at Sun Aug 14 13:17:16 AST 2016, Filter BAM file by mapping quality]

[ => SeqMule Execution Status: step 23 is finished at Sun Aug 14 13:17:16 AST 2016, Merge BAM by bwamem and NIST7035]

[ => SeqMule Execution Status: step 24 is finished at Sun Aug 14 13:17:16 AST 2016, Merge BAM by bwamem and NIST7086]

[ => SeqMule Execution Status: step 25 is finished at Sun Aug 14 13:17:16 AST 2016, gatklite realn]

Current version: 1.2.5 [ => SeqMule Execution Status: step 26 is finished at Sun Aug 14 13:17:16 AST 2016, gatklite realn]

[ => SeqMule Execution Status: step 27 is finished at Sun Aug 14 13:17:16 AST 2016, gatklite.multi-call variant calling]

[ => SeqMule Execution Status: step 28 is finished at Sun Aug 14 13:17:16 AST 2016, Copy results to other samples]

[ => SeqMule Execution Status: step 29 is finished at Sun Aug 14 13:17:16 AST 2016, gatklite.multi-call variant filtering]

[ => SeqMule Execution Status: step 30 is finished at Sun Aug 14 13:17:16 AST 2016, Copy results to other samples]

[ => SeqMule Execution Status: step 31 is finished at Sun Aug 14 13:17:16 AST 2016, samtools.multi-call variant calling]

[ => SeqMule Execution Status: step 32 is finished at Sun Aug 14 13:17:16 AST 2016, Copy results to other samples]

[ => SeqMule Execution Status: step 33 is finished at Sun Aug 14 13:17:16 AST 2016, freebayes.multi-call variant calling]

[ => SeqMule Execution Status: step 34 is finished at Sun Aug 14 13:17:16 AST 2016, Copy results to other samples]

[ => SeqMule Execution Status: step 35 is finished at Sun Aug 14 13:17:16 AST 2016, Extract variants in custom regions]

[ => SeqMule Execution Status: step 36 is finished at Sun Aug 14 13:17:16 AST 2016, Extract variants in custom regions]

[ => SeqMule Execution Status: step 37 is finished at Sun Aug 14 13:17:16 AST 2016, Extract variants in custom regions]

[ => SeqMule Execution Status: step 38 is finished at Sun Aug 14 13:17:16 AST 2016, Extract variants in custom regions]

[ => SeqMule Execution Status: step 39 is finished at Sun Aug 14 13:17:16 AST 2016, Extract variants in custom regions]

[ => SeqMule Execution Status: step 40 is finished at Sun Aug 14 13:17:16 AST 2016, Extract variants in custom regions]

[ => SeqMule Execution Status: step 41 is finished at Sun Aug 14 13:17:16 AST 2016, Extract variants in custom regions]

Check for upgrade ...

----------NOTICE---------- [ => SeqMule Execution Status: Running 43 of 51 steps: Extract consensus calls, at Sun Aug 14 13:17:17 AST 2016, Time Elapsed: 0 hr 0 min 2 s] Current version: 1.2.5 Check for upgrade ...

----------NOTICE---------- [ => SeqMule Execution Status: Running 44 of 51 steps: Generate Venn digram, at Sun Aug 14 13:17:18 AST 2016, Time Elapsed: 0 hr 0 min 3 s] Current version: 1.2.5 Check for upgrade ...

----------NOTICE---------- [ => SeqMule Execution Status: Running 45 of 51 steps: Generate alignment and coverage stat, at Sun Aug 14 13:17:19 AST 2016, Time Elapsed: 0 hr 0 min 4 s] Current version: 1.2.5 Check for upgrade ...

----------NOTICE---------- [ => SeqMule Execution Status: Running 47 of 51 steps: Generate Venn digram, at Sun Aug 14 13:17:20 AST 2016, Time Elapsed: 0 hr 0 min 5 s] Current version: 1.2.5 Check for upgrade ...

----------NOTICE---------- [ => SeqMule Execution Status: Running 48 of 51 steps: Generate alignment and coverage stat, at Sun Aug 14 13:17:21 AST 2016, Time Elapsed: 0 hr 0 min 6 s] Current version: 1.2.5 Check for upgrade ...

----------NOTICE---------- [ => SeqMule Execution Status: Running 50 of 51 steps: Remove intermediate files, at Sun Aug 14 13:17:22 AST 2016, Time Elapsed: 0 hr 0 min 7 s] [ => SeqMule Execution Status: step 50 is finished at Sun Aug 14 13:17:23 AST 2016, Remove intermediate files]

----------ERROR---------- [ => SeqMule Execution Status: step 42 FAILED at Sun Aug 14 13:17:32 AST 2016, Extract consensus calls] ERROR: command failed /work/rashidma/software/seqmule_by_admin/SeqMule-master/bin/secondary/../../bin/secondary/worker /work/rashidma/Exome_data/GIAB_NIST_exome_data/MyAnalysis/04_seqmule/seqmule.08112016.9339.logs 42 "/work/rashidma/software/seqmule_by_admin/SeqMule-master/bin/secondary/../../bin/seqmule stats -tmpdir /tmp -ref /work/rashidma/software/seqmule_by_admin/SeqMule-master/bin/secondary/../../database/human_g1k_v37.fasta -jmem 1750m -jexe java -t 2 -c-vcf NIST7035_result/NIST7035_bwamem.merge.realn.0_gatklite.multi-call.extract.vcf,NIST7035_result/NIST7035_bwamem.merge.0_freebayes.multi-call.extract.vcf,NIST7035_result/NIST7035_bwamem.merge.0_samtools.multi-call.extract.vcf -p NIST7035_result/NIST7035.extract " !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! After fixing the problem, please execute 'cd /work/rashidma/Exome_data/GIAB_NIST_exome_data/MyAnalysis/04_seqmule' and 'seqmule run NIST7035-NIST7086.script' to resume analysis. !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! NOTICE: unable to download '/work/rashidma/software/seqmule_by_admin/SeqMule-master/bin/secondary/../../version' from http://www.openbioinformatics.org/seqmule. Bad Internet connection? NOTICE: unable to check for updates. Results will not be affected. NOTICE: Counting non-empty lines in NIST7086_result/NIST7086_bwamem.merge.realn.0_gatklite.multi-call.extract.vcf NOTICE: Counting non-empty lines in /tmp/123345208.69531140168_nofiltered.vcf NOTICE: 2984 records removed because of being filtered. NOTICE: Counting non-empty lines in NIST7086_result/NIST7086_bwamem.merge.realn.0_gatklite.multi-call.extract.vcf NOTICE: Counting non-empty lines in /tmp/1233411431.7051270461_nofiltered.vcf NOTICE: 2984 records removed because of being filtered. NOTICE: Counting non-empty lines in NIST7086_result/NIST7086_bwamem.merge.0_freebayes.multi-call.extract.vcf NOTICE: Counting non-empty lines in /tmp/123341698.80131634328_nofiltered.vcf NOTICE: 0 records removed because of being filtered. NOTICE: Counting non-empty lines in NIST7086_result/NIST7086_bwamem.merge.0_samtools.multi-call.extract.vcf NOTICE: Counting non-empty lines in /tmp/123346028.90369492563_nofiltered.vcf NOTICE: 0 records removed because of being filtered. ERROR: not all VCFs have the same number of samples! NOTICE: Cleaning... NOTICE: unable to download '/work/rashidma/software/seqmule_by_admin/SeqMule-master/bin/secondary/../../version' from http://www.openbioinformatics.org/seqmule. Bad Internet connection? NOTICE: unable to check for updates. Results will not be affected. NOTICE: normalzing VCF with Vt... NOTICE: unable to download '/work/rashidma/software/seqmule_by_admin/SeqMule-master/bin/secondary/../../version' from http://www.openbioinformatics.org/seqmule. Bad Internet connection? NOTICE: unable to check for updates. Results will not be affected. NOTICE: Calculating coverage for NIST7035_result/NIST7035_bwamem.merge.bam. This takes a while NOTICE: checking contig(chromosome) name consistency in nexterarapidcapture_expandedexome_targetedregions.chrmod.nooverlap.bed and NIST7035_result/NIST7035_bwamem.merge.bam. NOTICE: unable to download '/work/rashidma/software/seqmule_by_admin/SeqMule-master/bin/secondary/../../version' from http://www.openbioinformatics.org/seqmule. Bad Internet connection? NOTICE: unable to check for updates. Results will not be affected. NOTICE: normalzing VCF with Vt... NOTICE: all chromosome names in BED can be found in BAM header. NOTICE: unable to download '/work/rashidma/software/seqmule_by_admin/SeqMule-master/bin/secondary/../../version' from http://www.openbioinformatics.org/seqmule. Bad Internet connection? NOTICE: unable to check for updates. Results will not be affected. NOTICE: Calculating coverage for NIST7086_result/NIST7086_bwamem.merge.bam. This takes a while NOTICE: checking contig(chromosome) name consistency in nexterarapidcapture_expandedexome_targetedregions.chrmod.nooverlap.bed and NIST7086_result/NIST7086_bwamem.merge.bam. NOTICE: done normalzing. NOTICE: Converting input file to ANNOVAR format, variants that failed to pass filtering will be ignored. NOTICE: Counting non-empty lines in /tmp/123531905.58289809473_normalized.vcf NOTICE: Counting non-empty lines in /tmp/1235311291.479818785_nofiltered.vcf NOTICE: 2983 records removed because of being filtered. NOTICE: output files will be written to /tmp/123531860.38965316354_annovar_raw..avinput NOTICE: Calculating coverage ... NOTICE: Start reading from SAMTools depth command ... NOTICE: all chromosome names in BED can be found in BAM header. NOTICE: Calculating coverage ... NOTICE: Start reading from SAMTools depth command ... NOTICE: Finished reading 54779 lines from VCF file NOTICE: A total of 54651 locus in VCF file passed QC threshold, representing 49556 SNPs (34950 transitions and 14606 transversions) and 5250 indels/substitutions NOTICE: Finished writting 97968 SNPs (69304 transitions and 28664 transversions) and 10375 indels/substitutions for 2 samples NOTICE: Converting input file to ANNOVAR format, variants that failed to pass filtering will be ignored. NOTICE: Counting non-empty lines in /tmp/12353766.953784874118_normalized.vcf NOTICE: Counting non-empty lines in /tmp/123534872.44339684713_nofiltered.vcf NOTICE: 0 records removed because of being filtered. NOTICE: output files will be written to /tmp/123533944.48215996573_annovar_raw..avinput NOTICE: Finished reading 53617 lines from VCF file NOTICE: A total of 53496 locus in VCF file passed QC threshold, representing 48758 SNPs (33513 transitions and 15245 transversions) and 5821 indels/substitutions NOTICE: Finished writting 94602 SNPs (66260 transitions and 28342 transversions) and 9256 indels/substitutions for 2 samples NOTICE: Converting input file to ANNOVAR format, variants that failed to pass filtering will be ignored. NOTICE: Counting non-empty lines in /tmp/1235311227.3700838786_normalized.vcf NOTICE: Counting non-empty lines in /tmp/123535082.36007615542_nofiltered.vcf NOTICE: 0 records removed because of being filtered. NOTICE: output files will be written to /tmp/123539675.61198998177_annovar_raw..avinput NOTICE: Finished reading 52571 lines from VCF file NOTICE: A total of 52514 locus in VCF file passed QC threshold, representing 47737 SNPs (33510 transitions and 14227 transversions) and 5059 indels/substitutions NOTICE: Finished writting 47510 SNPs (33447 transitions and 14063 transversions) and 5050 indels/substitutions for 1 samples NOTICE: done normalzing. NOTICE: Converting input file to ANNOVAR format, variants that failed to pass filtering will be ignored. NOTICE: Counting non-empty lines in /tmp/124176312.30427180976_normalized.vcf NOTICE: Counting non-empty lines in /tmp/1241710428.9039150244_nofiltered.vcf NOTICE: 2983 records removed because of being filtered. NOTICE: output files will be written to /tmp/124176746.48781616289_annovar_raw..avinput NOTICE: Generating Venn diagram... Loading required package: VennDiagram Loading required package: grid NOTICE: Finished reading 54779 lines from VCF file NOTICE: A total of 54651 locus in VCF file passed QC threshold, representing 49556 SNPs (34950 transitions and 14606 transversions) and 5250 indels/substitutions NOTICE: Finished writting 97968 SNPs (69304 transitions and 28664 transversions) and 10375 indels/substitutions for 2 samples NOTICE: Converting input file to ANNOVAR format, variants that failed to pass filtering will be ignored. NOTICE: Counting non-empty lines in /tmp/124172117.70279018037_normalized.vcf NOTICE: Counting non-empty lines in /tmp/124176961.42663685602_nofiltered.vcf NOTICE: 2983 records removed because of being filtered. NOTICE: output files will be written to /tmp/124172981.88346578824_annovar_raw..avinput [1] 1 NOTICE: Venn Digram plotted to NIST7035_result/NIST7035_SNV_venn.tiff and NIST7035_result/NIST7035_SNV_venn.jpg NOTICE: Generating Venn diagram... Loading required package: VennDiagram Loading required package: grid NOTICE: Finished reading 54779 lines from VCF file NOTICE: A total of 54651 locus in VCF file passed QC threshold, representing 49556 SNPs (34950 transitions and 14606 transversions) and 5250 indels/substitutions NOTICE: Finished writting 97968 SNPs (69304 transitions and 28664 transversions) and 10375 indels/substitutions for 2 samples NOTICE: Converting input file to ANNOVAR format, variants that failed to pass filtering will be ignored. [1] 1 NOTICE: Counting non-empty lines in /tmp/124175165.74573991909_normalized.vcf NOTICE: Counting non-empty lines in /tmp/1241711213.5758299978_nofiltered.vcf NOTICE: 0 records removed because of being filtered. NOTICE: output files will be written to /tmp/124179109.8503215057_annovar_raw..avinput NOTICE: Venn Digram plotted to NIST7035_result/NIST7035_NON-SNV_venn.tiff and NIST7035_result/NIST7035_NON-SNV_venn.jpg NOTICE: Cleaning... NOTICE: Finished reading 53617 lines from VCF file NOTICE: A total of 53496 locus in VCF file passed QC threshold, representing 48758 SNPs (33513 transitions and 15245 transversions) and 5821 indels/substitutions NOTICE: Finished writting 94602 SNPs (66260 transitions and 28342 transversions) and 9256 indels/substitutions for 2 samples NOTICE: Converting input file to ANNOVAR format, variants that failed to pass filtering will be ignored. NOTICE: Counting non-empty lines in /tmp/124176909.82132034195_normalized.vcf NOTICE: Counting non-empty lines in /tmp/124176244.624832915_nofiltered.vcf NOTICE: 0 records removed because of being filtered. NOTICE: output files will be written to /tmp/124179650.81970514196_annovar_raw..avinput NOTICE: Finished reading 52571 lines from VCF file NOTICE: A total of 52514 locus in VCF file passed QC threshold, representing 47737 SNPs (33510 transitions and 14227 transversions) and 5059 indels/substitutions NOTICE: Finished writting 47510 SNPs (33447 transitions and 14063 transversions) and 5050 indels/substitutions for 1 samples mv: cannot stat `/tmp/124179650.81970514196_annovar_raw.NIST7086.avinput': No such file or directory ERROR:Bad file descriptor NOTICE: Cleaning... NOTICE: nexterarapidcapture_expandedexome_targetedregions.chrmod.nooverlap.bed contains 62085286 base pairs Statistics written to NIST7035_result/NIST7035_cov_stat.txt and NIST7035_result/NIST7035_cov_stat_detail.txt NOTICE: coverage plot <<NIST7035_result/NIST7035_cov.jpg>> generated NOTICE: Extracting primary alignments NOTICE: nexterarapidcapture_expandedexome_targetedregions.chrmod.nooverlap.bed contains 62085286 base pairs Statistics written to NIST7086_result/NIST7086_cov_stat.txt and NIST7086_result/NIST7086_cov_stat_detail.txt NOTICE: coverage plot <<NIST7086_result/NIST7086_cov.jpg>> generated NOTICE: Extracting primary alignments Generating alignment stats Output written to NIST7035_result/NIST7035_aln_stat.txt NOTICE: Cleaning... Generating alignment stats Output written to NIST7086_result/NIST7086_aln_stat.txt NOTICE: Cleaning... Current version: 1.2.5 Check for upgrade ... NOTICE: unable to download '/work/rashidma/software/seqmule_by_admin/SeqMule-master/bin/secondary/../../version' from http://www.openbioinformatics.org/seqmule. Bad Internet connection? NOTICE: unable to check for updates. Results will not be affected. ****EXECUTION BEGINS at Mon Aug 15 08:29:28 AST 2016****

----------NOTICE---------- [ => SeqMule Execution Status: Running 42 of 51 steps: Extract consensus calls, at Mon Aug 15 08:29:29 AST 2016, Time Elapsed: 0 hr 0 min 1 s] [ => SeqMule Execution Status: step 1 is finished at Mon Aug 15 08:29:29 AST 2016, QC assesment on input]

[ => SeqMule Execution Status: step 2 is finished at Mon Aug 15 08:29:29 AST 2016, QC assesment on input]

[ => SeqMule Execution Status: step 3 is finished at Mon Aug 15 08:29:29 AST 2016, QC assesment on input]

[ => SeqMule Execution Status: step 4 is finished at Mon Aug 15 08:29:29 AST 2016, QC assesment on input]

[ => SeqMule Execution Status: step 5 is finished at Mon Aug 15 08:29:29 AST 2016, QC assesment on input]

[ => SeqMule Execution Status: step 6 is finished at Mon Aug 15 08:29:29 AST 2016, QC assesment on input]

[ => SeqMule Execution Status: step 7 is finished at Mon Aug 15 08:29:29 AST 2016, QC assesment on input]

[ => SeqMule Execution Status: step 8 is finished at Mon Aug 15 08:29:29 AST 2016, QC assesment on input]

[ => SeqMule Execution Status: step 9 is finished at Mon Aug 15 08:29:29 AST 2016, Generate QC stat]

[ => SeqMule Execution Status: step 10 is finished at Mon Aug 15 08:29:29 AST 2016, Generate QC stat]

[ => SeqMule Execution Status: step 11 is finished at Mon Aug 15 08:29:29 AST 2016, bwamem alignment]

[ => SeqMule Execution Status: step 12 is finished at Mon Aug 15 08:29:29 AST 2016, bwamem alignment]

[ => SeqMule Execution Status: step 13 is finished at Mon Aug 15 08:29:29 AST 2016, bwamem alignment]

[ => SeqMule Execution Status: step 14 is finished at Mon Aug 15 08:29:29 AST 2016, bwamem alignment]

[ => SeqMule Execution Status: step 15 is finished at Mon Aug 15 08:29:29 AST 2016, Remove duplicates in NIST7035_result/NIST7035.0.0_bwamem.sort.bam]

[ => SeqMule Execution Status: step 16 is finished at Mon Aug 15 08:29:29 AST 2016, Remove duplicates in NIST7035_result/NIST7035.1.0_bwamem.sort.bam]

[ => SeqMule Execution Status: step 17 is finished at Mon Aug 15 08:29:29 AST 2016, Remove duplicates in NIST7086_result/NIST7086.0.0_bwamem.sort.bam]

[ => SeqMule Execution Status: step 18 is finished at Mon Aug 15 08:29:29 AST 2016, Remove duplicates in NIST7086_result/NIST7086.1.0_bwamem.sort.bam]

[ => SeqMule Execution Status: step 19 is finished at Mon Aug 15 08:29:29 AST 2016, Filter BAM file by mapping quality]

[ => SeqMule Execution Status: step 20 is finished at Mon Aug 15 08:29:29 AST 2016, Filter BAM file by mapping quality]

[ => SeqMule Execution Status: step 21 is finished at Mon Aug 15 08:29:29 AST 2016, Filter BAM file by mapping quality]

[ => SeqMule Execution Status: step 22 is finished at Mon Aug 15 08:29:29 AST 2016, Filter BAM file by mapping quality]

[ => SeqMule Execution Status: step 23 is finished at Mon Aug 15 08:29:29 AST 2016, Merge BAM by bwamem and NIST7035]

[ => SeqMule Execution Status: step 24 is finished at Mon Aug 15 08:29:29 AST 2016, Merge BAM by bwamem and NIST7086]

[ => SeqMule Execution Status: step 25 is finished at Mon Aug 15 08:29:29 AST 2016, gatklite realn]

Current version: 1.2.5 [ => SeqMule Execution Status: step 26 is finished at Mon Aug 15 08:29:29 AST 2016, gatklite realn]

[ => SeqMule Execution Status: step 27 is finished at Mon Aug 15 08:29:29 AST 2016, gatklite.multi-call variant calling]

[ => SeqMule Execution Status: step 28 is finished at Mon Aug 15 08:29:29 AST 2016, Copy results to other samples]

[ => SeqMule Execution Status: step 29 is finished at Mon Aug 15 08:29:29 AST 2016, gatklite.multi-call variant filtering]

[ => SeqMule Execution Status: step 30 is finished at Mon Aug 15 08:29:29 AST 2016, Copy results to other samples]

[ => SeqMule Execution Status: step 31 is finished at Mon Aug 15 08:29:29 AST 2016, samtools.multi-call variant calling]

[ => SeqMule Execution Status: step 32 is finished at Mon Aug 15 08:29:29 AST 2016, Copy results to other samples]

[ => SeqMule Execution Status: step 33 is finished at Mon Aug 15 08:29:29 AST 2016, freebayes.multi-call variant calling]

[ => SeqMule Execution Status: step 34 is finished at Mon Aug 15 08:29:29 AST 2016, Copy results to other samples]

[ => SeqMule Execution Status: step 35 is finished at Mon Aug 15 08:29:29 AST 2016, Extract variants in custom regions]

[ => SeqMule Execution Status: step 36 is finished at Mon Aug 15 08:29:29 AST 2016, Extract variants in custom regions]

[ => SeqMule Execution Status: step 37 is finished at Mon Aug 15 08:29:29 AST 2016, Extract variants in custom regions]

[ => SeqMule Execution Status: step 38 is finished at Mon Aug 15 08:29:29 AST 2016, Extract variants in custom regions]

[ => SeqMule Execution Status: step 39 is finished at Mon Aug 15 08:29:29 AST 2016, Extract variants in custom regions]

[ => SeqMule Execution Status: step 40 is finished at Mon Aug 15 08:29:29 AST 2016, Extract variants in custom regions]

[ => SeqMule Execution Status: step 41 is finished at Mon Aug 15 08:29:29 AST 2016, Extract variants in custom regions]

Check for upgrade ...

----------NOTICE---------- [ => SeqMule Execution Status: Running 43 of 51 steps: Extract consensus calls, at Mon Aug 15 08:29:30 AST 2016, Time Elapsed: 0 hr 0 min 2 s] Current version: 1.2.5 Check for upgrade ...

----------NOTICE---------- [ => SeqMule Execution Status: Running 44 of 51 steps: Generate Venn digram, at Mon Aug 15 08:29:31 AST 2016, Time Elapsed: 0 hr 0 min 3 s] Current version: 1.2.5 Check for upgrade ...

----------NOTICE---------- [ => SeqMule Execution Status: Running 45 of 51 steps: Generate alignment and coverage stat, at Mon Aug 15 08:29:32 AST 2016, Time Elapsed: 0 hr 0 min 4 s] Current version: 1.2.5 Check for upgrade ...

----------NOTICE---------- [ => SeqMule Execution Status: Running 47 of 51 steps: Generate Venn digram, at Mon Aug 15 08:29:33 AST 2016, Time Elapsed: 0 hr 0 min 5 s] Current version: 1.2.5 Check for upgrade ...

----------NOTICE---------- [ => SeqMule Execution Status: Running 48 of 51 steps: Generate alignment and coverage stat, at Mon Aug 15 08:29:34 AST 2016, Time Elapsed: 0 hr 0 min 6 s] Current version: 1.2.5 Check for upgrade ...

----------NOTICE---------- [ => SeqMule Execution Status: Running 50 of 51 steps: Remove intermediate files, at Mon Aug 15 08:29:35 AST 2016, Time Elapsed: 0 hr 0 min 7 s] [ => SeqMule Execution Status: step 50 is finished at Mon Aug 15 08:29:37 AST 2016, Remove intermediate files]

rashidma commented 7 years ago

Hi, I just noticed in the _.log file of seqmule run -- the VCF " NIST7086_result/NIST7086_bwamem.merge.realn.0gatklite.multi-call.extract.vcf" has been mentioned twice. please see below colored in red. Is this creating proble?? Per sample there should be 4 VCF files (3 different aligner variant caller combinations and one consensus) BUT in case of NIST7086 why this is 5 VCF. *

_2 sample(s) in this analysis: NIST7035 NIST7086All input is merged on a per-sample basis (after alignment if FASTQ).Multi-sample variant calling ENABLED.Input is exome (or captured) sequencing data.4 variant caller(s) used: GATKLite_UnifiedGenotyper SAMtools FreeBayes Consensus1 aligner(s) used: bwamemFile used for caculating coverage statistics and extracting variants: nexterarapidcapture_expandedexome_targetedregions.nooverlap.bedReadgroup : READGROUP_NIST7035,READGROUP_NIST7086Library : LIBRARYSequencing platform: ILLUMINAReference genome build is hg19dbsnp138 is used for variant calling and recalibration (in GATK VQSR).Java memory usage is limited to 1750mjava executable path: javaMax number of processes: 12NOTICE: /tmp will be used for storing temporary files###################################################FINAL OUTPUT for NIST7035:###################################################NOTICE: Both consensus results and individual results are listed.WARNING: consensus results may not be available because some variant callers or aligners may fail to generate output.NIST7035_result/NIST7035.extract_consensus.vcfNIST7035_result/NIST7035_bwamem.merge.realn.0_gatklite.multi-call.extract.vcfNIST7035_result/NIST7035_bwamem.merge.0_samtools.multi-call.extract.vcfNIST7035_result/NIST7035_bwamem.merge.0freebayes.multi-call.extract.vcf###################################################FINAL OUTPUT for NIST7086:###################################################NOTICE: Both consensus results and individual results are listed.WARNING: consensus results may not be available because some variant callers or aligners may fail to generate output.

_NIST7086_result/NIST7086.extract_consensus.vcfNIST7086_result/NIST7086_bwamem.merge.realn.0_gatklite.multi-call.extract.vcfNIST7086_result/NIST7086_bwamem.merge.realn.0_gatklite.multi-call.extract.vcfNIST7086_result/NIST7086_bwamem.merge.0_samtools.multi-call.extract.vcfNIST7086_result/NIST7086_bwamem.merge.0freebayes.multi-call.extract.vcf

Thanks

On Tue, Aug 16, 2016 at 8:07 AM, Mamoon Rashid rashidmamoon@gmail.com wrote:

Hi Yunfei, I sent you the big nohup file (~8MB) and it failed to reach you. Now I am sending you the last 1000 lines of the nohup file. I can see there are errors at step 42 and 43. I thank you so much for your time. MR

On Mon, Aug 15, 2016 at 4:05 PM, Yunfei Guo notifications@github.com wrote:

Hi Rashid,

Could you please provide the contents of nohup.out file? I need to know where it stopped.

Thanks.

Best, Yunfei

On Mon, Aug 15, 2016 at 12:47 AM, rashidma notifications@github.com wrote:

Thanks a lot Yunfei, I successfully ran the test run. Now, I was running a real sample from NIST. Out of 51 steps 48 steps ran well. I am stuck in the last few steps. The seqmule was stopped many times due to system shutdown and couple of times I killed the process. But every time I restarted it with--

nohup seqmule run NIST7035-NIST7086.script &

Please let me know any troubleshooting. I will be thankful to you. Regards MR

On Thu, Aug 11, 2016 at 1:02 AM, Yunfei Guo notifications@github.com wrote:

Hi Mamoon,

Please use inst/hg19_exome.legacy.bed after updating SeqMule with seqmule update --git. Thank you!

Best, Yunfei

— You are receiving this because you authored the thread. Reply to this email directly, view it on GitHub https://github.com/WGLab/SeqMule/issues/146#issuecomment-239018748, or mute the thread https://github.com/notifications/unsubscribe-auth/ ATyiOAPu4as9L3bQ8R0fedRCtTQ976qQks5qekpygaJpZM4Jf64z .

Mamoon Rashid, PhD *Post-Doctoral Fellow Marine Microbial Ecology Lab Red Sea Research Center 4700 King Abdullah University of Science and Technology (KAUST) Room 2217-WS03, Ibn Al-Haytham Building (2) 4700 King Abdullah University of Science and Technology* Thuwal 23955-6900, Kingdom of Saudi Arabia Office: +966 (0) 2 808-2671

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Mamoon Rashid, PhD *Post-Doctoral Fellow Marine Microbial Ecology Lab Red Sea Research Center 4700 King Abdullah University of Science and Technology (KAUST) Room 2217-WS03, Ibn Al-Haytham Building (2) 4700 King Abdullah University of Science and Technology* Thuwal 23955-6900, Kingdom of Saudi Arabia Office: +966 (0) 2 808-2671

yunfeiguo commented 7 years ago

Hi MR,

I noticed that there was an error "ERROR: not all VCFs have the same number of samples!", could you check how many samples are there in each *multi-call.extract.vcf file? I don't know why there are two gatklite VCFs, though.

command to check # of samples:

for i in *multi-call.extract.vcf; do echo $i; perl -ne 'if (/^#CHROM/){print ((scalar (split /\t/,$_))-9),"\n"}' $i ; done

On Tue, Aug 16, 2016 at 4:33 AM, rashidma notifications@github.com wrote:

Hi, I just noticed in the _.log file of seqmule run -- the VCF " NIST7086_result/NIST7086_bwamem.merge.realn.0gatklite. multi-call.extract.vcf" has been mentioned twice. please see below colored in red. Is this creating proble?? Per sample there should be 4 VCF files (3 different aligner variant caller combinations and one consensus) BUT in case of NIST7086 why this is 5 VCF. *

_2 sample(s) in this analysis: NIST7035 NIST7086All input is merged on a per-sample basis (after alignment if FASTQ).Multi-sample variant calling ENABLED.Input is exome (or captured) sequencing data.4 variant caller(s) used: GATKLite_UnifiedGenotyper SAMtools FreeBayes Consensus1 aligner(s) used: bwamemFile used for caculating coverage statistics and extracting variants: nexterarapidcapture_expandedexome_targetedregions.nooverlap.bedReadgroup : READGROUP_NIST7035,READGROUP_NIST7086Library : LIBRARYSequencing platform: ILLUMINAReference genome build is hg19dbsnp138 is used for variant calling and recalibration (in GATK VQSR).Java memory usage is limited to 1750mjava executable path: javaMax number of processes: 12NOTICE: /tmp will be used for storing temporary files###################################################FINAL OUTPUT for NIST7035:###################################################NOTICE: Both consensus results and individual results are listed.WARNING: consensus results may not be available because some variant callers or aligners may fail to generate output.NIST7035_result/NIST7035.extract_consensus. vcfNIST7035_result/NIST7035_bwamem.merge.realn.0_gatklite. multi-call.extract.vcfNIST7035result/NIST7035 bwamem.merge.0samtools.multi-call.extract.vcfNIST7035 result/NIST7035_bwamem.merge.0freebayes.multi-call. extract.vcf###################################################FINAL OUTPUT for NIST7086:###################################################NOTICE: Both consensus results and individual results are listed.WARNING: consensus results may not be available because some variant callers or aligners may fail to generate output.

_NIST7086_result/NIST7086.extractconsensus.vcfNIST7086 result/NIST7086_bwamem.merge.realn.0_gatklite.multi-call. extract.vcfNIST7086_result/NIST7086bwamem.merge.realn.0 gatklite.multi-call.extract.vcfNIST7086result/NIST7086 bwamem.merge.0samtools.multi-call.extract.vcfNIST7086 result/NIST7086_bwamem.merge.0freebayes.multi-call.extract.vcf

Thanks

On Tue, Aug 16, 2016 at 8:07 AM, Mamoon Rashid rashidmamoon@gmail.com wrote:

Hi Yunfei, I sent you the big nohup file (~8MB) and it failed to reach you. Now I am sending you the last 1000 lines of the nohup file. I can see there are errors at step 42 and 43. I thank you so much for your time. MR

On Mon, Aug 15, 2016 at 4:05 PM, Yunfei Guo notifications@github.com wrote:

Hi Rashid,

Could you please provide the contents of nohup.out file? I need to know where it stopped.

Thanks.

Best, Yunfei

On Mon, Aug 15, 2016 at 12:47 AM, rashidma notifications@github.com wrote:

Thanks a lot Yunfei, I successfully ran the test run. Now, I was running a real sample from NIST. Out of 51 steps 48 steps ran well. I am stuck in the last few steps. The seqmule was stopped many times due to system shutdown and couple of times I killed the process. But every time I restarted it with--

nohup seqmule run NIST7035-NIST7086.script &

Please let me know any troubleshooting. I will be thankful to you. Regards MR

On Thu, Aug 11, 2016 at 1:02 AM, Yunfei Guo <notifications@github.com

wrote:

Hi Mamoon,

Please use inst/hg19_exome.legacy.bed after updating SeqMule with seqmule update --git. Thank you!

Best, Yunfei

— You are receiving this because you authored the thread. Reply to this email directly, view it on GitHub <https://github.com/WGLab/SeqMule/issues/146#issuecomment-239018748 , or mute the thread https://github.com/notifications/unsubscribe-auth/ ATyiOAPu4as9L3bQ8R0fedRCtTQ976qQks5qekpygaJpZM4Jf64z .

Mamoon Rashid, PhD *Post-Doctoral Fellow Marine Microbial Ecology Lab Red Sea Research Center 4700 King Abdullah University of Science and Technology (KAUST) Room 2217-WS03, Ibn Al-Haytham Building (2) 4700 King Abdullah University of Science and Technology* Thuwal 23955-6900, Kingdom of Saudi Arabia Office: +966 (0) 2 808-2671

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Mamoon Rashid, PhD *Post-Doctoral Fellow Marine Microbial Ecology Lab Red Sea Research Center 4700 King Abdullah University of Science and Technology (KAUST) Room 2217-WS03, Ibn Al-Haytham Building (2) 4700 King Abdullah University of Science and Technology* Thuwal 23955-6900, Kingdom of Saudi Arabia Office: +966 (0) 2 808-2671

Mamoon Rashid, PhD *Post-Doctoral Fellow Marine Microbial Ecology Lab Red Sea Research Center 4700 King Abdullah University of Science and Technology (KAUST) Room 2217-WS03, Ibn Al-Haytham Building (2) 4700 King Abdullah University of Science and Technology* Thuwal 23955-6900, Kingdom of Saudi Arabia Office: +966 (0) 2 808-2671

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rashidma commented 7 years ago

Hi I checked the VCF files for number of samples ---

VCFs number of samples NIST7035_bwamem.merge.0_freebayes.multi-call.extract.vcf 1 NIST7035_bwamem.merge.0_samtools.multi-call.extract.vcf 2 NIST7035_bwamem.merge.realn.0_gatklite.multi-call.extract.vcf 2

NIST7086_bwamem.merge.0_freebayes.multi-call.extract.vcf 1 NIST7086_bwamem.merge.0_samtools.multi-call.extract.vcf 2 NIST7086_bwamem.merge.realn.0_gatklite.multi-call.extract.vcf 2

I noticed frrebayes processed only one sample. Is this a known bug in freebayes??

I am trying to understand the system well so that I can use seqmule on my large number of samples. Best regards Mamoon

On Tue, Aug 16, 2016 at 5:46 PM, Yunfei Guo notifications@github.com wrote:

Hi MR,

I noticed that there was an error "ERROR: not all VCFs have the same number of samples!", could you check how many samples are there in each *multi-call.extract.vcf file? I don't know why there are two gatklite VCFs, though.

command to check # of samples:

for i in *multi-call.extract.vcf; do echo $i; perl -ne 'if (/^#CHROM/){print ((scalar (split /\t/,$_))-9),"\n"}' $i ; done

On Tue, Aug 16, 2016 at 4:33 AM, rashidma notifications@github.com wrote:

Hi, I just noticed in the _.log file of seqmule run -- the VCF " NIST7086_result/NIST7086_bwamem.merge.realn.0gatklite. multi-call.extract.vcf" has been mentioned twice. please see below colored in red. Is this creating proble?? Per sample there should be 4 VCF files (3 different aligner variant caller combinations and one consensus) BUT in case of NIST7086 why this is 5 VCF. *

_2 sample(s) in this analysis: NIST7035 NIST7086All input is merged on a per-sample basis (after alignment if FASTQ).Multi-sample variant calling ENABLED.Input is exome (or captured) sequencing data.4 variant caller(s) used: GATKLite_UnifiedGenotyper SAMtools FreeBayes Consensus1 aligner(s) used: bwamemFile used for caculating coverage statistics and extracting variants: nexterarapidcapture_expandedexome_targetedregions.nooverlap.bedReadgroup : READGROUP_NIST7035,READGROUP_NIST7086Library : LIBRARYSequencing platform: ILLUMINAReference genome build is hg19dbsnp138 is used for variant calling and recalibration (in GATK VQSR).Java memory usage is limited to 1750mjava executable path: javaMax number of processes: 12NOTICE: /tmp will be used for storing temporary files###################################################FINAL OUTPUT for NIST7035:###################################################NOTICE: Both consensus results and individual results are listed.WARNING: consensus results may not be available because some variant callers or aligners may fail to generate output.NIST7035_result/NIST7035.extract_consensus. vcfNIST7035_result/NIST7035_bwamem.merge.realn.0_gatklite. multi-call.extract.vcfNIST7035result/NIST7035 bwamem.merge.0samtools.multi-call.extract.vcfNIST7035 result/NIST7035_bwamem.merge.0freebayes.multi-call. extract.vcf###################################################FINAL OUTPUT for NIST7086:###################################################NOTICE: Both consensus results and individual results are listed.WARNING: consensus results may not be available because some variant callers or aligners may fail to generate output.

_NIST7086_result/NIST7086.extractconsensus.vcfNIST7086 result/NIST7086_bwamem.merge.realn.0_gatklite.multi-call. extract.vcfNIST7086_result/NIST7086bwamem.merge.realn.0 gatklite.multi-call.extract.vcfNIST7086result/NIST7086 bwamem.merge.0samtools.multi-call.extract.vcfNIST7086 result/NIST7086_bwamem.merge.0freebayes.multi-call.extract.vcf

Thanks

On Tue, Aug 16, 2016 at 8:07 AM, Mamoon Rashid rashidmamoon@gmail.com wrote:

Hi Yunfei, I sent you the big nohup file (~8MB) and it failed to reach you. Now I am sending you the last 1000 lines of the nohup file. I can see there are errors at step 42 and 43. I thank you so much for your time. MR

On Mon, Aug 15, 2016 at 4:05 PM, Yunfei Guo notifications@github.com wrote:

Hi Rashid,

Could you please provide the contents of nohup.out file? I need to know where it stopped.

Thanks.

Best, Yunfei

On Mon, Aug 15, 2016 at 12:47 AM, rashidma notifications@github.com wrote:

Thanks a lot Yunfei, I successfully ran the test run. Now, I was running a real sample from NIST. Out of 51 steps 48 steps ran well. I am stuck in the last few steps. The seqmule was stopped many times due to system shutdown and couple of times I killed the process. But every time I restarted it with--

nohup seqmule run NIST7035-NIST7086.script &

Please let me know any troubleshooting. I will be thankful to you. Regards MR

On Thu, Aug 11, 2016 at 1:02 AM, Yunfei Guo < notifications@github.com

wrote:

Hi Mamoon,

Please use inst/hg19_exome.legacy.bed after updating SeqMule with seqmule update --git. Thank you!

Best, Yunfei

— You are receiving this because you authored the thread. Reply to this email directly, view it on GitHub <https://github.com/WGLab/SeqMule/issues/146# issuecomment-239018748 , or mute the thread https://github.com/notifications/unsubscribe-auth/ ATyiOAPu4as9L3bQ8R0fedRCtTQ976qQks5qekpygaJpZM4Jf64z .

Mamoon Rashid, PhD *Post-Doctoral Fellow Marine Microbial Ecology Lab Red Sea Research Center 4700 King Abdullah University of Science and Technology (KAUST) Room 2217-WS03, Ibn Al-Haytham Building (2) 4700 King Abdullah University of Science and Technology* Thuwal 23955-6900, Kingdom of Saudi Arabia Office: +966 (0) 2 808-2671

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.

Mamoon Rashid, PhD *Post-Doctoral Fellow Marine Microbial Ecology Lab Red Sea Research Center 4700 King Abdullah University of Science and Technology (KAUST) Room 2217-WS03, Ibn Al-Haytham Building (2) 4700 King Abdullah University of Science and Technology* Thuwal 23955-6900, Kingdom of Saudi Arabia Office: +966 (0) 2 808-2671

Mamoon Rashid, PhD *Post-Doctoral Fellow Marine Microbial Ecology Lab Red Sea Research Center 4700 King Abdullah University of Science and Technology (KAUST) Room 2217-WS03, Ibn Al-Haytham Building (2) 4700 King Abdullah University of Science and Technology* Thuwal 23955-6900, Kingdom of Saudi Arabia Office: +966 (0) 2 808-2671

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.

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yunfeiguo commented 7 years ago

Thanks, Mamoon. Let me test the latest freebayes and get back to you later.

On Wed, Aug 17, 2016 at 10:05 PM, rashidma notifications@github.com wrote:

Hi I checked the VCF files for number of samples ---

VCFs number of samples NIST7035_bwamem.merge.0_freebayes.multi-call.extract.vcf 1 NIST7035_bwamem.merge.0_samtools.multi-call.extract.vcf 2 NIST7035_bwamem.merge.realn.0_gatklite.multi-call.extract.vcf 2

NIST7086_bwamem.merge.0_freebayes.multi-call.extract.vcf 1 NIST7086_bwamem.merge.0_samtools.multi-call.extract.vcf 2 NIST7086_bwamem.merge.realn.0_gatklite.multi-call.extract.vcf 2

I noticed frrebayes processed only one sample. Is this a known bug in freebayes??

I am trying to understand the system well so that I can use seqmule on my large number of samples. Best regards Mamoon

On Tue, Aug 16, 2016 at 5:46 PM, Yunfei Guo notifications@github.com wrote:

Hi MR,

I noticed that there was an error "ERROR: not all VCFs have the same number of samples!", could you check how many samples are there in each *multi-call.extract.vcf file? I don't know why there are two gatklite VCFs, though.

command to check # of samples:

for i in *multi-call.extract.vcf; do echo $i; perl -ne 'if (/^#CHROM/){print ((scalar (split /\t/,$_))-9),"\n"}' $i ; done

On Tue, Aug 16, 2016 at 4:33 AM, rashidma notifications@github.com wrote:

Hi, I just noticed in the _.log file of seqmule run -- the VCF " NIST7086_result/NIST7086_bwamem.merge.realn.0gatklite. multi-call.extract.vcf" has been mentioned twice. please see below colored in red. Is this creating proble?? Per sample there should be 4 VCF files (3 different aligner variant caller combinations and one consensus) BUT in case of NIST7086 why this is 5 VCF. *

_2 sample(s) in this analysis: NIST7035 NIST7086All input is merged on a per-sample basis (after alignment if FASTQ).Multi-sample variant calling ENABLED.Input is exome (or captured) sequencing data.4 variant caller(s) used: GATKLite_UnifiedGenotyper SAMtools FreeBayes Consensus1 aligner(s) used: bwamemFile used for caculating coverage statistics and extracting variants: nexterarapidcapture_expandedexome_targetedregions. nooverlap.bedReadgroup : READGROUP_NIST7035,READGROUP_NIST7086Library : LIBRARYSequencing platform: ILLUMINAReference genome build is hg19dbsnp138 is used for variant calling and recalibration (in GATK VQSR).Java memory usage is limited to 1750mjava executable path: javaMax number of processes: 12NOTICE: /tmp will be used for storing temporary files###################################################FINAL OUTPUT for NIST7035:###################################################NOTICE: Both consensus results and individual results are listed.WARNING: consensus results may not be available because some variant callers or aligners may fail to generate output.NIST7035_result/NIST7035.extract_consensus. vcfNIST7035_result/NIST7035_bwamem.merge.realn.0_gatklite. multi-call.extract.vcfNIST7035result/NIST7035 bwamem.merge.0samtools.multi-call.extract.vcfNIST7035 result/NIST7035_bwamem.merge.0freebayes.multi-call. extract.vcf###################################################FINAL OUTPUT for NIST7086:###################################################NOTICE: Both consensus results and individual results are listed.WARNING: consensus results may not be available because some variant callers or aligners may fail to generate output.

_NIST7086_result/NIST7086.extractconsensus.vcfNIST7086 result/NIST7086_bwamem.merge.realn.0_gatklite.multi-call. extract.vcfNIST7086_result/NIST7086bwamem.merge.realn.0 gatklite.multi-call.extract.vcfNIST7086result/NIST7086 bwamem.merge.0samtools.multi-call.extract.vcfNIST7086 result/NIST7086_bwamem.merge.0freebayes.multi-call.extract.vcf

Thanks

On Tue, Aug 16, 2016 at 8:07 AM, Mamoon Rashid <rashidmamoon@gmail.com

wrote:

Hi Yunfei, I sent you the big nohup file (~8MB) and it failed to reach you. Now I am sending you the last 1000 lines of the nohup file. I can see there are errors at step 42 and 43. I thank you so much for your time. MR

On Mon, Aug 15, 2016 at 4:05 PM, Yunfei Guo < notifications@github.com> wrote:

Hi Rashid,

Could you please provide the contents of nohup.out file? I need to know where it stopped.

Thanks.

Best, Yunfei

On Mon, Aug 15, 2016 at 12:47 AM, rashidma < notifications@github.com> wrote:

Thanks a lot Yunfei, I successfully ran the test run. Now, I was running a real sample from NIST. Out of 51 steps 48 steps ran well. I am stuck in the last few steps. The seqmule was stopped many times due to system shutdown and couple of times I killed the process. But every time I restarted it with--

nohup seqmule run NIST7035-NIST7086.script &

Please let me know any troubleshooting. I will be thankful to you. Regards MR

On Thu, Aug 11, 2016 at 1:02 AM, Yunfei Guo < notifications@github.com

wrote:

Hi Mamoon,

Please use inst/hg19_exome.legacy.bed after updating SeqMule with seqmule update --git. Thank you!

Best, Yunfei

— You are receiving this because you authored the thread. Reply to this email directly, view it on GitHub <https://github.com/WGLab/SeqMule/issues/146# issuecomment-239018748 , or mute the thread https://github.com/notifications/unsubscribe-auth/ ATyiOAPu4as9L3bQ8R0fedRCtTQ976qQks5qekpygaJpZM4Jf64z .

Mamoon Rashid, PhD *Post-Doctoral Fellow Marine Microbial Ecology Lab Red Sea Research Center 4700 King Abdullah University of Science and Technology (KAUST) Room 2217-WS03, Ibn Al-Haytham Building (2) 4700 King Abdullah University of Science and Technology* Thuwal 23955-6900, Kingdom of Saudi Arabia Office: +966 (0) 2 808-2671

— You are receiving this because you modified the open/close state. Reply to this email directly, view it on GitHub <https://github.com/WGLab/SeqMule/issues/146# issuecomment-239746634 , or mute the thread https://github.com/notifications/unsubscribe-auth/ AHYPzsknYKT-MSQOex9rMD0_O_MHhoqLks5qgBmcgaJpZM4Jf64z .

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.

Mamoon Rashid, PhD *Post-Doctoral Fellow Marine Microbial Ecology Lab Red Sea Research Center 4700 King Abdullah University of Science and Technology (KAUST) Room 2217-WS03, Ibn Al-Haytham Building (2) 4700 King Abdullah University of Science and Technology* Thuwal 23955-6900, Kingdom of Saudi Arabia Office: +966 (0) 2 808-2671

Mamoon Rashid, PhD *Post-Doctoral Fellow Marine Microbial Ecology Lab Red Sea Research Center 4700 King Abdullah University of Science and Technology (KAUST) Room 2217-WS03, Ibn Al-Haytham Building (2) 4700 King Abdullah University of Science and Technology* Thuwal 23955-6900, Kingdom of Saudi Arabia Office: +966 (0) 2 808-2671

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Mamoon Rashid, PhD *Post-Doctoral Fellow Marine Microbial Ecology Lab Red Sea Research Center 4700 King Abdullah University of Science and Technology (KAUST) Room 2217-WS03, Ibn Al-Haytham Building (2) 4700 King Abdullah University of Science and Technology* Thuwal 23955-6900, Kingdom of Saudi Arabia Office: +966 (0) 2 808-2671

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rashidma commented 7 years ago

Thanks a lot for your reply. I started another run with bwa-gatk-samtools-varscan and I found that it is also incomplete.

The log file contains the *gatk-multicall-extract.vcf twice for second sample. For first sample it is ok.

I could not understand the behavior. Lets first troubleshoot the first run that we are discussing.

regards MR

On Thu, Aug 18, 2016 at 5:43 PM, Yunfei Guo notifications@github.com wrote:

Thanks, Mamoon. Let me test the latest freebayes and get back to you later.

On Wed, Aug 17, 2016 at 10:05 PM, rashidma notifications@github.com wrote:

Hi I checked the VCF files for number of samples ---

VCFs number of samples NIST7035_bwamem.merge.0_freebayes.multi-call.extract.vcf 1 NIST7035_bwamem.merge.0_samtools.multi-call.extract.vcf 2 NIST7035_bwamem.merge.realn.0_gatklite.multi-call.extract.vcf 2

NIST7086_bwamem.merge.0_freebayes.multi-call.extract.vcf 1 NIST7086_bwamem.merge.0_samtools.multi-call.extract.vcf 2 NIST7086_bwamem.merge.realn.0_gatklite.multi-call.extract.vcf 2

I noticed frrebayes processed only one sample. Is this a known bug in freebayes??

I am trying to understand the system well so that I can use seqmule on my large number of samples. Best regards Mamoon

On Tue, Aug 16, 2016 at 5:46 PM, Yunfei Guo notifications@github.com wrote:

Hi MR,

I noticed that there was an error "ERROR: not all VCFs have the same number of samples!", could you check how many samples are there in each *multi-call.extract.vcf file? I don't know why there are two gatklite VCFs, though.

command to check # of samples:

for i in *multi-call.extract.vcf; do echo $i; perl -ne 'if (/^#CHROM/){print ((scalar (split /\t/,$_))-9),"\n"}' $i ; done

On Tue, Aug 16, 2016 at 4:33 AM, rashidma notifications@github.com wrote:

Hi, I just noticed in the _.log file of seqmule run -- the VCF " NIST7086_result/NIST7086_bwamem.merge.realn.0gatklite. multi-call.extract.vcf" has been mentioned twice. please see below colored in red. Is this creating proble?? Per sample there should be 4 VCF files (3 different aligner variant caller combinations and one consensus) BUT in case of NIST7086 why this is 5 VCF. *

_2 sample(s) in this analysis: NIST7035 NIST7086All input is merged on a per-sample basis (after alignment if FASTQ).Multi-sample variant calling ENABLED.Input is exome (or captured) sequencing data.4 variant caller(s) used: GATKLite_UnifiedGenotyper SAMtools FreeBayes Consensus1 aligner(s) used: bwamemFile used for caculating coverage statistics and extracting variants: nexterarapidcapture_expandedexome_targetedregions. nooverlap.bedReadgroup : READGROUP_NIST7035,READGROUP_NIST7086Library : LIBRARYSequencing platform: ILLUMINAReference genome build is hg19dbsnp138 is used for variant calling and recalibration (in GATK VQSR).Java memory usage is limited to 1750mjava executable path: javaMax number of processes: 12NOTICE: /tmp will be used for storing temporary files###################################################FINAL OUTPUT for NIST7035:###################################################NOTICE: Both consensus results and individual results are listed.WARNING: consensus results may not be available because some variant callers or aligners may fail to generate output.NIST7035_result/NIST7035.extract_consensus. vcfNIST7035_result/NIST7035_bwamem.merge.realn.0_gatklite. multi-call.extract.vcfNIST7035result/NIST7035 bwamem.merge.0samtools.multi-call.extract.vcfNIST7035 result/NIST7035_bwamem.merge.0freebayes.multi-call. extract.vcf###################################################FINAL OUTPUT for NIST7086:###################################################NOTICE: Both consensus results and individual results are listed.WARNING: consensus results may not be available because some variant callers or aligners may fail to generate output.

_NIST7086_result/NIST7086.extractconsensus.vcfNIST7086 result/NIST7086_bwamem.merge.realn.0_gatklite.multi-call. extract.vcfNIST7086_result/NIST7086bwamem.merge.realn.0 gatklite.multi-call.extract.vcfNIST7086result/NIST7086 bwamem.merge.0samtools.multi-call.extract.vcfNIST7086 result/NIST7086_bwamem.merge.0freebayes.multi-call.extract.vcf

Thanks

On Tue, Aug 16, 2016 at 8:07 AM, Mamoon Rashid < rashidmamoon@gmail.com

wrote:

Hi Yunfei, I sent you the big nohup file (~8MB) and it failed to reach you. Now I am sending you the last 1000 lines of the nohup file. I can see there are errors at step 42 and 43. I thank you so much for your time. MR

On Mon, Aug 15, 2016 at 4:05 PM, Yunfei Guo < notifications@github.com> wrote:

Hi Rashid,

Could you please provide the contents of nohup.out file? I need to know where it stopped.

Thanks.

Best, Yunfei

On Mon, Aug 15, 2016 at 12:47 AM, rashidma < notifications@github.com> wrote:

Thanks a lot Yunfei, I successfully ran the test run. Now, I was running a real sample from NIST. Out of 51 steps 48 steps ran well. I am stuck in the last few steps. The seqmule was stopped many times due to system shutdown and couple of times I killed the process. But every time I restarted it with--

nohup seqmule run NIST7035-NIST7086.script &

Please let me know any troubleshooting. I will be thankful to you. Regards MR

On Thu, Aug 11, 2016 at 1:02 AM, Yunfei Guo < notifications@github.com

wrote:

Hi Mamoon,

Please use inst/hg19_exome.legacy.bed after updating SeqMule with seqmule update --git. Thank you!

Best, Yunfei

— You are receiving this because you authored the thread. Reply to this email directly, view it on GitHub <https://github.com/WGLab/SeqMule/issues/146# issuecomment-239018748 , or mute the thread https://github.com/notifications/unsubscribe-auth/ ATyiOAPu4as9L3bQ8R0fedRCtTQ976qQks5qekpygaJpZM4Jf64z .

Mamoon Rashid, PhD *Post-Doctoral Fellow Marine Microbial Ecology Lab Red Sea Research Center 4700 King Abdullah University of Science and Technology (KAUST) Room 2217-WS03, Ibn Al-Haytham Building (2) 4700 King Abdullah University of Science and Technology* Thuwal 23955-6900, Kingdom of Saudi Arabia Office: +966 (0) 2 808-2671

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.

Mamoon Rashid, PhD *Post-Doctoral Fellow Marine Microbial Ecology Lab Red Sea Research Center 4700 King Abdullah University of Science and Technology (KAUST) Room 2217-WS03, Ibn Al-Haytham Building (2) 4700 King Abdullah University of Science and Technology* Thuwal 23955-6900, Kingdom of Saudi Arabia Office: +966 (0) 2 808-2671

Mamoon Rashid, PhD *Post-Doctoral Fellow Marine Microbial Ecology Lab Red Sea Research Center 4700 King Abdullah University of Science and Technology (KAUST) Room 2217-WS03, Ibn Al-Haytham Building (2) 4700 King Abdullah University of Science and Technology* Thuwal 23955-6900, Kingdom of Saudi Arabia Office: +966 (0) 2 808-2671

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Mamoon Rashid, PhD *Post-Doctoral Fellow Marine Microbial Ecology Lab Red Sea Research Center 4700 King Abdullah University of Science and Technology (KAUST) Room 2217-WS03, Ibn Al-Haytham Building (2) 4700 King Abdullah University of Science and Technology* Thuwal 23955-6900, Kingdom of Saudi Arabia Office: +966 (0) 2 808-2671

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yunfeiguo commented 7 years ago

Is it because you tried to rerun the analysis?

On Aug 18, 2016, at 8:06 AM, rashidma notifications@github.com wrote:

Thanks a lot for your reply. I started another run with bwa-gatk-samtools-varscan and I found that it is also incomplete.

The log file contains the *gatk-multicall-extract.vcf twice for second sample. For first sample it is ok.

I could not understand the behavior. Lets first troubleshoot the first run that we are discussing.

regards MR

On Thu, Aug 18, 2016 at 5:43 PM, Yunfei Guo notifications@github.com wrote:

Thanks, Mamoon. Let me test the latest freebayes and get back to you later.

On Wed, Aug 17, 2016 at 10:05 PM, rashidma notifications@github.com wrote:

Hi I checked the VCF files for number of samples ---

VCFs number of samples NIST7035_bwamem.merge.0_freebayes.multi-call.extract.vcf 1 NIST7035_bwamem.merge.0_samtools.multi-call.extract.vcf 2 NIST7035_bwamem.merge.realn.0_gatklite.multi-call.extract.vcf 2

NIST7086_bwamem.merge.0_freebayes.multi-call.extract.vcf 1 NIST7086_bwamem.merge.0_samtools.multi-call.extract.vcf 2 NIST7086_bwamem.merge.realn.0_gatklite.multi-call.extract.vcf 2

I noticed frrebayes processed only one sample. Is this a known bug in freebayes??

I am trying to understand the system well so that I can use seqmule on my large number of samples. Best regards Mamoon

On Tue, Aug 16, 2016 at 5:46 PM, Yunfei Guo notifications@github.com wrote:

Hi MR,

I noticed that there was an error "ERROR: not all VCFs have the same number of samples!", could you check how many samples are there in each *multi-call.extract.vcf file? I don't know why there are two gatklite VCFs, though.

command to check # of samples:

for i in *multi-call.extract.vcf; do echo $i; perl -ne 'if (/^#CHROM/){print ((scalar (split /\t/,$_))-9),"\n"}' $i ; done

On Tue, Aug 16, 2016 at 4:33 AM, rashidma notifications@github.com wrote:

Hi, I just noticed in the _.log file of seqmule run -- the VCF " NIST7086_result/NIST7086_bwamem.merge.realn.0gatklite. multi-call.extract.vcf" has been mentioned twice. please see below colored in red. Is this creating proble?? Per sample there should be 4 VCF files (3 different aligner variant caller combinations and one consensus) BUT in case of NIST7086 why this is 5 VCF. *

_2 sample(s) in this analysis: NIST7035 NIST7086All input is merged on a per-sample basis (after alignment if FASTQ).Multi-sample variant calling ENABLED.Input is exome (or captured) sequencing data.4 variant caller(s) used: GATKLite_UnifiedGenotyper SAMtools FreeBayes Consensus1 aligner(s) used: bwamemFile used for caculating coverage statistics and extracting variants: nexterarapidcapture_expandedexome_targetedregions. nooverlap.bedReadgroup : READGROUP_NIST7035,READGROUP_NIST7086Library : LIBRARYSequencing platform: ILLUMINAReference genome build is hg19dbsnp138 is used for variant calling and recalibration (in GATK VQSR).Java memory usage is limited to 1750mjava executable path: javaMax number of processes: 12NOTICE: /tmp will be used for storing temporary files###################################################FINAL OUTPUT for NIST7035:###################################################NOTICE: Both consensus results and individual results are listed.WARNING: consensus results may not be available because some variant callers or aligners may fail to generate output.NIST7035_result/NIST7035.extract_consensus. vcfNIST7035_result/NIST7035_bwamem.merge.realn.0_gatklite. multi-call.extract.vcfNIST7035result/NIST7035 bwamem.merge.0samtools.multi-call.extract.vcfNIST7035 result/NIST7035_bwamem.merge.0freebayes.multi-call. extract.vcf###################################################FINAL OUTPUT for NIST7086:###################################################NOTICE: Both consensus results and individual results are listed.WARNING: consensus results may not be available because some variant callers or aligners may fail to generate output.

_NIST7086_result/NIST7086.extractconsensus.vcfNIST7086 result/NIST7086_bwamem.merge.realn.0_gatklite.multi-call. extract.vcfNIST7086_result/NIST7086bwamem.merge.realn.0 gatklite.multi-call.extract.vcfNIST7086result/NIST7086 bwamem.merge.0samtools.multi-call.extract.vcfNIST7086 result/NIST7086_bwamem.merge.0freebayes.multi-call.extract.vcf

Thanks

On Tue, Aug 16, 2016 at 8:07 AM, Mamoon Rashid < rashidmamoon@gmail.com

wrote:

Hi Yunfei, I sent you the big nohup file (~8MB) and it failed to reach you. Now I am sending you the last 1000 lines of the nohup file. I can see there are errors at step 42 and 43. I thank you so much for your time. MR

On Mon, Aug 15, 2016 at 4:05 PM, Yunfei Guo < notifications@github.com> wrote:

Hi Rashid,

Could you please provide the contents of nohup.out file? I need to know where it stopped.

Thanks.

Best, Yunfei

On Mon, Aug 15, 2016 at 12:47 AM, rashidma < notifications@github.com> wrote:

Thanks a lot Yunfei, I successfully ran the test run. Now, I was running a real sample from NIST. Out of 51 steps 48 steps ran well. I am stuck in the last few steps. The seqmule was stopped many times due to system shutdown and couple of times I killed the process. But every time I restarted it with--

nohup seqmule run NIST7035-NIST7086.script &

Please let me know any troubleshooting. I will be thankful to you. Regards MR

On Thu, Aug 11, 2016 at 1:02 AM, Yunfei Guo < notifications@github.com

wrote:

Hi Mamoon,

Please use inst/hg19_exome.legacy.bed after updating SeqMule with seqmule update --git. Thank you!

Best, Yunfei

— You are receiving this because you authored the thread. Reply to this email directly, view it on GitHub <https://github.com/WGLab/SeqMule/issues/146# issuecomment-239018748 , or mute the thread https://github.com/notifications/unsubscribe-auth/ ATyiOAPu4as9L3bQ8R0fedRCtTQ976qQks5qekpygaJpZM4Jf64z .

Mamoon Rashid, PhD *Post-Doctoral Fellow Marine Microbial Ecology Lab Red Sea Research Center 4700 King Abdullah University of Science and Technology (KAUST) Room 2217-WS03, Ibn Al-Haytham Building (2) 4700 King Abdullah University of Science and Technology* Thuwal 23955-6900, Kingdom of Saudi Arabia Office: +966 (0) 2 808-2671

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.

Mamoon Rashid, PhD *Post-Doctoral Fellow Marine Microbial Ecology Lab Red Sea Research Center 4700 King Abdullah University of Science and Technology (KAUST) Room 2217-WS03, Ibn Al-Haytham Building (2) 4700 King Abdullah University of Science and Technology* Thuwal 23955-6900, Kingdom of Saudi Arabia Office: +966 (0) 2 808-2671

Mamoon Rashid, PhD *Post-Doctoral Fellow Marine Microbial Ecology Lab Red Sea Research Center 4700 King Abdullah University of Science and Technology (KAUST) Room 2217-WS03, Ibn Al-Haytham Building (2) 4700 King Abdullah University of Science and Technology* Thuwal 23955-6900, Kingdom of Saudi Arabia Office: +966 (0) 2 808-2671

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rashidma commented 7 years ago

I re-run the analysis in a completely new directory. So both runs should not influence each other.

Thanks MR On 18 Aug 2016 18:41, "Yunfei Guo" notifications@github.com wrote:

Is it because you tried to rerun the analysis?

On Aug 18, 2016, at 8:06 AM, rashidma notifications@github.com wrote:

Thanks a lot for your reply. I started another run with bwa-gatk-samtools-varscan and I found that it is also incomplete.

The log file contains the *gatk-multicall-extract.vcf twice for second sample. For first sample it is ok.

I could not understand the behavior. Lets first troubleshoot the first run that we are discussing.

regards MR

On Thu, Aug 18, 2016 at 5:43 PM, Yunfei Guo notifications@github.com wrote:

Thanks, Mamoon. Let me test the latest freebayes and get back to you later.

On Wed, Aug 17, 2016 at 10:05 PM, rashidma notifications@github.com wrote:

Hi I checked the VCF files for number of samples ---

VCFs number of samples NIST7035_bwamem.merge.0_freebayes.multi-call.extract.vcf 1 NIST7035_bwamem.merge.0_samtools.multi-call.extract.vcf 2 NIST7035_bwamem.merge.realn.0_gatklite.multi-call.extract.vcf 2

NIST7086_bwamem.merge.0_freebayes.multi-call.extract.vcf 1 NIST7086_bwamem.merge.0_samtools.multi-call.extract.vcf 2 NIST7086_bwamem.merge.realn.0_gatklite.multi-call.extract.vcf 2

I noticed frrebayes processed only one sample. Is this a known bug in freebayes??

I am trying to understand the system well so that I can use seqmule on my large number of samples. Best regards Mamoon

On Tue, Aug 16, 2016 at 5:46 PM, Yunfei Guo < notifications@github.com> wrote:

Hi MR,

I noticed that there was an error "ERROR: not all VCFs have the same number of samples!", could you check how many samples are there in each *multi-call.extract.vcf file? I don't know why there are two gatklite VCFs, though.

command to check # of samples:

for i in *multi-call.extract.vcf; do echo $i; perl -ne 'if (/^#CHROM/){print ((scalar (split /\t/,$_))-9),"\n"}' $i ; done

On Tue, Aug 16, 2016 at 4:33 AM, rashidma < notifications@github.com> wrote:

Hi, I just noticed in the _.log file of seqmule run -- the VCF " NIST7086_result/NIST7086_bwamem.merge.realn.0gatklite. multi-call.extract.vcf" has been mentioned twice. please see below colored in red. Is this creating proble?? Per sample there should be 4 VCF files (3 different aligner variant caller combinations and one consensus) BUT in case of NIST7086 why this is 5 VCF. *

_2 sample(s) in this analysis: NIST7035 NIST7086All input is merged on a per-sample basis (after alignment if FASTQ).Multi-sample variant calling ENABLED.Input is exome (or captured) sequencing data.4 variant caller(s) used: GATKLite_UnifiedGenotyper SAMtools FreeBayes Consensus1 aligner(s) used: bwamemFile used for caculating coverage statistics and extracting variants: nexterarapidcapture_expandedexome_targetedregions. nooverlap.bedReadgroup : READGROUP_NIST7035,READGROUP_NIST7086Library : LIBRARYSequencing platform: ILLUMINAReference genome build is hg19dbsnp138 is used for variant calling and recalibration (in GATK VQSR).Java memory usage is limited to 1750mjava executable path: javaMax number of processes: 12NOTICE: /tmp will be used for storing temporary files###################################################FINAL OUTPUT for NIST7035:################################################### NOTICE: Both consensus results and individual results are listed.WARNING: consensus results may not be available because some variant callers or aligners may fail to generate output.NIST7035_result/NIST7035.extract_consensus. vcfNIST7035_result/NIST7035_bwamem.merge.realn.0_gatklite. multi-call.extract.vcfNIST7035result/NIST7035 bwamem.merge.0samtools.multi-call.extract.vcfNIST7035 result/NIST7035_bwamem.merge.0_freebayes.multi-call. extract.vcf#################################################

FINAL

OUTPUT for NIST7086:################################################### NOTICE: Both consensus results and individual results are listed.WARNING: consensus results may not be available because some variant callers or aligners may fail to generate output._

_NIST7086_result/NIST7086.extractconsensus.vcfNIST7086 result/NIST7086_bwamem.merge.realn.0_gatklite.multi-call. extract.vcfNIST7086_result/NIST7086bwamem.merge.realn.0 gatklite.multi-call.extract.vcfNIST7086result/NIST7086 bwamem.merge.0samtools.multi-call.extract.vcfNIST7086 result/NIST7086_bwamem.merge.0freebayes.multi-call.extract.vcf

Thanks

On Tue, Aug 16, 2016 at 8:07 AM, Mamoon Rashid < rashidmamoon@gmail.com

wrote:

Hi Yunfei, I sent you the big nohup file (~8MB) and it failed to reach you. Now I am sending you the last 1000 lines of the nohup file. I can see there are errors at step 42 and 43. I thank you so much for your time. MR

On Mon, Aug 15, 2016 at 4:05 PM, Yunfei Guo < notifications@github.com> wrote:

Hi Rashid,

Could you please provide the contents of nohup.out file? I need to know where it stopped.

Thanks.

Best, Yunfei

On Mon, Aug 15, 2016 at 12:47 AM, rashidma < notifications@github.com> wrote:

Thanks a lot Yunfei, I successfully ran the test run. Now, I was running a real sample from NIST. Out of 51 steps 48 steps ran well. I am stuck in the last few steps. The seqmule was stopped many times due to system shutdown and couple of times I killed the process. But every time I restarted it with--

nohup seqmule run NIST7035-NIST7086.script &

Please let me know any troubleshooting. I will be thankful to you. Regards MR

On Thu, Aug 11, 2016 at 1:02 AM, Yunfei Guo < notifications@github.com

wrote:

Hi Mamoon,

Please use inst/hg19_exome.legacy.bed after updating SeqMule with seqmule update --git. Thank you!

Best, Yunfei

— You are receiving this because you authored the thread. Reply to this email directly, view it on GitHub <https://github.com/WGLab/SeqMule/issues/146# issuecomment-239018748 , or mute the thread https://github.com/notifications/unsubscribe-auth/ ATyiOAPu4as9L3bQ8R0fedRCtTQ976qQks5qekpygaJpZM4Jf64z .

Mamoon Rashid, PhD *Post-Doctoral Fellow Marine Microbial Ecology Lab Red Sea Research Center 4700 King Abdullah University of Science and Technology (KAUST) Room 2217-WS03, Ibn Al-Haytham Building (2) 4700 King Abdullah University of Science and Technology* Thuwal 23955-6900, Kingdom of Saudi Arabia Office: +966 (0) 2 808-2671

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.

Mamoon Rashid, PhD *Post-Doctoral Fellow Marine Microbial Ecology Lab Red Sea Research Center 4700 King Abdullah University of Science and Technology (KAUST) Room 2217-WS03, Ibn Al-Haytham Building (2) 4700 King Abdullah University of Science and Technology* Thuwal 23955-6900, Kingdom of Saudi Arabia Office: +966 (0) 2 808-2671

Mamoon Rashid, PhD *Post-Doctoral Fellow Marine Microbial Ecology Lab Red Sea Research Center 4700 King Abdullah University of Science and Technology (KAUST) Room 2217-WS03, Ibn Al-Haytham Building (2) 4700 King Abdullah University of Science and Technology* Thuwal 23955-6900, Kingdom of Saudi Arabia Office: +966 (0) 2 808-2671

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yunfeiguo commented 7 years ago

I mean for each analysis, did you run it twice (probably with 'seqmule run' after 'seqmule pipeline')?

On Thu, Aug 18, 2016 at 7:13 PM, rashidma notifications@github.com wrote:

I re-run the analysis in a completely new directory. So both runs should not influence each other.

Thanks MR

On 18 Aug 2016 18:41, "Yunfei Guo" notifications@github.com wrote:

Is it because you tried to rerun the analysis?

On Aug 18, 2016, at 8:06 AM, rashidma notifications@github.com wrote:

Thanks a lot for your reply. I started another run with bwa-gatk-samtools-varscan and I found that it is also incomplete.

The log file contains the *gatk-multicall-extract.vcf twice for second sample. For first sample it is ok.

I could not understand the behavior. Lets first troubleshoot the first run that we are discussing.

regards MR

On Thu, Aug 18, 2016 at 5:43 PM, Yunfei Guo notifications@github.com wrote:

Thanks, Mamoon. Let me test the latest freebayes and get back to you later.

On Wed, Aug 17, 2016 at 10:05 PM, rashidma <notifications@github.com

wrote:

Hi I checked the VCF files for number of samples ---

VCFs number of samples NIST7035_bwamem.merge.0_freebayes.multi-call.extract.vcf 1 NIST7035_bwamem.merge.0_samtools.multi-call.extract.vcf 2 NIST7035_bwamem.merge.realn.0_gatklite.multi-call.extract.vcf 2

NIST7086_bwamem.merge.0_freebayes.multi-call.extract.vcf 1 NIST7086_bwamem.merge.0_samtools.multi-call.extract.vcf 2 NIST7086_bwamem.merge.realn.0_gatklite.multi-call.extract.vcf 2

I noticed frrebayes processed only one sample. Is this a known bug in freebayes??

I am trying to understand the system well so that I can use seqmule on my large number of samples. Best regards Mamoon

On Tue, Aug 16, 2016 at 5:46 PM, Yunfei Guo < notifications@github.com> wrote:

Hi MR,

I noticed that there was an error "ERROR: not all VCFs have the same number of samples!", could you check how many samples are there in each *multi-call.extract.vcf file? I don't know why there are two gatklite VCFs, though.

command to check # of samples:

for i in *multi-call.extract.vcf; do echo $i; perl -ne 'if (/^#CHROM/){print ((scalar (split /\t/,$_))-9),"\n"}' $i ; done

On Tue, Aug 16, 2016 at 4:33 AM, rashidma < notifications@github.com> wrote:

Hi, I just noticed in the _.log file of seqmule run -- the VCF " NIST7086_result/NIST7086_bwamem.merge.realn.0gatklite. multi-call.extract.vcf" has been mentioned twice. please see below colored in red. Is this creating proble?? Per sample there should be 4 VCF files (3 different aligner variant caller combinations and one consensus) BUT in case of NIST7086 why this is 5 VCF. *

_2 sample(s) in this analysis: NIST7035 NIST7086All input is merged on a per-sample basis (after alignment if FASTQ).Multi-sample variant calling ENABLED.Input is exome (or captured) sequencing data.4 variant caller(s) used: GATKLite_UnifiedGenotyper SAMtools FreeBayes Consensus1 aligner(s) used: bwamemFile used for caculating coverage statistics and extracting variants: nexterarapidcapture_expandedexome_targetedregions. nooverlap.bedReadgroup : READGROUP_NIST7035,READGROUP_NIST7086Library : LIBRARYSequencing platform: ILLUMINAReference genome build is hg19dbsnp138 is used for variant calling and recalibration (in GATK VQSR).Java memory usage is limited to 1750mjava executable path: javaMax number of processes: 12NOTICE: /tmp will be used for storing temporary files###################################################FINAL OUTPUT for NIST7035:################################################### NOTICE: Both consensus results and individual results are listed.WARNING: consensus results may not be available because some variant callers or aligners may fail to generate output.NIST7035_result/NIST7035.extract_consensus. vcfNIST7035_result/NIST7035_bwamem.merge.realn.0_gatklite. multi-call.extract.vcfNIST7035result/NIST7035 bwamem.merge.0samtools.multi-call.extract.vcfNIST7035 result/NIST7035_bwamem.merge.0_freebayes.multi-call. extract.vcf#################################################

FINAL

OUTPUT for NIST7086:################################################### NOTICE: Both consensus results and individual results are listed.WARNING: consensus results may not be available because some variant callers or aligners may fail to generate output._

_NIST7086_result/NIST7086.extractconsensus.vcfNIST7086 result/NIST7086_bwamem.merge.realn.0_gatklite.multi-call. extract.vcfNIST7086_result/NIST7086bwamem.merge.realn.0 gatklite.multi-call.extract.vcfNIST7086result/NIST7086 bwamem.merge.0samtools.multi-call.extract.vcfNIST7086 result/NIST7086_bwamem.merge.0freebayes.multi-call. extract.vcf

Thanks

On Tue, Aug 16, 2016 at 8:07 AM, Mamoon Rashid < rashidmamoon@gmail.com

wrote:

Hi Yunfei, I sent you the big nohup file (~8MB) and it failed to reach you. Now I am sending you the last 1000 lines of the nohup file. I can see there are errors at step 42 and 43. I thank you so much for your time. MR

On Mon, Aug 15, 2016 at 4:05 PM, Yunfei Guo < notifications@github.com> wrote:

Hi Rashid,

Could you please provide the contents of nohup.out file? I need to know where it stopped.

Thanks.

Best, Yunfei

On Mon, Aug 15, 2016 at 12:47 AM, rashidma < notifications@github.com> wrote:

Thanks a lot Yunfei, I successfully ran the test run. Now, I was running a real sample from NIST. Out of 51 steps 48 steps ran well. I am stuck in the last few steps. The seqmule was stopped many times due to system shutdown and couple of times I killed the process. But every time I restarted it with--

nohup seqmule run NIST7035-NIST7086.script &

Please let me know any troubleshooting. I will be thankful to you. Regards MR

On Thu, Aug 11, 2016 at 1:02 AM, Yunfei Guo < notifications@github.com

wrote:

Hi Mamoon,

Please use inst/hg19_exome.legacy.bed after updating SeqMule with seqmule update --git. Thank you!

Best, Yunfei

— You are receiving this because you authored the thread. Reply to this email directly, view it on GitHub <https://github.com/WGLab/SeqMule/issues/146# issuecomment-239018748 , or mute the thread https://github.com/notifications/unsubscribe-auth/ ATyiOAPu4as9L3bQ8R0fedRCtTQ976qQks5qekpygaJpZM4Jf64z .

Mamoon Rashid, PhD *Post-Doctoral Fellow Marine Microbial Ecology Lab Red Sea Research Center 4700 King Abdullah University of Science and Technology (KAUST) Room 2217-WS03, Ibn Al-Haytham Building (2) 4700 King Abdullah University of Science and Technology* Thuwal 23955-6900, Kingdom of Saudi Arabia Office: +966 (0) 2 808-2671

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.

Mamoon Rashid, PhD *Post-Doctoral Fellow Marine Microbial Ecology Lab Red Sea Research Center 4700 King Abdullah University of Science and Technology (KAUST) Room 2217-WS03, Ibn Al-Haytham Building (2) 4700 King Abdullah University of Science and Technology* Thuwal 23955-6900, Kingdom of Saudi Arabia Office: +966 (0) 2 808-2671

Mamoon Rashid, PhD *Post-Doctoral Fellow Marine Microbial Ecology Lab Red Sea Research Center 4700 King Abdullah University of Science and Technology (KAUST) Room 2217-WS03, Ibn Al-Haytham Building (2) 4700 King Abdullah University of Science and Technology* Thuwal 23955-6900, Kingdom of Saudi Arabia Office: +966 (0) 2 808-2671

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rashidma commented 7 years ago

Yes I did run the analysis multiple times with "seqmule run *.script" in the same directory.

The analysis takes a lot of time so seqmule run saves a lot of time.

Does it affect the result after rerunning seqmule?

Thanks MR On 19 Aug 2016 17:43, "Yunfei Guo" notifications@github.com wrote:

I mean for each analysis, did you run it twice (probably with 'seqmule run' after 'seqmule pipeline')?

On Thu, Aug 18, 2016 at 7:13 PM, rashidma notifications@github.com wrote:

I re-run the analysis in a completely new directory. So both runs should not influence each other.

Thanks MR

On 18 Aug 2016 18:41, "Yunfei Guo" notifications@github.com wrote:

Is it because you tried to rerun the analysis?

On Aug 18, 2016, at 8:06 AM, rashidma notifications@github.com wrote:

Thanks a lot for your reply. I started another run with bwa-gatk-samtools-varscan and I found that it is also incomplete.

The log file contains the *gatk-multicall-extract.vcf twice for second sample. For first sample it is ok.

I could not understand the behavior. Lets first troubleshoot the first run that we are discussing.

regards MR

On Thu, Aug 18, 2016 at 5:43 PM, Yunfei Guo < notifications@github.com> wrote:

Thanks, Mamoon. Let me test the latest freebayes and get back to you later.

On Wed, Aug 17, 2016 at 10:05 PM, rashidma < notifications@github.com

wrote:

Hi I checked the VCF files for number of samples ---

VCFs number of samples NIST7035_bwamem.merge.0_freebayes.multi-call.extract.vcf 1 NIST7035_bwamem.merge.0_samtools.multi-call.extract.vcf 2 NIST7035_bwamem.merge.realn.0_gatklite.multi-call.extract.vcf 2

NIST7086_bwamem.merge.0_freebayes.multi-call.extract.vcf 1 NIST7086_bwamem.merge.0_samtools.multi-call.extract.vcf 2 NIST7086_bwamem.merge.realn.0_gatklite.multi-call.extract.vcf 2

I noticed frrebayes processed only one sample. Is this a known bug in freebayes??

I am trying to understand the system well so that I can use seqmule on my large number of samples. Best regards Mamoon

On Tue, Aug 16, 2016 at 5:46 PM, Yunfei Guo < notifications@github.com> wrote:

Hi MR,

I noticed that there was an error "ERROR: not all VCFs have the same number of samples!", could you check how many samples are there in each *multi-call.extract.vcf file? I don't know why there are two gatklite VCFs, though.

command to check # of samples:

for i in *multi-call.extract.vcf; do echo $i; perl -ne 'if (/^#CHROM/){print ((scalar (split /\t/,$_))-9),"\n"}' $i ; done

On Tue, Aug 16, 2016 at 4:33 AM, rashidma < notifications@github.com> wrote:

Hi, I just noticed in the _.log file of seqmule run -- the VCF " NIST7086_result/NIST7086_bwamem.merge.realn.0gatklite. multi-call.extract.vcf" has been mentioned twice. please see below colored in red. Is this creating proble?? Per sample there should be 4 VCF files (3 different aligner variant caller combinations and one consensus) BUT in case of NIST7086 why this is 5 VCF. *

_2 sample(s) in this analysis: NIST7035 NIST7086All input is merged on a per-sample basis (after alignment if FASTQ).Multi-sample variant calling ENABLED.Input is exome (or captured) sequencing data.4 variant caller(s) used: GATKLite_UnifiedGenotyper SAMtools FreeBayes Consensus1 aligner(s) used: bwamemFile used for caculating coverage statistics and extracting variants: nexterarapidcapture_expandedexome_targetedregions. nooverlap.bedReadgroup : READGROUP_NIST7035,READGROUP_NIST7086Library : LIBRARYSequencing platform: ILLUMINAReference genome build is hg19dbsnp138 is used for variant calling and recalibration (in GATK VQSR).Java memory usage is limited to 1750mjava executable path: javaMax number of processes: 12NOTICE: /tmp will be used for storing temporary files################################################### FINAL OUTPUT for NIST7035:################################################### NOTICE: Both consensus results and individual results are listed.WARNING: consensus results may not be available because some variant callers or aligners may fail to generate output.NIST7035_result/NIST7035.extract_consensus. vcfNIST7035_result/NIST7035_bwamem.merge.realn.0_gatklite. multi-call.extract.vcfNIST7035result/NIST7035 bwamem.merge.0samtools.multi-call.extract.vcfNIST7035 result/NIST7035_bwamem.merge.0_freebayes.multi-call. extract.vcf#################################################

FINAL

OUTPUT for NIST7086:################################################### NOTICE: Both consensus results and individual results are listed.WARNING: consensus results may not be available because some variant callers or aligners may fail to generate output._

_NIST7086_result/NIST7086.extractconsensus.vcfNIST7086 result/NIST7086_bwamem.merge.realn.0_gatklite.multi-call. extract.vcfNIST7086_result/NIST7086bwamem.merge.realn.0 gatklite.multi-call.extract.vcfNIST7086result/NIST7086 bwamem.merge.0samtools.multi-call.extract.vcfNIST7086 result/NIST7086_bwamem.merge.0freebayes.multi-call. extract.vcf

Thanks

On Tue, Aug 16, 2016 at 8:07 AM, Mamoon Rashid < rashidmamoon@gmail.com

wrote:

Hi Yunfei, I sent you the big nohup file (~8MB) and it failed to reach you. Now I am sending you the last 1000 lines of the nohup file. I can see there are errors at step 42 and 43. I thank you so much for your time. MR

On Mon, Aug 15, 2016 at 4:05 PM, Yunfei Guo < notifications@github.com> wrote:

Hi Rashid,

Could you please provide the contents of nohup.out file? I need to know where it stopped.

Thanks.

Best, Yunfei

On Mon, Aug 15, 2016 at 12:47 AM, rashidma < notifications@github.com> wrote:

Thanks a lot Yunfei, I successfully ran the test run. Now, I was running a real sample from NIST. Out of 51 steps 48 steps ran well. I am stuck in the last few steps. The seqmule was stopped many times due to system shutdown and couple of times I killed the process. But every time I restarted it with--

nohup seqmule run NIST7035-NIST7086.script &

Please let me know any troubleshooting. I will be thankful to you. Regards MR

On Thu, Aug 11, 2016 at 1:02 AM, Yunfei Guo < notifications@github.com

wrote:

Hi Mamoon,

Please use inst/hg19_exome.legacy.bed after updating SeqMule with seqmule update --git. Thank you!

Best, Yunfei

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Mamoon Rashid, PhD *Post-Doctoral Fellow Marine Microbial Ecology Lab Red Sea Research Center 4700 King Abdullah University of Science and Technology (KAUST) Room 2217-WS03, Ibn Al-Haytham Building (2) 4700 King Abdullah University of Science and Technology* Thuwal 23955-6900, Kingdom of Saudi Arabia Office: +966 (0) 2 808-2671

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yunfeiguo commented 7 years ago

This should not affect the results, however, it may be the reason why you see some vcf files more than once in the runtime information.

On Fri, Aug 19, 2016 at 7:56 AM, rashidma notifications@github.com wrote:

Yes I did run the analysis multiple times with "seqmule run *.script" in the same directory.

The analysis takes a lot of time so seqmule run saves a lot of time.

Does it affect the result after rerunning seqmule?

Thanks MR

On 19 Aug 2016 17:43, "Yunfei Guo" notifications@github.com wrote:

I mean for each analysis, did you run it twice (probably with 'seqmule run' after 'seqmule pipeline')?

On Thu, Aug 18, 2016 at 7:13 PM, rashidma notifications@github.com wrote:

I re-run the analysis in a completely new directory. So both runs should not influence each other.

Thanks MR

On 18 Aug 2016 18:41, "Yunfei Guo" notifications@github.com wrote:

Is it because you tried to rerun the analysis?

On Aug 18, 2016, at 8:06 AM, rashidma notifications@github.com wrote:

Thanks a lot for your reply. I started another run with bwa-gatk-samtools-varscan and I found that it is also incomplete.

The log file contains the *gatk-multicall-extract.vcf twice for second sample. For first sample it is ok.

I could not understand the behavior. Lets first troubleshoot the first run that we are discussing.

regards MR

On Thu, Aug 18, 2016 at 5:43 PM, Yunfei Guo < notifications@github.com> wrote:

Thanks, Mamoon. Let me test the latest freebayes and get back to you later.

On Wed, Aug 17, 2016 at 10:05 PM, rashidma < notifications@github.com

wrote:

Hi I checked the VCF files for number of samples ---

VCFs number of samples NIST7035_bwamem.merge.0_freebayes.multi-call.extract.vcf 1 NIST7035_bwamem.merge.0_samtools.multi-call.extract.vcf 2 NIST7035_bwamem.merge.realn.0_gatklite.multi-call.extract.vcf 2

NIST7086_bwamem.merge.0_freebayes.multi-call.extract.vcf 1 NIST7086_bwamem.merge.0_samtools.multi-call.extract.vcf 2 NIST7086_bwamem.merge.realn.0_gatklite.multi-call.extract.vcf 2

I noticed frrebayes processed only one sample. Is this a known bug in freebayes??

I am trying to understand the system well so that I can use seqmule on my large number of samples. Best regards Mamoon

On Tue, Aug 16, 2016 at 5:46 PM, Yunfei Guo < notifications@github.com> wrote:

Hi MR,

I noticed that there was an error "ERROR: not all VCFs have the same number of samples!", could you check how many samples are there in each *multi-call.extract.vcf file? I don't know why there are two gatklite VCFs, though.

command to check # of samples:

for i in *multi-call.extract.vcf; do echo $i; perl -ne 'if (/^#CHROM/){print ((scalar (split /\t/,$_))-9),"\n"}' $i ; done

On Tue, Aug 16, 2016 at 4:33 AM, rashidma < notifications@github.com> wrote:

Hi, I just noticed in the _.log file of seqmule run -- the VCF " NIST7086_result/NIST7086_bwamem.merge.realn.0gatklite. multi-call.extract.vcf" has been mentioned twice. please see below colored in red. Is this creating proble?? Per sample there should be 4 VCF files (3 different aligner variant caller combinations and one consensus) BUT in case of NIST7086 why this is 5 VCF. *

_2 sample(s) in this analysis: NIST7035 NIST7086All input is merged on a per-sample basis (after alignment if FASTQ).Multi-sample variant calling ENABLED.Input is exome (or captured) sequencing data.4 variant caller(s) used: GATKLite_UnifiedGenotyper SAMtools FreeBayes Consensus1 aligner(s) used: bwamemFile used for caculating coverage statistics and extracting variants: nexterarapidcapture_expandedexome_targetedregions. nooverlap.bedReadgroup : READGROUP_NIST7035,READGROUP_NIST7086Library : LIBRARYSequencing platform: ILLUMINAReference genome build is hg19dbsnp138 is used for variant calling and recalibration (in GATK VQSR).Java memory usage is limited to 1750mjava executable path: javaMax number of processes: 12NOTICE: /tmp will be used for storing temporary files################################################### FINAL OUTPUT for NIST7035:##################### ############################## NOTICE: Both consensus results and individual results are listed.WARNING: consensus results may not be available because some variant callers or aligners may fail to generate output.NIST7035_result/NIST7035.extract_consensus. vcfNIST7035_result/NIST7035_bwamem.merge.realn.0_gatklite. multi-call.extract.vcfNIST7035result/NIST7035 bwamem.merge.0samtools.multi-call.extract.vcfNIST7035 result/NIST7035_bwamem.merge.0_freebayes.multi-call. extract.vcf################### ##############################

FINAL

OUTPUT for NIST7086:##################### ############################## NOTICE: Both consensus results and individual results are listed.WARNING: consensus results may not be available because some variant callers or aligners may fail to generate output._

_NIST7086_result/NIST7086.extractconsensus.vcfNIST7086 result/NIST7086_bwamem.merge.realn.0_gatklite.multi-call. extract.vcfNIST7086_result/NIST7086bwamem.merge.realn.0 gatklite.multi-call.extract.vcfNIST7086result/NIST7086 bwamem.merge.0samtools.multi-call.extract.vcfNIST7086 result/NIST7086_bwamem.merge.0freebayes.multi-call. extract.vcf

Thanks

On Tue, Aug 16, 2016 at 8:07 AM, Mamoon Rashid < rashidmamoon@gmail.com

wrote:

Hi Yunfei, I sent you the big nohup file (~8MB) and it failed to reach you. Now I am sending you the last 1000 lines of the nohup file. I can see there are errors at step 42 and 43. I thank you so much for your time. MR

On Mon, Aug 15, 2016 at 4:05 PM, Yunfei Guo < notifications@github.com> wrote:

Hi Rashid,

Could you please provide the contents of nohup.out file? I need to know where it stopped.

Thanks.

Best, Yunfei

On Mon, Aug 15, 2016 at 12:47 AM, rashidma < notifications@github.com> wrote:

Thanks a lot Yunfei, I successfully ran the test run. Now, I was running a real sample from NIST. Out of 51 steps 48 steps ran well. I am stuck in the last few steps. The seqmule was stopped many times due to system shutdown and couple of times I killed the process. But every time I restarted it with--

nohup seqmule run NIST7035-NIST7086.script &

Please let me know any troubleshooting. I will be thankful to you. Regards MR

On Thu, Aug 11, 2016 at 1:02 AM, Yunfei Guo < notifications@github.com

wrote:

Hi Mamoon,

Please use inst/hg19_exome.legacy.bed after updating SeqMule with seqmule update --git. Thank you!

Best, Yunfei

— You are receiving this because you authored the thread. Reply to this email directly, view it on GitHub <https://github.com/WGLab/SeqMule/issues/146# issuecomment-239018748 , or mute the thread https://github.com/notifications/unsubscribe-auth/ ATyiOAPu4as9L3bQ8R0fedRCtTQ976qQks5qekpygaJpZM4Jf64z .

Mamoon Rashid, PhD *Post-Doctoral Fellow Marine Microbial Ecology Lab Red Sea Research Center 4700 King Abdullah University of Science and Technology (KAUST) Room 2217-WS03, Ibn Al-Haytham Building (2) 4700 King Abdullah University of Science and Technology* Thuwal 23955-6900, Kingdom of Saudi Arabia Office: +966 (0) 2 808-2671

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Mamoon Rashid, PhD *Post-Doctoral Fellow Marine Microbial Ecology Lab Red Sea Research Center 4700 King Abdullah University of Science and Technology (KAUST) Room 2217-WS03, Ibn Al-Haytham Building (2) 4700 King Abdullah University of Science and Technology* Thuwal 23955-6900, Kingdom of Saudi Arabia Office: +966 (0) 2 808-2671

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rashidma commented 7 years ago

Hi Yunfei, Did you test the Freebayes? In my seqmule run it failed to call SNPs for multiple samples (as we discussed in the last email). Thanks Mamoon

On Fri, Aug 19, 2016 at 7:53 PM, Yunfei Guo notifications@github.com wrote:

This should not affect the results, however, it may be the reason why you see some vcf files more than once in the runtime information.

On Fri, Aug 19, 2016 at 7:56 AM, rashidma notifications@github.com wrote:

Yes I did run the analysis multiple times with "seqmule run *.script" in the same directory.

The analysis takes a lot of time so seqmule run saves a lot of time.

Does it affect the result after rerunning seqmule?

Thanks MR

On 19 Aug 2016 17:43, "Yunfei Guo" notifications@github.com wrote:

I mean for each analysis, did you run it twice (probably with 'seqmule run' after 'seqmule pipeline')?

On Thu, Aug 18, 2016 at 7:13 PM, rashidma notifications@github.com wrote:

I re-run the analysis in a completely new directory. So both runs should not influence each other.

Thanks MR

On 18 Aug 2016 18:41, "Yunfei Guo" notifications@github.com wrote:

Is it because you tried to rerun the analysis?

On Aug 18, 2016, at 8:06 AM, rashidma notifications@github.com wrote:

Thanks a lot for your reply. I started another run with bwa-gatk-samtools-varscan and I found that it is also incomplete.

The log file contains the *gatk-multicall-extract.vcf twice for second sample. For first sample it is ok.

I could not understand the behavior. Lets first troubleshoot the first run that we are discussing.

regards MR

On Thu, Aug 18, 2016 at 5:43 PM, Yunfei Guo < notifications@github.com> wrote:

Thanks, Mamoon. Let me test the latest freebayes and get back to you later.

On Wed, Aug 17, 2016 at 10:05 PM, rashidma < notifications@github.com

wrote:

Hi I checked the VCF files for number of samples ---

VCFs number of samples NIST7035_bwamem.merge.0_freebayes.multi-call.extract.vcf 1 NIST7035_bwamem.merge.0_samtools.multi-call.extract.vcf 2 NIST7035_bwamem.merge.realn.0_gatklite.multi-call.extract. vcf 2

NIST7086_bwamem.merge.0_freebayes.multi-call.extract.vcf 1 NIST7086_bwamem.merge.0_samtools.multi-call.extract.vcf 2 NIST7086_bwamem.merge.realn.0_gatklite.multi-call.extract. vcf 2

I noticed frrebayes processed only one sample. Is this a known bug in freebayes??

I am trying to understand the system well so that I can use seqmule on my large number of samples. Best regards Mamoon

On Tue, Aug 16, 2016 at 5:46 PM, Yunfei Guo < notifications@github.com> wrote:

Hi MR,

I noticed that there was an error "ERROR: not all VCFs have the same number of samples!", could you check how many samples are there in each *multi-call.extract.vcf file? I don't know why there are two gatklite VCFs, though.

command to check # of samples:

for i in *multi-call.extract.vcf; do echo $i; perl -ne 'if (/^#CHROM/){print ((scalar (split /\t/,$_))-9),"\n"}' $i ; done

On Tue, Aug 16, 2016 at 4:33 AM, rashidma < notifications@github.com> wrote:

Hi, I just noticed in the _.log file of seqmule run -- the VCF " NIST7086_result/NIST7086_bwamem.merge.realn.0gatklite. multi-call.extract.vcf" has been mentioned twice. please see below colored in red. Is this creating proble?? Per sample there should be 4 VCF files (3 different aligner variant caller combinations and one consensus) BUT in case of NIST7086 why this is 5 VCF. *

_2 sample(s) in this analysis: NIST7035 NIST7086All input is merged on a per-sample basis (after alignment if FASTQ).Multi-sample variant calling ENABLED.Input is exome (or captured) sequencing data.4 variant caller(s) used: GATKLite_UnifiedGenotyper SAMtools FreeBayes Consensus1 aligner(s) used: bwamemFile used for caculating coverage statistics and extracting variants: nexterarapidcapture_expandedexome_targetedregions. nooverlap.bedReadgroup : READGROUP_NIST7035,READGROUP_NIST7086Library : LIBRARYSequencing platform: ILLUMINAReference genome build is hg19dbsnp138 is used for variant calling and recalibration (in GATK VQSR).Java memory usage is limited to 1750mjava executable path: javaMax number of processes: 12NOTICE: /tmp will be used for storing temporary files################################################### FINAL OUTPUT for NIST7035:##################### ############################## NOTICE: Both consensus results and individual results are listed.WARNING: consensus results may not be available because some variant callers or aligners may fail to generate output.NIST7035_result/NIST7035.extract_consensus. vcfNIST7035result/NIST7035 bwamem.merge.realn.0_gatklite. multi-call.extract.vcfNIST7035result/NIST7035 bwamem.merge.0samtools.multi-call.extract.vcfNIST7035 result/NIST7035_bwamem.merge.0_freebayes.multi-call. extract.vcf################### ##############################

FINAL

OUTPUT for NIST7086:##################### ############################## NOTICE: Both consensus results and individual results are listed.WARNING: consensus results may not be available because some variant callers or aligners may fail to generate output._

_NIST7086_result/NIST7086.extractconsensus.vcfNIST7086 result/NIST7086_bwamem.merge. realn.0_gatklite.multi-call. extract.vcfNIST7086_result/ NIST7086bwamem.merge.realn.0 gatklite.multi-call.extract.vcfNIST7086result/NIST7086 bwamem.merge.0samtools.multi-call.extract.vcfNIST7086 result/NIST7086_bwamem.merge.0freebayes.multi-call. extract.vcf

Thanks

On Tue, Aug 16, 2016 at 8:07 AM, Mamoon Rashid < rashidmamoon@gmail.com

wrote:

Hi Yunfei, I sent you the big nohup file (~8MB) and it failed to reach you. Now I am sending you the last 1000 lines of the nohup file. I can see there are errors at step 42 and 43. I thank you so much for your time. MR

On Mon, Aug 15, 2016 at 4:05 PM, Yunfei Guo < notifications@github.com> wrote:

Hi Rashid,

Could you please provide the contents of nohup.out file? I need to know where it stopped.

Thanks.

Best, Yunfei

On Mon, Aug 15, 2016 at 12:47 AM, rashidma < notifications@github.com> wrote:

Thanks a lot Yunfei, I successfully ran the test run. Now, I was running a real sample from NIST. Out of 51 steps 48 steps ran well. I am stuck in the last few steps. The seqmule was stopped many times due to system shutdown and couple of times I killed the process. But every time I restarted it with--

nohup seqmule run NIST7035-NIST7086.script &

Please let me know any troubleshooting. I will be thankful to you. Regards MR

On Thu, Aug 11, 2016 at 1:02 AM, Yunfei Guo < notifications@github.com

wrote:

Hi Mamoon,

Please use inst/hg19_exome.legacy.bed after updating SeqMule with seqmule update --git. Thank you!

Best, Yunfei

— You are receiving this because you authored the thread. Reply to this email directly, view it on GitHub <https://github.com/WGLab/SeqMule/issues/146# issuecomment-239018748 , or mute the thread https://github.com/notifications/unsubscribe- auth/ ATyiOAPu4as9L3bQ8R0fedRCtTQ976 qQks5qekpygaJpZM4Jf64z .

Mamoon Rashid, PhD *Post-Doctoral Fellow Marine Microbial Ecology Lab Red Sea Research Center 4700 King Abdullah University of Science and Technology (KAUST) Room 2217-WS03, Ibn Al-Haytham Building (2) 4700 King Abdullah University of Science and Technology* Thuwal 23955-6900, Kingdom of Saudi Arabia Office: +966 (0) 2 808-2671

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Mamoon Rashid, PhD *Post-Doctoral Fellow Marine Microbial Ecology Lab Red Sea Research Center 4700 King Abdullah University of Science and Technology (KAUST) Room 2217-WS03, Ibn Al-Haytham Building (2) 4700 King Abdullah University of Science and Technology* Thuwal 23955-6900, Kingdom of Saudi Arabia Office: +966 (0) 2 808-2671

Mamoon Rashid, PhD *Post-Doctoral Fellow Marine Microbial Ecology Lab Red Sea Research Center 4700 King Abdullah University of Science and Technology (KAUST) Room 2217-WS03, Ibn Al-Haytham Building (2) 4700 King Abdullah University of Science and Technology* Thuwal 23955-6900, Kingdom of Saudi Arabia Office: +966 (0) 2 808-2671

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Mamoon Rashid, PhD *Post-Doctoral Fellow Marine Microbial Ecology Lab Red Sea Research Center 4700 King Abdullah University of Science and Technology (KAUST) Room 2217-WS03, Ibn Al-Haytham Building (2) 4700 King Abdullah University of Science and Technology* Thuwal 23955-6900, Kingdom of Saudi Arabia Office: +966 (0) 2 808-2671

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yunfeiguo commented 7 years ago

Hi Mamoon,

Sorry, our servers are temporarily unavailable right now. We are still trying to fix them. Could you please try install freebayes yourself and run seqmule from the freebayes step?

Instructions:

go to seqmule directory
move exe/freebayes folder to somewhere outside seqmule folder
download freebayes by "git clone --recursive git:// github.com/ekg/freebayes.git exe/freebayes"
cd exe/freebayes
make
run seqmule from the step where freebayes is executed (look for the step number for freebayes variant calling in *.script)

Thanks.

On Sun, Aug 21, 2016 at 4:28 AM, rashidma notifications@github.com wrote:

Hi Yunfei, Did you test the Freebayes? In my seqmule run it failed to call SNPs for multiple samples (as we discussed in the last email). Thanks Mamoon

On Fri, Aug 19, 2016 at 7:53 PM, Yunfei Guo notifications@github.com

wrote:

This should not affect the results, however, it may be the reason why you see some vcf files more than once in the runtime information.

On Fri, Aug 19, 2016 at 7:56 AM, rashidma notifications@github.com wrote:

Yes I did run the analysis multiple times with "seqmule run *.script" in the same directory.

The analysis takes a lot of time so seqmule run saves a lot of time.

Does it affect the result after rerunning seqmule?

Thanks MR

On 19 Aug 2016 17:43, "Yunfei Guo" notifications@github.com wrote:

I mean for each analysis, did you run it twice (probably with 'seqmule run' after 'seqmule pipeline')?

On Thu, Aug 18, 2016 at 7:13 PM, rashidma notifications@github.com wrote:

I re-run the analysis in a completely new directory. So both runs should not influence each other.

Thanks MR

On 18 Aug 2016 18:41, "Yunfei Guo" notifications@github.com wrote:

Is it because you tried to rerun the analysis?

On Aug 18, 2016, at 8:06 AM, rashidma < notifications@github.com> wrote:

Thanks a lot for your reply. I started another run with bwa-gatk-samtools-varscan and I found that it is also incomplete.

The log file contains the *gatk-multicall-extract.vcf twice for second sample. For first sample it is ok.

I could not understand the behavior. Lets first troubleshoot the first run that we are discussing.

regards MR

On Thu, Aug 18, 2016 at 5:43 PM, Yunfei Guo < notifications@github.com> wrote:

Thanks, Mamoon. Let me test the latest freebayes and get back to you later.

On Wed, Aug 17, 2016 at 10:05 PM, rashidma < notifications@github.com

wrote:

Hi I checked the VCF files for number of samples ---

VCFs number of samples NIST7035_bwamem.merge.0_freebayes.multi-call.extract.vcf 1 NIST7035_bwamem.merge.0_samtools.multi-call.extract.vcf 2 NIST7035_bwamem.merge.realn.0_gatklite.multi-call.extract. vcf 2

NIST7086_bwamem.merge.0_freebayes.multi-call.extract.vcf 1 NIST7086_bwamem.merge.0_samtools.multi-call.extract.vcf 2 NIST7086_bwamem.merge.realn.0_gatklite.multi-call.extract. vcf 2

I noticed frrebayes processed only one sample. Is this a known bug in freebayes??

I am trying to understand the system well so that I can use seqmule on my large number of samples. Best regards Mamoon

On Tue, Aug 16, 2016 at 5:46 PM, Yunfei Guo < notifications@github.com> wrote:

Hi MR,

I noticed that there was an error "ERROR: not all VCFs have the same number of samples!", could you check how many samples are there in each *multi-call.extract.vcf file? I don't know why there are two gatklite VCFs, though.

command to check # of samples:

for i in *multi-call.extract.vcf; do echo $i; perl -ne 'if (/^#CHROM/){print ((scalar (split /\t/,$_))-9),"\n"}' $i ; done

On Tue, Aug 16, 2016 at 4:33 AM, rashidma < notifications@github.com> wrote:

Hi, I just noticed in the _.log file of seqmule run -- the VCF " NIST7086result/NIST7086 bwamem.merge.realn.0gatklite. multi-call.extract.vcf" has been mentioned twice. please see below colored in red. Is this creating proble?? Per sample there should be 4 VCF files (3 different aligner variant caller combinations and one consensus) BUT in case of NIST7086 why this is 5 VCF. *

_2 sample(s) in this analysis: NIST7035 NIST7086All input is merged on a per-sample basis (after alignment if FASTQ).Multi-sample variant calling ENABLED.Input is exome (or captured) sequencing data.4 variant caller(s) used: GATKLite_UnifiedGenotyper SAMtools FreeBayes Consensus1 aligner(s) used: bwamemFile used for caculating coverage statistics and extracting variants: nexterarapidcapture_expandedexome_targetedregions. nooverlap.bedReadgroup : READGROUP_NIST7035,READGROUP_NIST7086Library : LIBRARYSequencing platform: ILLUMINAReference genome build is hg19dbsnp138 is used for variant calling and recalibration (in GATK VQSR).Java memory usage is limited to 1750mjava executable path: javaMax number of processes: 12NOTICE: /tmp will be used for storing temporary files######################### ########################## FINAL OUTPUT for NIST7035:##################### ############################## NOTICE: Both consensus results and individual results are listed.WARNING: consensus results may not be available because some variant callers or aligners may fail to generate output.NIST7035_result/NIST7035.extract_consensus. vcfNIST7035result/NIST7035 bwamem.merge.realn.0_gatklite. multi-call.extract.vcfNIST7035result/NIST7035 bwamem.merge.0samtools.multi- call.extract.vcfNIST7035 result/NIST7035_bwamem.merge.0_freebayes.multi-call. extract.vcf################### ##############################

FINAL

OUTPUT for NIST7086:##################### ############################## NOTICE: Both consensus results and individual results are listed.WARNING: consensus results may not be available because some variant callers or aligners may fail to generate output._

_NIST7086_result/NIST7086. extractconsensus.vcfNIST7086 result/NIST7086_bwamem.merge. realn.0_gatklite.multi-call. extract.vcfNIST7086_result/ NIST7086bwamem.merge.realn.0 gatklite.multi-call.extract. vcfNIST7086result/NIST7086 bwamem.merge.0samtools.multi- call.extract.vcfNIST7086 result/NIST7086_bwamem.merge.0freebayes.multi-call. extract.vcf

Thanks

On Tue, Aug 16, 2016 at 8:07 AM, Mamoon Rashid < rashidmamoon@gmail.com

wrote:

Hi Yunfei, I sent you the big nohup file (~8MB) and it failed to reach you. Now I am sending you the last 1000 lines of the nohup file. I can see there are errors at step 42 and 43. I thank you so much for your time. MR

On Mon, Aug 15, 2016 at 4:05 PM, Yunfei Guo < notifications@github.com> wrote:

Hi Rashid,

Could you please provide the contents of nohup.out file? I need to know where it stopped.

Thanks.

Best, Yunfei

On Mon, Aug 15, 2016 at 12:47 AM, rashidma < notifications@github.com> wrote:

Thanks a lot Yunfei, I successfully ran the test run. Now, I was running a real sample from NIST. Out of 51 steps 48 steps ran well. I am stuck in the last few steps. The seqmule was stopped many times due to system shutdown and couple of times I killed the process. But every time I restarted it with--

nohup seqmule run NIST7035-NIST7086.script &

Please let me know any troubleshooting. I will be thankful to you. Regards MR

On Thu, Aug 11, 2016 at 1:02 AM, Yunfei Guo < notifications@github.com

wrote:

Hi Mamoon,

Please use inst/hg19_exome.legacy.bed after updating SeqMule with seqmule update --git. Thank you!

Best, Yunfei

— You are receiving this because you authored the thread. Reply to this email directly, view it on GitHub <https://github.com/WGLab/SeqMule/issues/146# issuecomment-239018748 , or mute the thread https://github.com/notifications/unsubscribe- auth/ ATyiOAPu4as9L3bQ8R0fedRCtTQ976 qQks5qekpygaJpZM4Jf64z .

Mamoon Rashid, PhD *Post-Doctoral Fellow Marine Microbial Ecology Lab Red Sea Research Center 4700 King Abdullah University of Science and Technology (KAUST) Room 2217-WS03, Ibn Al-Haytham Building (2) 4700 King Abdullah University of Science and Technology* Thuwal 23955-6900, Kingdom of Saudi Arabia Office: +966 (0) 2 808-2671

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Mamoon Rashid, PhD *Post-Doctoral Fellow Marine Microbial Ecology Lab Red Sea Research Center 4700 King Abdullah University of Science and Technology (KAUST) Room 2217-WS03, Ibn Al-Haytham Building (2) 4700 King Abdullah University of Science and Technology* Thuwal 23955-6900, Kingdom of Saudi Arabia Office: +966 (0) 2 808-2671

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rashidma commented 7 years ago

I installed freebayes from github as you mentioned. Please let me know how to run seqmule from the step where freebayes is executed? I looked at *.script file and found ---

_[33]JOBID=0PID=14673command=/work/rashidma/software/seqmule_by_admin/SeqMule-master/bin/secondary/../../bin/secondary/worker /work/rashidma/Exome_data/GIAB_NIST_exome_data/MyAnalysis/04_seqmule_qsub-ucschg19/seqmule.08162016.23639.logs 33 "/work/rashidma/software/seqmule_by_admin/SeqMule-master/bin/secondary/runFREEBAYES -advanced NIST7035-NIST7086.config -n 9 -freebayes /work/rashidma/software/seqmule_by_admin/SeqMule-master/exe/freebayes/bin/freebayes -vcfsorter /work/rashidma/software/seqmule_by_admin/SeqMule-master/bin/secondary/../../bin/secondary/vcfsorter -ref /work/rashidma/Databases/hg19/UCSC/ucsc.hg19.fa -samtools /work/rashidma/software/seqmule_by_admin/SeqMule-master/exe/samtools/samtools -tmpdir /tmp -vcf NIST7035_result/NIST7035_bwamem.merge.0_freebayes.multi-call.vcf -bed nexterarapidcapture_expandedexome_targetedregions.nooverlap.bed -bam NIST7035_result/NIST7035_bwamem.merge.bam -bam NIST7086_result/NIST7086_bwamem.merge.bam"dependency=23,24message=freebayes.multi-call variant callingnCPUrequested=12status=finished

What should i do now? Thanks MR

On Mon, Aug 22, 2016 at 2:15 AM, Yunfei Guo notifications@github.com wrote:

Hi Mamoon,

Sorry, our servers are temporarily unavailable right now. We are still trying to fix them. Could you please try install freebayes yourself and run seqmule from the freebayes step?

Instructions:

go to seqmule directory

move exe/freebayes folder to somewhere outside seqmule folder

download freebayes by "git clone --recursive git:// github.com/ekg/freebayes.git exe/freebayes"

cd exe/freebayes

make

run seqmule from the step where freebayes is executed (look for the step number for freebayes variant calling in *.script)

Thanks.

On Sun, Aug 21, 2016 at 4:28 AM, rashidma notifications@github.com wrote:

Hi Yunfei, Did you test the Freebayes? In my seqmule run it failed to call SNPs for multiple samples (as we discussed in the last email). Thanks Mamoon

On Fri, Aug 19, 2016 at 7:53 PM, Yunfei Guo notifications@github.com

wrote:

This should not affect the results, however, it may be the reason why you see some vcf files more than once in the runtime information.

On Fri, Aug 19, 2016 at 7:56 AM, rashidma notifications@github.com wrote:

Yes I did run the analysis multiple times with "seqmule run *.script" in the same directory.

The analysis takes a lot of time so seqmule run saves a lot of time.

Does it affect the result after rerunning seqmule?

Thanks MR

On 19 Aug 2016 17:43, "Yunfei Guo" notifications@github.com wrote:

I mean for each analysis, did you run it twice (probably with 'seqmule run' after 'seqmule pipeline')?

On Thu, Aug 18, 2016 at 7:13 PM, rashidma < notifications@github.com> wrote:

I re-run the analysis in a completely new directory. So both runs should not influence each other.

Thanks MR

On 18 Aug 2016 18:41, "Yunfei Guo" notifications@github.com wrote:

Is it because you tried to rerun the analysis?

On Aug 18, 2016, at 8:06 AM, rashidma < notifications@github.com> wrote:

Thanks a lot for your reply. I started another run with bwa-gatk-samtools-varscan and I found that it is also incomplete.

The log file contains the *gatk-multicall-extract.vcf twice for second sample. For first sample it is ok.

I could not understand the behavior. Lets first troubleshoot the first run that we are discussing.

regards MR

On Thu, Aug 18, 2016 at 5:43 PM, Yunfei Guo < notifications@github.com> wrote:

Thanks, Mamoon. Let me test the latest freebayes and get back to you later.

On Wed, Aug 17, 2016 at 10:05 PM, rashidma < notifications@github.com

wrote:

Hi I checked the VCF files for number of samples ---

VCFs number of samples NIST7035_bwamem.merge.0_freebayes.multi-call.extract.vcf 1 NIST7035_bwamem.merge.0_samtools.multi-call.extract.vcf 2 NIST7035bwamem.merge.realn.0 gatklite.multi-call.extract. vcf 2

NIST7086_bwamem.merge.0_freebayes.multi-call.extract.vcf 1 NIST7086_bwamem.merge.0_samtools.multi-call.extract.vcf 2 NIST7086bwamem.merge.realn.0 gatklite.multi-call.extract. vcf 2

I noticed frrebayes processed only one sample. Is this a known bug in freebayes??

I am trying to understand the system well so that I can use seqmule on my large number of samples. Best regards Mamoon

On Tue, Aug 16, 2016 at 5:46 PM, Yunfei Guo < notifications@github.com> wrote:

Hi MR,

I noticed that there was an error "ERROR: not all VCFs have the same number of samples!", could you check how many samples are there in each *multi-call.extract.vcf file? I don't know why there are two gatklite VCFs, though.

command to check # of samples:

for i in *multi-call.extract.vcf; do echo $i; perl -ne 'if (/^#CHROM/){print ((scalar (split /\t/,$_))-9),"\n"}' $i ; done

On Tue, Aug 16, 2016 at 4:33 AM, rashidma < notifications@github.com> wrote:

Hi, I just noticed in the _.log file of seqmule run -- the VCF " NIST7086result/NIST7086 bwamem.merge.realn.0gatklite. multi-call.extract.vcf" has been mentioned twice. please see below colored in red. Is this creating proble?? Per sample there should be 4 VCF files (3 different aligner variant caller combinations and one consensus) BUT in case of NIST7086 why this is 5 VCF. *

_2 sample(s) in this analysis: NIST7035 NIST7086All input is merged on a per-sample basis (after alignment if FASTQ).Multi-sample variant calling ENABLED.Input is exome (or captured) sequencing data.4 variant caller(s) used: GATKLite_UnifiedGenotyper SAMtools FreeBayes Consensus1 aligner(s) used: bwamemFile used for caculating coverage statistics and extracting variants: nexterarapidcapture_expandedexome_targetedregions. nooverlap.bedReadgroup : READGROUP_NIST7035,READGROUP_NIST7086Library : LIBRARYSequencing platform: ILLUMINAReference genome build is hg19dbsnp138 is used for variant calling and recalibration (in GATK VQSR).Java memory usage is limited to 1750mjava executable path: javaMax number of processes: 12NOTICE: /tmp will be used for storing temporary files######################### ########################## FINAL OUTPUT for NIST7035:##################### ############################## NOTICE: Both consensus results and individual results are listed.WARNING: consensus results may not be available because some variant callers or aligners may fail to generate output.NIST7035_result/NIST7035.extract_consensus. vcfNIST7035result/NIST7035 bwamem.merge.realn.0_gatklite. multi-call.extract.vcfNIST7035result/NIST7035 bwamem.merge.0samtools.multi- call.extract.vcfNIST7035 result/NIST7035_bwamem.merge.0_freebayes.multi-call. extract.vcf################### ##############################

FINAL

OUTPUT for NIST7086:##################### ############################## NOTICE: Both consensus results and individual results are listed.WARNING: consensus results may not be available because some variant callers or aligners may fail to generate output._

_NIST7086_result/NIST7086. extractconsensus.vcfNIST7086 result/NIST7086_bwamem.merge. realn.0_gatklite.multi-call. extract.vcfNIST7086_result/ NIST7086bwamem.merge.realn.0 gatklite.multi-call.extract. vcfNIST7086result/NIST7086 bwamem.merge.0samtools.multi- call.extract.vcfNIST7086 result/NIST7086_bwamem.merge.0freebayes.multi-call. extract.vcf

Thanks

On Tue, Aug 16, 2016 at 8:07 AM, Mamoon Rashid < rashidmamoon@gmail.com

wrote:

Hi Yunfei, I sent you the big nohup file (~8MB) and it failed to reach you. Now I am sending you the last 1000 lines of the nohup file. I can see there are errors at step 42 and 43. I thank you so much for your time. MR

On Mon, Aug 15, 2016 at 4:05 PM, Yunfei Guo < notifications@github.com> wrote:

Hi Rashid,

Could you please provide the contents of nohup.out file? I need to know where it stopped.

Thanks.

Best, Yunfei

On Mon, Aug 15, 2016 at 12:47 AM, rashidma < notifications@github.com> wrote:

Thanks a lot Yunfei, I successfully ran the test run. Now, I was running a real sample from NIST. Out of 51 steps 48 steps ran well. I am stuck in the last few steps. The seqmule was stopped many times due to system shutdown and couple of times I killed the process. But every time I restarted it with--

nohup seqmule run NIST7035-NIST7086.script &

Please let me know any troubleshooting. I will be thankful to you. Regards MR

On Thu, Aug 11, 2016 at 1:02 AM, Yunfei Guo < notifications@github.com

wrote:

Hi Mamoon,

Please use inst/hg19_exome.legacy.bed after updating SeqMule with seqmule update --git. Thank you!

Best, Yunfei

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yunfeiguo commented 7 years ago

33 is the step number.

seqmule run -n 33 yourseqmule.script

On Mon, Aug 22, 2016 at 6:27 AM, rashidma notifications@github.com wrote:

I installed freebayes from github as you mentioned. Please let me know how to run seqmule from the step where freebayes is executed? I looked at *.script file and found ---

_[33]JOBID=0PID=14673command=/work/rashidma/software/ seqmule_by_admin/SeqMule-master/bin/secondary/../../bin/secondary/worker /work/rashidma/Exome_data/GIAB_NIST_exome_data/MyAnalysis/04_seqmule_qsub- ucschg19/seqmule.08162016.23639.logs 33 "/work/rashidma/software/seqmule_by_admin/SeqMule-master/bin/secondary/ runFREEBAYES -advanced NIST7035-NIST7086.config -n 9 -freebayes /work/rashidma/software/seqmule_by_admin/SeqMule-master/exe/freebayes/bin/ freebayes -vcfsorter /work/rashidma/software/seqmule_by_admin/SeqMule- master/bin/secondary/../../bin/secondary/vcfsorter -ref /work/rashidma/Databases/hg19/UCSC/ucsc.hg19.fa -samtools /work/rashidma/software/seqmule_by_admin/SeqMule- master/exe/samtools/samtools -tmpdir /tmp -vcf NIST7035_result/NIST7035_bwamem.merge.0_freebayes.multi-call.vcf -bed nexterarapidcapture_expandedexome_targetedregions.nooverlap.bed -bam NIST7035_result/NIST7035_bwamem.merge.bam -bam NIST7086_result/NIST7086_bwamem.merge.bam"dependency= 23,24message=freebayes.multi-call variant callingnCPUrequested=12status=finished

What should i do now? Thanks MR

On Mon, Aug 22, 2016 at 2:15 AM, Yunfei Guo notifications@github.com wrote:

Hi Mamoon,

Sorry, our servers are temporarily unavailable right now. We are still trying to fix them. Could you please try install freebayes yourself and run seqmule from the freebayes step?

Instructions:

go to seqmule directory

move exe/freebayes folder to somewhere outside seqmule folder

download freebayes by "git clone --recursive git:// github.com/ekg/freebayes.git exe/freebayes"

cd exe/freebayes

make

run seqmule from the step where freebayes is executed (look for the step number for freebayes variant calling in *.script)

Thanks.

On Sun, Aug 21, 2016 at 4:28 AM, rashidma notifications@github.com wrote:

Hi Yunfei, Did you test the Freebayes? In my seqmule run it failed to call SNPs for multiple samples (as we discussed in the last email). Thanks Mamoon

On Fri, Aug 19, 2016 at 7:53 PM, Yunfei Guo notifications@github.com

wrote:

This should not affect the results, however, it may be the reason why you see some vcf files more than once in the runtime information.

On Fri, Aug 19, 2016 at 7:56 AM, rashidma notifications@github.com wrote:

Yes I did run the analysis multiple times with "seqmule run *.script" in the same directory.

The analysis takes a lot of time so seqmule run saves a lot of time.

Does it affect the result after rerunning seqmule?

Thanks MR

On 19 Aug 2016 17:43, "Yunfei Guo" notifications@github.com wrote:

I mean for each analysis, did you run it twice (probably with 'seqmule run' after 'seqmule pipeline')?

On Thu, Aug 18, 2016 at 7:13 PM, rashidma < notifications@github.com> wrote:

I re-run the analysis in a completely new directory. So both runs should not influence each other.

Thanks MR

On 18 Aug 2016 18:41, "Yunfei Guo" notifications@github.com wrote:

Is it because you tried to rerun the analysis?

On Aug 18, 2016, at 8:06 AM, rashidma < notifications@github.com> wrote:

Thanks a lot for your reply. I started another run with bwa-gatk-samtools-varscan and I found that it is also incomplete.

The log file contains the *gatk-multicall-extract.vcf twice for second sample. For first sample it is ok.

I could not understand the behavior. Lets first troubleshoot the first run that we are discussing.

regards MR

On Thu, Aug 18, 2016 at 5:43 PM, Yunfei Guo < notifications@github.com> wrote:

Thanks, Mamoon. Let me test the latest freebayes and get back to you later.

On Wed, Aug 17, 2016 at 10:05 PM, rashidma < notifications@github.com

wrote:

Hi I checked the VCF files for number of samples ---

VCFs number of samples NIST7035_bwamem.merge.0_freebayes.multi-call.extract. vcf 1 NIST7035_bwamem.merge.0_samtools.multi-call.extract. vcf 2 NIST7035bwamem.merge.realn.0 gatklite.multi-call.extract. vcf 2

NIST7086_bwamem.merge.0_freebayes.multi-call.extract. vcf 1 NIST7086_bwamem.merge.0_samtools.multi-call.extract. vcf 2 NIST7086bwamem.merge.realn.0 gatklite.multi-call.extract. vcf 2

I noticed frrebayes processed only one sample. Is this a known bug in freebayes??

I am trying to understand the system well so that I can use seqmule on my large number of samples. Best regards Mamoon

On Tue, Aug 16, 2016 at 5:46 PM, Yunfei Guo < notifications@github.com> wrote:

Hi MR,

I noticed that there was an error "ERROR: not all VCFs have the same number of samples!", could you check how many samples are there in each *multi-call.extract.vcf file? I don't know why there are two gatklite VCFs, though.

command to check # of samples:

for i in *multi-call.extract.vcf; do echo $i; perl -ne 'if (/^#CHROM/){print ((scalar (split /\t/,$_))-9),"\n"}' $i ; done

On Tue, Aug 16, 2016 at 4:33 AM, rashidma < notifications@github.com> wrote:

Hi, I just noticed in the _.log file of seqmule run -- the VCF " NIST7086result/NIST7086 bwamem.merge.realn.0gatklite. multi-call.extract.vcf" has been mentioned twice. please see below colored in red. Is this creating proble?? Per sample there should be 4 VCF files (3 different aligner variant caller combinations and one consensus) BUT in case of NIST7086 why this is 5 VCF. *

_2 sample(s) in this analysis: NIST7035 NIST7086All input is merged on a per-sample basis (after alignment if FASTQ).Multi-sample variant calling ENABLED.Input is exome (or captured) sequencing data.4 variant caller(s) used: GATKLite_UnifiedGenotyper SAMtools FreeBayes Consensus1 aligner(s) used: bwamemFile used for caculating coverage statistics and extracting variants: nexterarapidcapture_expandedexome_targetedregions. nooverlap.bedReadgroup : READGROUP_NIST7035,READGROUP_NIST7086Library : LIBRARYSequencing platform: ILLUMINAReference genome build is hg19dbsnp138 is used for variant calling and recalibration (in GATK VQSR).Java memory usage is limited to 1750mjava executable path: javaMax number of processes: 12NOTICE: /tmp will be used for storing temporary files######################### ########################## FINAL OUTPUT for NIST7035:##################### ############################## NOTICE: Both consensus results and individual results are listed.WARNING: consensus results may not be available because some variant callers or aligners may fail to generate output.NIST7035_result/NIST7035.extract_consensus. vcfNIST7035result/NIST7035 bwamem.merge.realn.0_gatklite. multi-call.extract.vcfNIST7035result/NIST7035 bwamem.merge.0samtools.multi- call.extract.vcfNIST7035 result/NIST7035_bwamem.merge. 0_freebayes.multi-call. extract.vcf################### ##############################

FINAL

OUTPUT for NIST7086:##################### ############################## NOTICE: Both consensus results and individual results are listed.WARNING: consensus results may not be available because some variant callers or aligners may fail to generate output._

_NIST7086_result/NIST7086. extractconsensus.vcfNIST7086 result/NIST7086_bwamem.merge. realn.0_gatklite.multi-call. extract.vcfNIST7086_result/ NIST7086bwamem.merge.realn.0 gatklite.multi-call.extract. vcfNIST7086result/NIST7086 bwamem.merge.0samtools.multi- call.extract.vcfNIST7086 result/NIST7086_bwamem.merge. 0freebayes.multi-call. extract.vcf

Thanks

On Tue, Aug 16, 2016 at 8:07 AM, Mamoon Rashid < rashidmamoon@gmail.com

wrote:

Hi Yunfei, I sent you the big nohup file (~8MB) and it failed to reach you. Now I am sending you the last 1000 lines of the nohup file. I can see there are errors at step 42 and 43. I thank you so much for your time. MR

On Mon, Aug 15, 2016 at 4:05 PM, Yunfei Guo < notifications@github.com> wrote:

Hi Rashid,

Could you please provide the contents of nohup.out file? I need to know where it stopped.

Thanks.

Best, Yunfei

On Mon, Aug 15, 2016 at 12:47 AM, rashidma < notifications@github.com> wrote:

Thanks a lot Yunfei, I successfully ran the test run. Now, I was running a real sample from NIST. Out of 51 steps 48 steps ran well. I am stuck in the last few steps. The seqmule was stopped many times due to system shutdown and couple of times I killed the process. But every time I restarted it with--

nohup seqmule run NIST7035-NIST7086.script &

Please let me know any troubleshooting. I will be thankful to you. Regards MR

On Thu, Aug 11, 2016 at 1:02 AM, Yunfei Guo < notifications@github.com

wrote:

Hi Mamoon,

Please use inst/hg19_exome.legacy.bed after updating SeqMule with seqmule update --git. Thank you!

Best, Yunfei

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rashidma commented 7 years ago

Hi Yunfei, It is running now.

I would like to know --

which /misc/predefined_config file is used in default mode by seqmule?
Is it possible to pass singletons reads in seqmule? I have a considerable amount of reads which are unpaired.

Thanks you so much for your time. Mamoon

On Mon, Aug 22, 2016 at 7:53 PM, Yunfei Guo notifications@github.com wrote:

33 is the step number.

seqmule run -n 33 yourseqmule.script

On Mon, Aug 22, 2016 at 6:27 AM, rashidma notifications@github.com wrote:

I installed freebayes from github as you mentioned. Please let me know how to run seqmule from the step where freebayes is executed? I looked at *.script file and found ---

_[33]JOBID=0PID=14673command=/work/rashidma/software/ seqmule_by_admin/SeqMule-master/bin/secondary/../../bin/secondary/worker /work/rashidma/Exome_data/GIAB_NIST_exome_data/ MyAnalysis/04_seqmule_qsub- ucschg19/seqmule.08162016.23639.logs 33 "/work/rashidma/software/seqmule_by_admin/SeqMule-master/bin/secondary/ runFREEBAYES -advanced NIST7035-NIST7086.config -n 9 -freebayes /work/rashidma/software/seqmule_by_admin/SeqMule- master/exe/freebayes/bin/ freebayes -vcfsorter /work/rashidma/software/seqmule_by_admin/SeqMule- master/bin/secondary/../../bin/secondary/vcfsorter -ref /work/rashidma/Databases/hg19/UCSC/ucsc.hg19.fa -samtools /work/rashidma/software/seqmule_by_admin/SeqMule- master/exe/samtools/samtools -tmpdir /tmp -vcf NIST7035_result/NIST7035_bwamem.merge.0_freebayes.multi-call.vcf -bed nexterarapidcapture_expandedexome_targetedregions.nooverlap.bed -bam NIST7035_result/NIST7035_bwamem.merge.bam -bam NIST7086_result/NIST7086_bwamem.merge.bam"dependency= 23,24message=freebayes.multi-call variant callingnCPUrequested=12status=finished

What should i do now? Thanks MR

On Mon, Aug 22, 2016 at 2:15 AM, Yunfei Guo notifications@github.com wrote:

Hi Mamoon,

Sorry, our servers are temporarily unavailable right now. We are still trying to fix them. Could you please try install freebayes yourself and run seqmule from the freebayes step?

Instructions:

go to seqmule directory

move exe/freebayes folder to somewhere outside seqmule folder

download freebayes by "git clone --recursive git:// github.com/ekg/freebayes.git exe/freebayes"

cd exe/freebayes

make

run seqmule from the step where freebayes is executed (look for the step number for freebayes variant calling in *.script)

Thanks.

On Sun, Aug 21, 2016 at 4:28 AM, rashidma notifications@github.com wrote:

Hi Yunfei, Did you test the Freebayes? In my seqmule run it failed to call SNPs for multiple samples (as we discussed in the last email). Thanks Mamoon

On Fri, Aug 19, 2016 at 7:53 PM, Yunfei Guo < notifications@github.com>

wrote:

This should not affect the results, however, it may be the reason why you see some vcf files more than once in the runtime information.

On Fri, Aug 19, 2016 at 7:56 AM, rashidma < notifications@github.com> wrote:

Yes I did run the analysis multiple times with "seqmule run *.script" in the same directory.

The analysis takes a lot of time so seqmule run saves a lot of time.

Does it affect the result after rerunning seqmule?

Thanks MR

On 19 Aug 2016 17:43, "Yunfei Guo" notifications@github.com wrote:

I mean for each analysis, did you run it twice (probably with 'seqmule run' after 'seqmule pipeline')?

On Thu, Aug 18, 2016 at 7:13 PM, rashidma < notifications@github.com> wrote:

I re-run the analysis in a completely new directory. So both runs should not influence each other.

Thanks MR

On 18 Aug 2016 18:41, "Yunfei Guo" <notifications@github.com

wrote:

Is it because you tried to rerun the analysis?

On Aug 18, 2016, at 8:06 AM, rashidma < notifications@github.com> wrote:

Thanks a lot for your reply. I started another run with bwa-gatk-samtools-varscan and I found that it is also incomplete.

The log file contains the *gatk-multicall-extract.vcf twice for second sample. For first sample it is ok.

I could not understand the behavior. Lets first troubleshoot the first run that we are discussing.

regards MR

On Thu, Aug 18, 2016 at 5:43 PM, Yunfei Guo < notifications@github.com> wrote:

Thanks, Mamoon. Let me test the latest freebayes and get back to you later.

On Wed, Aug 17, 2016 at 10:05 PM, rashidma < notifications@github.com

wrote:

Hi I checked the VCF files for number of samples ---

VCFs number of samples NIST7035bwamem.merge.0 freebayes.multi-call.extract. vcf 1 NIST7035_bwamem.merge.0_samtools.multi-call.extract. vcf 2 NIST7035bwamem.merge.realn.0 gatklite.multi-call.extract. vcf 2

NIST7086bwamem.merge.0 freebayes.multi-call.extract. vcf 1 NIST7086_bwamem.merge.0_samtools.multi-call.extract. vcf 2 NIST7086bwamem.merge.realn.0 gatklite.multi-call.extract. vcf 2

I noticed frrebayes processed only one sample. Is this a known bug in freebayes??

I am trying to understand the system well so that I can use seqmule on my large number of samples. Best regards Mamoon

On Tue, Aug 16, 2016 at 5:46 PM, Yunfei Guo < notifications@github.com> wrote:

Hi MR,

I noticed that there was an error "ERROR: not all VCFs have the same number of samples!", could you check how many samples are there in each *multi-call.extract.vcf file? I don't know why there are two gatklite VCFs, though.

command to check # of samples:

for i in *multi-call.extract.vcf; do echo $i; perl -ne 'if (/^#CHROM/){print ((scalar (split /\t/,$_))-9),"\n"}' $i ; done

On Tue, Aug 16, 2016 at 4:33 AM, rashidma < notifications@github.com> wrote:

Hi,

I just noticed in the *.log file of seqmule run

the VCF " _NIST7086result/NIST7086 bwamem.merge.realn.0gatklite. multi-call.extract.vcf" has been mentioned twice. please see below colored in red. Is this creating proble?? Per sample there should be 4 VCF files (3 different aligner variant caller combinations and one consensus) BUT in case of NIST7086 why this is 5 VCF.

_2 sample(s) in this analysis: NIST7035 NIST7086All input is merged on a per-sample basis (after alignment if FASTQ).Multi-sample variant calling ENABLED.Input is exome (or captured) sequencing data.4 variant caller(s) used: GATKLiteUnifiedGenotyper SAMtools FreeBayes Consensus1 aligner(s) used: bwamemFile used for caculating coverage statistics and extracting variants: nexterarapidcapture expandedexome_targetedregions. nooverlap.bedReadgroup : READGROUP_NIST7035,READGROUP_NIST7086Library : LIBRARYSequencing platform: ILLUMINAReference genome build is hg19dbsnp138 is used for variant calling and recalibration (in GATK VQSR).Java memory usage is limited to 1750mjava executable path: javaMax number of processes: 12NOTICE: /tmp will be used for storing temporary files######################### ########################## FINAL OUTPUT for NIST7035:##################### ############################## NOTICE: Both consensus results and individual results are listed.WARNING: consensus results may not be available because some variant callers or aligners may fail to generate output.NIST7035_result/ NIST7035.extract_consensus. vcfNIST7035result/NIST7035 bwamem.merge.realn.0_gatklite. multi-call.extract.vcfNIST7035result/NIST7035 bwamem.merge.0samtools.multi- call.extract.vcfNIST7035 result/NIST7035_bwamem.merge. 0_freebayes.multi-call. extract.vcf################### ##############################

FINAL

OUTPUT for NIST7086:##################### ############################## NOTICE: Both consensus results and individual results are listed.WARNING: consensus results may not be available because some variant callers or aligners may fail to generate output._

_NIST7086_result/NIST7086. extractconsensus.vcfNIST7086 result/NIST7086_bwamem.merge. realn.0_gatklite.multi-call. extract.vcfNIST7086_result/ NIST7086bwamem.merge.realn.0 gatklite.multi-call.extract. vcfNIST7086result/NIST7086 bwamem.merge.0samtools.multi- call.extract.vcfNIST7086 result/NIST7086_bwamem.merge. 0freebayes.multi-call. extract.vcf

Thanks

On Tue, Aug 16, 2016 at 8:07 AM, Mamoon Rashid < rashidmamoon@gmail.com

wrote:

Hi Yunfei, I sent you the big nohup file (~8MB) and it failed to reach you. Now I am sending you the last 1000 lines of the nohup file. I can see there are errors at step 42 and 43. I thank you so much for your time. MR

On Mon, Aug 15, 2016 at 4:05 PM, Yunfei Guo < notifications@github.com> wrote:

Hi Rashid,

Could you please provide the contents of nohup.out file? I need to know where it stopped.

Thanks.

Best, Yunfei

On Mon, Aug 15, 2016 at 12:47 AM, rashidma < notifications@github.com> wrote:

Thanks a lot Yunfei, I successfully ran the test run. Now, I was running a real sample from NIST. Out of 51 steps 48 steps ran well. I am stuck in the last few steps. The seqmule was stopped many times due to system shutdown and couple of times I killed the process. But every time I restarted it with--

nohup seqmule run NIST7035-NIST7086.script &

Please let me know any troubleshooting. I will be thankful to you. Regards MR

On Thu, Aug 11, 2016 at 1:02 AM, Yunfei Guo < notifications@github.com

wrote:

Hi Mamoon,

Please use inst/hg19_exome.legacy.bed after updating SeqMule with seqmule update --git. Thank you!

Best, Yunfei

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yunfeiguo commented 7 years ago

Hi Mamoon,

Please refer to http://seqmule.openbioinformatics.org/en/latest/Miscellaneous/FAQ/#which-file-is-used-as-default-argument-for-advanced
Yes. Instead of specifying -a and -b at the same time, just specify -a in seqmule pipeline. Note, however, seqmule does not support mixing single-end data with pair-end data.

Thanks.

On Mon, Aug 22, 2016 at 10:23 PM, rashidma notifications@github.com wrote:

Hi Yunfei, It is running now.

I would like to know --

which /misc/predefined_config file is used in default mode by seqmule?

Is it possible to pass singletons reads in seqmule? I have a considerable amount of reads which are unpaired.

Thanks you so much for your time. Mamoon

On Mon, Aug 22, 2016 at 7:53 PM, Yunfei Guo notifications@github.com wrote:

33 is the step number.

seqmule run -n 33 yourseqmule.script

On Mon, Aug 22, 2016 at 6:27 AM, rashidma notifications@github.com wrote:

I installed freebayes from github as you mentioned. Please let me know how to run seqmule from the step where freebayes is executed? I looked at *.script file and found ---

_[33]JOBID=0PID=14673command=/work/rashidma/software/ seqmule_by_admin/SeqMule-master/bin/secondary/../../ bin/secondary/worker /work/rashidma/Exome_data/GIAB_NIST_exome_data/ MyAnalysis/04_seqmule_qsub- ucschg19/seqmule.08162016.23639.logs 33 "/work/rashidma/software/seqmule_by_admin/SeqMule- master/bin/secondary/ runFREEBAYES -advanced NIST7035-NIST7086.config -n 9 -freebayes /work/rashidma/software/seqmule_by_admin/SeqMule- master/exe/freebayes/bin/ freebayes -vcfsorter /work/rashidma/software/seqmule_by_admin/SeqMule- master/bin/secondary/../../bin/secondary/vcfsorter -ref /work/rashidma/Databases/hg19/UCSC/ucsc.hg19.fa -samtools /work/rashidma/software/seqmule_by_admin/SeqMule- master/exe/samtools/samtools -tmpdir /tmp -vcf NIST7035_result/NIST7035_bwamem.merge.0_freebayes.multi-call.vcf -bed nexterarapidcapture_expandedexome_targetedregions.nooverlap.bed -bam NIST7035_result/NIST7035_bwamem.merge.bam -bam NIST7086_result/NIST7086_bwamem.merge.bam"dependency= 23,24message=freebayes.multi-call variant callingnCPUrequested=12status=finished

What should i do now? Thanks MR

On Mon, Aug 22, 2016 at 2:15 AM, Yunfei Guo notifications@github.com wrote:

Hi Mamoon,

Sorry, our servers are temporarily unavailable right now. We are still trying to fix them. Could you please try install freebayes yourself and run seqmule from the freebayes step?

Instructions:

go to seqmule directory

move exe/freebayes folder to somewhere outside seqmule folder

download freebayes by "git clone --recursive git:// github.com/ekg/freebayes.git exe/freebayes"

cd exe/freebayes

make

run seqmule from the step where freebayes is executed (look for the step number for freebayes variant calling in *.script)

Thanks.

On Sun, Aug 21, 2016 at 4:28 AM, rashidma notifications@github.com wrote:

Hi Yunfei, Did you test the Freebayes? In my seqmule run it failed to call SNPs for multiple samples (as we discussed in the last email). Thanks Mamoon

On Fri, Aug 19, 2016 at 7:53 PM, Yunfei Guo < notifications@github.com>

wrote:

This should not affect the results, however, it may be the reason why you see some vcf files more than once in the runtime information.

On Fri, Aug 19, 2016 at 7:56 AM, rashidma < notifications@github.com> wrote:

Yes I did run the analysis multiple times with "seqmule run *.script" in the same directory.

The analysis takes a lot of time so seqmule run saves a lot of time.

Does it affect the result after rerunning seqmule?

Thanks MR

On 19 Aug 2016 17:43, "Yunfei Guo" notifications@github.com wrote:

I mean for each analysis, did you run it twice (probably with 'seqmule run' after 'seqmule pipeline')?

On Thu, Aug 18, 2016 at 7:13 PM, rashidma < notifications@github.com> wrote:

I re-run the analysis in a completely new directory. So both runs should not influence each other.

Thanks MR

On 18 Aug 2016 18:41, "Yunfei Guo" < notifications@github.com

wrote:

Is it because you tried to rerun the analysis?

On Aug 18, 2016, at 8:06 AM, rashidma < notifications@github.com> wrote:

Thanks a lot for your reply. I started another run with bwa-gatk-samtools-varscan and I found that it is also incomplete.

The log file contains the *gatk-multicall-extract.vcf twice for second sample. For first sample it is ok.

I could not understand the behavior. Lets first troubleshoot the first run that we are discussing.

regards MR

On Thu, Aug 18, 2016 at 5:43 PM, Yunfei Guo < notifications@github.com> wrote:

Thanks, Mamoon. Let me test the latest freebayes and get back to you later.

On Wed, Aug 17, 2016 at 10:05 PM, rashidma < notifications@github.com

wrote:

Hi I checked the VCF files for number of samples ---

VCFs number of samples NIST7035bwamem.merge.0 freebayes.multi-call.extract. vcf 1 NIST7035bwamem.merge.0 samtools.multi-call.extract. vcf 2 NIST7035bwamem.merge.realn.0 gatklite.multi-call.extract. vcf 2

NIST7086bwamem.merge.0 freebayes.multi-call.extract. vcf 1 NIST7086bwamem.merge.0 samtools.multi-call.extract. vcf 2 NIST7086bwamem.merge.realn.0 gatklite.multi-call.extract. vcf 2

I noticed frrebayes processed only one sample. Is this a known bug in freebayes??

I am trying to understand the system well so that I can use seqmule on my large number of samples. Best regards Mamoon

On Tue, Aug 16, 2016 at 5:46 PM, Yunfei Guo < notifications@github.com> wrote:

Hi MR,

I noticed that there was an error "ERROR: not all VCFs have the same number of samples!", could you check how many samples are there in each *multi-call.extract.vcf file? I don't know why there are two gatklite VCFs, though.

command to check # of samples:

for i in *multi-call.extract.vcf; do echo $i; perl -ne 'if (/^#CHROM/){print ((scalar (split /\t/,$_))-9),"\n"}' $i ; done

On Tue, Aug 16, 2016 at 4:33 AM, rashidma < notifications@github.com> wrote:

Hi,

I just noticed in the *.log file of seqmule run

the VCF " _NIST7086result/NIST7086 bwamem.merge.realn.0gatklite. multi-call.extract.vcf" has been mentioned twice. please see below colored in red. Is this creating proble?? Per sample there should be 4 VCF files (3 different aligner variant caller combinations and one consensus) BUT in case of NIST7086 why this is 5 VCF.

_2 sample(s) in this analysis: NIST7035 NIST7086All input is merged on a per-sample basis (after alignment if FASTQ).Multi-sample variant calling ENABLED.Input is exome (or captured) sequencing data.4 variant caller(s) used: GATKLiteUnifiedGenotyper SAMtools FreeBayes Consensus1 aligner(s) used: bwamemFile used for caculating coverage statistics and extracting variants: nexterarapidcapture expandedexome_targetedregions. nooverlap.bedReadgroup : READGROUP_NIST7035,READGROUP_NIST7086Library : LIBRARYSequencing platform: ILLUMINAReference genome build is hg19dbsnp138 is used for variant calling and recalibration (in GATK VQSR).Java memory usage is limited to 1750mjava executable path: javaMax number of processes: 12NOTICE: /tmp will be used for storing temporary files######################### ########################## FINAL OUTPUT for NIST7035:##################### ############################## NOTICE: Both consensus results and individual results are listed.WARNING: consensus results may not be available because some variant callers or aligners may fail to generate output.NIST7035_result/ NIST7035.extract_consensus. vcfNIST7035result/NIST7035 bwamem.merge.realn.0_gatklite. multi-call.extract. vcfNIST7035result/NIST7035 bwamem.merge.0samtools.multi- call.extract.vcfNIST7035 result/NIST7035_bwamem.merge. 0_freebayes.multi-call. extract.vcf################### ##############################

FINAL

OUTPUT for NIST7086:##################### ############################## NOTICE: Both consensus results and individual results are listed.WARNING: consensus results may not be available because some variant callers or aligners may fail to generate output._

_NIST7086_result/NIST7086. extractconsensus.vcfNIST7086 result/NIST7086_bwamem.merge. realn.0_gatklite.multi-call. extract.vcfNIST7086_result/ NIST7086bwamem.merge.realn.0 gatklite.multi-call.extract. vcfNIST7086result/NIST7086 bwamem.merge.0samtools.multi- call.extract.vcfNIST7086 result/NIST7086_bwamem.merge. 0freebayes.multi-call. extract.vcf

Thanks

On Tue, Aug 16, 2016 at 8:07 AM, Mamoon Rashid < rashidmamoon@gmail.com

wrote:

Hi Yunfei, I sent you the big nohup file (~8MB) and it failed to reach you. Now I am sending you the last 1000 lines of the nohup file. I can see there are errors at step 42 and 43. I thank you so much for your time. MR

On Mon, Aug 15, 2016 at 4:05 PM, Yunfei Guo < notifications@github.com> wrote:

Hi Rashid,

Could you please provide the contents of nohup.out file? I need to know where it stopped.

Thanks.

Best, Yunfei

On Mon, Aug 15, 2016 at 12:47 AM, rashidma < notifications@github.com> wrote:

Thanks a lot Yunfei, I successfully ran the test run. Now, I was running a real sample from NIST. Out of 51 steps 48 steps ran well. I am stuck in the last few steps. The seqmule was stopped many times due to system shutdown and couple of times I killed the process. But every time I restarted it with--

nohup seqmule run NIST7035-NIST7086.script &

Please let me know any troubleshooting. I will be thankful to you. Regards MR

On Thu, Aug 11, 2016 at 1:02 AM, Yunfei Guo < notifications@github.com

wrote:

Hi Mamoon,

Please use inst/hg19_exome.legacy.bed after updating SeqMule with seqmule update --git. Thank you!

Best, Yunfei

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rashidma commented 7 years ago

Thanks a ton Even after installing Freebayes from github, my seqmule run could not complete. Again freebayes could not call multiple-sample variant calling. Anyway, I am running it with varscan, samtools, and gatk.

Does seqmule support parallel processing using MPI?

Thanks Mamoon

On Tue, Aug 23, 2016 at 8:42 PM, Yunfei Guo notifications@github.com wrote:

Hi Mamoon,

Please refer to http://seqmule.openbioinformatics.org/en/latest/Miscellaneous/FAQ/# which-file-is-used-as-default-argument-for-advanced

Yes. Instead of specifying -a and -b at the same time, just specify -a in seqmule pipeline. Note, however, seqmule does not support mixing single-end data with pair-end data.

Thanks.

On Mon, Aug 22, 2016 at 10:23 PM, rashidma notifications@github.com wrote:

Hi Yunfei, It is running now.

I would like to know --

which /misc/predefined_config file is used in default mode by seqmule?

Is it possible to pass singletons reads in seqmule? I have a considerable amount of reads which are unpaired.

Thanks you so much for your time. Mamoon

On Mon, Aug 22, 2016 at 7:53 PM, Yunfei Guo notifications@github.com wrote:

33 is the step number.

seqmule run -n 33 yourseqmule.script

On Mon, Aug 22, 2016 at 6:27 AM, rashidma notifications@github.com wrote:

I installed freebayes from github as you mentioned. Please let me know how to run seqmule from the step where freebayes is executed? I looked at *.script file and found ---

_[33]JOBID=0PID=14673command=/work/rashidma/software/ seqmule_by_admin/SeqMule-master/bin/secondary/../../ bin/secondary/worker /work/rashidma/Exome_data/GIAB_NIST_exome_data/ MyAnalysis/04_seqmule_qsub- ucschg19/seqmule.08162016.23639.logs 33 "/work/rashidma/software/seqmule_by_admin/SeqMule- master/bin/secondary/ runFREEBAYES -advanced NIST7035-NIST7086.config -n 9 -freebayes /work/rashidma/software/seqmule_by_admin/SeqMule- master/exe/freebayes/bin/ freebayes -vcfsorter /work/rashidma/software/seqmule_by_admin/SeqMule- master/bin/secondary/../../bin/secondary/vcfsorter -ref /work/rashidma/Databases/hg19/UCSC/ucsc.hg19.fa -samtools /work/rashidma/software/seqmule_by_admin/SeqMule- master/exe/samtools/samtools -tmpdir /tmp -vcf NIST7035_result/NIST7035_bwamem.merge.0_freebayes.multi-call.vcf -bed nexterarapidcapture_expandedexome_targetedregions.nooverlap.bed -bam NIST7035_result/NIST7035_bwamem.merge.bam -bam NIST7086_result/NIST7086_bwamem.merge.bam"dependency= 23,24message=freebayes.multi-call variant callingnCPUrequested=12status=finished

What should i do now? Thanks MR

On Mon, Aug 22, 2016 at 2:15 AM, Yunfei Guo < notifications@github.com> wrote:

Hi Mamoon,

Sorry, our servers are temporarily unavailable right now. We are still trying to fix them. Could you please try install freebayes yourself and run seqmule from the freebayes step?

Instructions:

go to seqmule directory

move exe/freebayes folder to somewhere outside seqmule folder

download freebayes by "git clone --recursive git:// github.com/ekg/freebayes.git exe/freebayes"

cd exe/freebayes

make

run seqmule from the step where freebayes is executed (look for the step number for freebayes variant calling in *.script)

Thanks.

On Sun, Aug 21, 2016 at 4:28 AM, rashidma < notifications@github.com> wrote:

Hi Yunfei, Did you test the Freebayes? In my seqmule run it failed to call SNPs for multiple samples (as we discussed in the last email). Thanks Mamoon

On Fri, Aug 19, 2016 at 7:53 PM, Yunfei Guo < notifications@github.com>

wrote:

This should not affect the results, however, it may be the reason why you see some vcf files more than once in the runtime information.

On Fri, Aug 19, 2016 at 7:56 AM, rashidma < notifications@github.com> wrote:

Yes I did run the analysis multiple times with "seqmule run *.script" in the same directory.

The analysis takes a lot of time so seqmule run saves a lot of time.

Does it affect the result after rerunning seqmule?

Thanks MR

On 19 Aug 2016 17:43, "Yunfei Guo" <notifications@github.com

wrote:

I mean for each analysis, did you run it twice (probably with 'seqmule run' after 'seqmule pipeline')?

On Thu, Aug 18, 2016 at 7:13 PM, rashidma < notifications@github.com> wrote:

I re-run the analysis in a completely new directory. So both runs should not influence each other.

Thanks MR

On 18 Aug 2016 18:41, "Yunfei Guo" < notifications@github.com

wrote:

Is it because you tried to rerun the analysis?

On Aug 18, 2016, at 8:06 AM, rashidma < notifications@github.com> wrote:

Thanks a lot for your reply. I started another run with bwa-gatk-samtools-varscan and I found that it is also incomplete.

The log file contains the *gatk-multicall-extract.vcf twice for second sample. For first sample it is ok.

I could not understand the behavior. Lets first troubleshoot the first run that we are discussing.

regards MR

On Thu, Aug 18, 2016 at 5:43 PM, Yunfei Guo < notifications@github.com> wrote:

Thanks, Mamoon. Let me test the latest freebayes and get back to you later.

On Wed, Aug 17, 2016 at 10:05 PM, rashidma < notifications@github.com

wrote:

Hi I checked the VCF files for number of samples ---

VCFs number of samples NIST7035bwamem.merge.0 freebayes.multi-call.extract. vcf 1 NIST7035bwamem.merge.0 samtools.multi-call.extract. vcf 2 NIST7035bwamem.merge.realn.0 gatklite.multi-call.extract. vcf 2

NIST7086bwamem.merge.0 freebayes.multi-call.extract. vcf 1 NIST7086bwamem.merge.0 samtools.multi-call.extract. vcf 2 NIST7086bwamem.merge.realn.0 gatklite.multi-call.extract. vcf 2

I noticed frrebayes processed only one sample. Is this a known bug in freebayes??

I am trying to understand the system well so that I can use seqmule on my large number of samples. Best regards Mamoon

On Tue, Aug 16, 2016 at 5:46 PM, Yunfei Guo < notifications@github.com> wrote:

Hi MR,

I noticed that there was an error "ERROR: not all VCFs have the same number of samples!", could you check how many samples are there in each *multi-call.extract.vcf file? I don't know why there are two gatklite VCFs, though.

command to check # of samples:

for i in *multi-call.extract.vcf; do echo $i; perl -ne 'if (/^#CHROM/){print ((scalar (split /\t/,$_))-9),"\n"}' $i ; done

On Tue, Aug 16, 2016 at 4:33 AM, rashidma < notifications@github.com> wrote:

Hi, I just noticed in the *.log file of seqmule

run

the VCF " _NIST7086result/NIST7086 bwamem.merge.realn.0gatklite. multi-call.extract.vcf" has been mentioned twice. please see below colored in red. Is this creating proble?? Per sample there should be 4 VCF files (3 different aligner variant caller combinations and one consensus) BUT in case of NIST7086 why this is 5 VCF.

_2 sample(s) in this analysis: NIST7035 NIST7086All input is merged on a per-sample basis (after alignment if FASTQ).Multi-sample variant calling ENABLED.Input is exome (or captured) sequencing data.4 variant caller(s) used: GATKLiteUnifiedGenotyper SAMtools FreeBayes Consensus1 aligner(s) used: bwamemFile used for caculating coverage statistics and extracting variants: nexterarapidcapture expandedexome_targetedregions. nooverlap.bedReadgroup : READGROUP_NIST7035,READGROUP_NIST7086Library : LIBRARYSequencing platform: ILLUMINAReference genome build is hg19dbsnp138 is used for variant calling and recalibration (in GATK VQSR).Java memory usage is limited to 1750mjava executable path: javaMax number of processes: 12NOTICE: /tmp will be used for storing temporary files######################### ########################## FINAL OUTPUT for NIST7035:##################### ############################## NOTICE: Both consensus results and individual results are listed.WARNING: consensus results may not be available because some variant callers or aligners may fail to generate output.NIST7035_result/ NIST7035.extract_consensus. vcfNIST7035result/NIST7035 bwamem.merge.realn.0_gatklite. multi-call.extract. vcfNIST7035result/NIST7035 bwamem.merge.0samtools.multi- call.extract.vcfNIST7035 result/NIST7035_bwamem.merge. 0_freebayes.multi-call. extract.vcf################### ##############################

FINAL

OUTPUT for NIST7086:##################### ############################## NOTICE: Both consensus results and individual results are listed.WARNING: consensus results may not be available because some variant callers or aligners may fail to generate output._

_NIST7086_result/NIST7086. extractconsensus.vcfNIST7086 result/NIST7086_bwamem.merge. realn.0_gatklite.multi-call. extract.vcfNIST7086_result/ NIST7086bwamem.merge.realn.0 gatklite.multi-call.extract. vcfNIST7086result/NIST7086 bwamem.merge.0samtools.multi- call.extract.vcfNIST7086 result/NIST7086_bwamem.merge. 0freebayes.multi-call. extract.vcf

Thanks

On Tue, Aug 16, 2016 at 8:07 AM, Mamoon Rashid < rashidmamoon@gmail.com

wrote:

Hi Yunfei, I sent you the big nohup file (~8MB) and it failed to reach you. Now I am sending you the last 1000 lines of the nohup file. I can see there are errors at step 42 and 43. I thank you so much for your time. MR

On Mon, Aug 15, 2016 at 4:05 PM, Yunfei Guo < notifications@github.com> wrote:

Hi Rashid,

Could you please provide the contents of nohup.out file? I need to know where it stopped.

Thanks.

Best, Yunfei

On Mon, Aug 15, 2016 at 12:47 AM, rashidma < notifications@github.com> wrote:

Thanks a lot Yunfei, I successfully ran the test run. Now, I was running a real sample from NIST. Out of 51 steps 48 steps ran well. I am stuck in the last few steps. The seqmule was stopped many times due to system shutdown and couple of times I killed the process. But every time I restarted it with--

nohup seqmule run NIST7035-NIST7086.script &

Please let me know any troubleshooting. I will be thankful to you. Regards MR

On Thu, Aug 11, 2016 at 1:02 AM, Yunfei Guo < notifications@github.com

wrote:

Hi Mamoon,

Please use inst/hg19_exome.legacy.bed after updating SeqMule with seqmule update --git. Thank you!

Best, Yunfei

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yunfeiguo commented 7 years ago

Hi Mamoon,

That is weird. If you could provide a small portion of your bam file so that I can reproduce the error, I can try to figure out what is going on.

SeqMule does not support MPI.

Best, Yunfei

On Tue, Aug 23, 2016 at 12:30 PM, rashidma notifications@github.com wrote:

Thanks a ton Even after installing Freebayes from github, my seqmule run could not complete. Again freebayes could not call multiple-sample variant calling. Anyway, I am running it with varscan, samtools, and gatk.

Does seqmule support parallel processing using MPI?

Thanks Mamoon

On Tue, Aug 23, 2016 at 8:42 PM, Yunfei Guo notifications@github.com wrote:

Hi Mamoon,

Please refer to http://seqmule.openbioinformatics.org/en/latest/Miscellaneous/FAQ/# which-file-is-used-as-default-argument-for-advanced

Yes. Instead of specifying -a and -b at the same time, just specify -a in seqmule pipeline. Note, however, seqmule does not support mixing single-end data with pair-end data.

Thanks.

On Mon, Aug 22, 2016 at 10:23 PM, rashidma notifications@github.com wrote:

Hi Yunfei, It is running now.

I would like to know --

which /misc/predefined_config file is used in default mode by seqmule?

Is it possible to pass singletons reads in seqmule? I have a considerable amount of reads which are unpaired.

Thanks you so much for your time. Mamoon

On Mon, Aug 22, 2016 at 7:53 PM, Yunfei Guo notifications@github.com wrote:

33 is the step number.

seqmule run -n 33 yourseqmule.script

On Mon, Aug 22, 2016 at 6:27 AM, rashidma notifications@github.com wrote:

I installed freebayes from github as you mentioned. Please let me know how to run seqmule from the step where freebayes is executed? I looked at *.script file and found ---

_[33]JOBID=0PID=14673command=/work/rashidma/software/ seqmule_by_admin/SeqMule-master/bin/secondary/../../ bin/secondary/worker /work/rashidma/Exome_data/GIAB_NIST_exome_data/ MyAnalysis/04_seqmule_qsub- ucschg19/seqmule.08162016.23639.logs 33 "/work/rashidma/software/seqmule_by_admin/SeqMule- master/bin/secondary/ runFREEBAYES -advanced NIST7035-NIST7086.config -n 9 -freebayes /work/rashidma/software/seqmule_by_admin/SeqMule- master/exe/freebayes/bin/ freebayes -vcfsorter /work/rashidma/software/seqmule_by_admin/SeqMule- master/bin/secondary/../../bin/secondary/vcfsorter -ref /work/rashidma/Databases/hg19/UCSC/ucsc.hg19.fa -samtools /work/rashidma/software/seqmule_by_admin/SeqMule- master/exe/samtools/samtools -tmpdir /tmp -vcf NIST7035_result/NIST7035_bwamem.merge.0_freebayes.multi-call.vcf -bed nexterarapidcapture_expandedexome_targetedregions.nooverlap.bed -bam NIST7035_result/NIST7035_bwamem.merge.bam -bam NIST7086_result/NIST7086_bwamem.merge.bam"dependency= 23,24message=freebayes.multi-call variant callingnCPUrequested=12status=finished

What should i do now? Thanks MR

On Mon, Aug 22, 2016 at 2:15 AM, Yunfei Guo < notifications@github.com> wrote:

Hi Mamoon,

Sorry, our servers are temporarily unavailable right now. We are still trying to fix them. Could you please try install freebayes yourself and run seqmule from the freebayes step?

Instructions:

go to seqmule directory

move exe/freebayes folder to somewhere outside seqmule folder

download freebayes by "git clone --recursive git:// github.com/ekg/freebayes.git exe/freebayes"

cd exe/freebayes

make

run seqmule from the step where freebayes is executed (look for the step number for freebayes variant calling in *.script)

Thanks.

On Sun, Aug 21, 2016 at 4:28 AM, rashidma < notifications@github.com> wrote:

Hi Yunfei, Did you test the Freebayes? In my seqmule run it failed to call SNPs for multiple samples (as we discussed in the last email). Thanks Mamoon

On Fri, Aug 19, 2016 at 7:53 PM, Yunfei Guo < notifications@github.com>

wrote:

This should not affect the results, however, it may be the reason why you see some vcf files more than once in the runtime information.

On Fri, Aug 19, 2016 at 7:56 AM, rashidma < notifications@github.com> wrote:

Yes I did run the analysis multiple times with "seqmule run *.script" in the same directory.

The analysis takes a lot of time so seqmule run saves a lot of time.

Does it affect the result after rerunning seqmule?

Thanks MR

On 19 Aug 2016 17:43, "Yunfei Guo" < notifications@github.com

wrote:

I mean for each analysis, did you run it twice (probably with 'seqmule run' after 'seqmule pipeline')?

On Thu, Aug 18, 2016 at 7:13 PM, rashidma < notifications@github.com> wrote:

I re-run the analysis in a completely new directory. So both runs should not influence each other.

Thanks MR

On 18 Aug 2016 18:41, "Yunfei Guo" < notifications@github.com

wrote:

Is it because you tried to rerun the analysis?

On Aug 18, 2016, at 8:06 AM, rashidma < notifications@github.com> wrote:

Thanks a lot for your reply. I started another run with bwa-gatk-samtools-varscan and I found that it is also incomplete.

The log file contains the *gatk-multicall-extract.vcf twice for second sample. For first sample it is ok.

I could not understand the behavior. Lets first troubleshoot the first run that we are discussing.

regards MR

On Thu, Aug 18, 2016 at 5:43 PM, Yunfei Guo < notifications@github.com> wrote:

Thanks, Mamoon. Let me test the latest freebayes and get back to you later.

On Wed, Aug 17, 2016 at 10:05 PM, rashidma < notifications@github.com

wrote:

Hi

I checked the VCF files for number of samples

VCFs number of samples NIST7035bwamem.merge.0 freebayes.multi-call.extract. vcf 1 NIST7035bwamem.merge.0 samtools.multi-call.extract. vcf 2 NIST7035bwamem.merge.realn.0 gatklite.multi-call.extract. vcf 2

NIST7086bwamem.merge.0 freebayes.multi-call.extract. vcf 1 NIST7086bwamem.merge.0 samtools.multi-call.extract. vcf 2 NIST7086bwamem.merge.realn.0 gatklite.multi-call.extract. vcf 2

I noticed frrebayes processed only one sample. Is this a known bug in freebayes??

I am trying to understand the system well so that I can use seqmule on my large number of samples. Best regards Mamoon

On Tue, Aug 16, 2016 at 5:46 PM, Yunfei Guo < notifications@github.com> wrote:

Hi MR,

I noticed that there was an error "ERROR: not all VCFs have the same number of samples!", could you check how many samples are there in each *multi-call.extract.vcf file? I don't know why there are two gatklite VCFs, though.

command to check # of samples:

for i in *multi-call.extract.vcf; do echo $i; perl -ne 'if (/^#CHROM/){print ((scalar (split /\t/,$_))-9),"\n"}' $i ; done

On Tue, Aug 16, 2016 at 4:33 AM, rashidma < notifications@github.com> wrote:

Hi, I just noticed in the *.log file of seqmule

run

the VCF " _NIST7086result/NIST7086 bwamem.merge.realn.0gatklite. multi-call.extract.vcf" has been mentioned twice. please see below colored in red. Is this creating proble?? Per sample there should be 4 VCF files (3 different aligner variant caller combinations and one consensus) BUT in case of NIST7086 why this is 5 VCF.

_2 sample(s) in this analysis: NIST7035 NIST7086All input is merged on a per-sample basis (after alignment if FASTQ).Multi-sample variant calling ENABLED.Input is exome (or captured) sequencing data.4 variant caller(s) used: GATKLiteUnifiedGenotyper SAMtools FreeBayes Consensus1 aligner(s) used: bwamemFile used for caculating coverage statistics and extracting variants: nexterarapidcapture expandedexome_targetedregions. nooverlap.bedReadgroup : READGROUPNIST7035,READGROUP NIST7086Library : LIBRARYSequencing platform: ILLUMINAReference genome build is hg19dbsnp138 is used for variant calling and recalibration (in GATK VQSR).Java memory usage is limited to 1750mjava executable path: javaMax number of processes: 12NOTICE: /tmp will be used for storing temporary files######################### ########################## FINAL OUTPUT for NIST7035:##################### ############################## NOTICE: Both consensus results and individual results are listed.WARNING: consensus results may not be available because some variant callers or aligners may fail to generate output.NIST7035_result/ NIST7035.extract_consensus. vcfNIST7035result/NIST7035 bwamem.merge.realn.0_gatklite. multi-call.extract. vcfNIST7035result/NIST7035 bwamem.merge.0samtools.multi- call.extract.vcfNIST7035 result/NIST7035_bwamem.merge. 0_freebayes.multi-call. extract.vcf################### ##############################

FINAL

OUTPUT for NIST7086:##################### ############################## NOTICE: Both consensus results and individual results are listed.WARNING: consensus results may not be available because some variant callers or aligners may fail to generate output._

_NIST7086_result/NIST7086. extractconsensus.vcfNIST7086 result/NIST7086_bwamem.merge. realn.0_gatklite.multi-call. extract.vcfNIST7086_result/ NIST7086bwamem.merge.realn.0 gatklite.multi-call.extract. vcfNIST7086result/NIST7086 bwamem.merge.0samtools.multi- call.extract.vcfNIST7086 result/NIST7086_bwamem.merge. 0freebayes.multi-call. extract.vcf

Thanks

On Tue, Aug 16, 2016 at 8:07 AM, Mamoon Rashid < rashidmamoon@gmail.com

wrote:

Hi Yunfei, I sent you the big nohup file (~8MB) and it failed to reach you. Now I am sending you the last 1000 lines of the nohup file. I can see there are errors at step 42 and 43. I thank you so much for your time. MR

On Mon, Aug 15, 2016 at 4:05 PM, Yunfei Guo < notifications@github.com> wrote:

Hi Rashid,

Could you please provide the contents of nohup.out file? I need to know where it stopped.

Thanks.

Best, Yunfei

On Mon, Aug 15, 2016 at 12:47 AM, rashidma < notifications@github.com> wrote:

Thanks a lot Yunfei, I successfully ran the test run. Now, I was running a real sample from NIST. Out of 51 steps 48 steps ran well. I am stuck in the last few steps. The seqmule was stopped many times due to system shutdown and couple of times I killed the process. But every time I restarted it with--

nohup seqmule run NIST7035-NIST7086.script &

Please let me know any troubleshooting. I will be thankful to you. Regards MR

On Thu, Aug 11, 2016 at 1:02 AM, Yunfei Guo < notifications@github.com

wrote:

Hi Mamoon,

Please use inst/hg19_exome.legacy.bed after updating SeqMule with seqmule update --git. Thank you!

Best, Yunfei

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rashidma commented 7 years ago

Hi Yunfei, Sorry for the late reply. I have run many instance of seqmule with different configurations. SOme of them run successfully for exome trio data. But i learned the following lessons-

With custom reference database the GATK_RECAL step fails.
In default mode one aligner bwamem and three variant callers (GATK, samtools, freebayes) are used by seqmule BUT freebayes FAILS on multi-sample variant calling. And the final results are not produced in this case.
One may have great precaution using custom database. You have to keep them in a separate directory and provide absolute path to the ref files in command line.
I used gatk, samtools, and varscan and i realised that varscan failed to call any variants BUT the final results are produced by seqmule.

Now, i want to know about the "average coverage in target region". Is this the width of coverage (that is prcentage of bases in target region that is covered by atleast a single base or it is the depth of coverage)?? please clarify this. I want to know that how many bases of target region not covered in the alignment.

waiting for reply Many thanks Mamoon

On Wed, Aug 24, 2016 at 6:09 AM, Yunfei Guo notifications@github.com wrote:

Hi Mamoon,

That is weird. If you could provide a small portion of your bam file so that I can reproduce the error, I can try to figure out what is going on.

SeqMule does not support MPI.

Best, Yunfei

On Tue, Aug 23, 2016 at 12:30 PM, rashidma notifications@github.com wrote:

Thanks a ton Even after installing Freebayes from github, my seqmule run could not complete. Again freebayes could not call multiple-sample variant calling. Anyway, I am running it with varscan, samtools, and gatk.

Does seqmule support parallel processing using MPI?

Thanks Mamoon

On Tue, Aug 23, 2016 at 8:42 PM, Yunfei Guo notifications@github.com wrote:

Hi Mamoon,

Please refer to http://seqmule.openbioinformatics.org/en/latest/Miscellaneous/FAQ/# which-file-is-used-as-default-argument-for-advanced

Yes. Instead of specifying -a and -b at the same time, just specify -a in seqmule pipeline. Note, however, seqmule does not support mixing single-end data with pair-end data.

Thanks.

On Mon, Aug 22, 2016 at 10:23 PM, rashidma notifications@github.com wrote:

Hi Yunfei, It is running now.

I would like to know --

which /misc/predefined_config file is used in default mode by seqmule?

Is it possible to pass singletons reads in seqmule? I have a considerable amount of reads which are unpaired.

Thanks you so much for your time. Mamoon

On Mon, Aug 22, 2016 at 7:53 PM, Yunfei Guo < notifications@github.com> wrote:

33 is the step number.

seqmule run -n 33 yourseqmule.script

On Mon, Aug 22, 2016 at 6:27 AM, rashidma < notifications@github.com> wrote:

I installed freebayes from github as you mentioned. Please let me know how to run seqmule from the step where freebayes is executed? I looked at *.script file and found ---

_[33]JOBID=0PID=14673command=/work/rashidma/software/ seqmule_by_admin/SeqMule-master/bin/secondary/../../ bin/secondary/worker /work/rashidma/Exome_data/GIAB_NIST_exome_data/ MyAnalysis/04_seqmule_qsub- ucschg19/seqmule.08162016.23639.logs 33 "/work/rashidma/software/seqmule_by_admin/SeqMule- master/bin/secondary/ runFREEBAYES -advanced NIST7035-NIST7086.config -n 9 -freebayes /work/rashidma/software/seqmule_by_admin/SeqMule- master/exe/freebayes/bin/ freebayes -vcfsorter /work/rashidma/software/seqmule_by_admin/SeqMule- master/bin/secondary/../../bin/secondary/vcfsorter -ref /work/rashidma/Databases/hg19/UCSC/ucsc.hg19.fa -samtools /work/rashidma/software/seqmule_by_admin/SeqMule- master/exe/samtools/samtools -tmpdir /tmp -vcf NIST7035_result/NIST7035_bwamem.merge.0_freebayes.multi-call.vcf -bed nexterarapidcapture_expandedexome_targetedregions.nooverlap.bed -bam NIST7035_result/NIST7035_bwamem.merge.bam -bam NIST7086_result/NIST7086_bwamem.merge.bam"dependency= 23,24message=freebayes.multi-call variant callingnCPUrequested=12status=finished

What should i do now? Thanks MR

On Mon, Aug 22, 2016 at 2:15 AM, Yunfei Guo < notifications@github.com> wrote:

Hi Mamoon,

Sorry, our servers are temporarily unavailable right now. We are still trying to fix them. Could you please try install freebayes yourself and run seqmule from the freebayes step?

Instructions:

go to seqmule directory

move exe/freebayes folder to somewhere outside seqmule folder

download freebayes by "git clone --recursive git:// github.com/ekg/freebayes.git exe/freebayes"

cd exe/freebayes

make

run seqmule from the step where freebayes is executed (look for the step number for freebayes variant calling in *.script)

Thanks.

On Sun, Aug 21, 2016 at 4:28 AM, rashidma < notifications@github.com> wrote:

Hi Yunfei, Did you test the Freebayes? In my seqmule run it failed to call SNPs for multiple samples (as we discussed in the last email). Thanks Mamoon

On Fri, Aug 19, 2016 at 7:53 PM, Yunfei Guo < notifications@github.com>

wrote:

This should not affect the results, however, it may be the reason why you see some vcf files more than once in the runtime information.

On Fri, Aug 19, 2016 at 7:56 AM, rashidma < notifications@github.com> wrote:

Yes I did run the analysis multiple times with "seqmule run *.script" in the same directory.

The analysis takes a lot of time so seqmule run saves a lot of time.

Does it affect the result after rerunning seqmule?

Thanks MR

On 19 Aug 2016 17:43, "Yunfei Guo" < notifications@github.com

wrote:

I mean for each analysis, did you run it twice (probably with 'seqmule run' after 'seqmule pipeline')?

On Thu, Aug 18, 2016 at 7:13 PM, rashidma < notifications@github.com> wrote:

I re-run the analysis in a completely new directory. So both runs should not influence each other.

Thanks MR

On 18 Aug 2016 18:41, "Yunfei Guo" < notifications@github.com

wrote:

Is it because you tried to rerun the analysis?

On Aug 18, 2016, at 8:06 AM, rashidma < notifications@github.com> wrote:

Thanks a lot for your reply. I started another run with bwa-gatk-samtools-varscan and I found that it is also incomplete.

The log file contains the *gatk-multicall-extract.vcf twice for second sample. For first sample it is ok.

I could not understand the behavior. Lets first troubleshoot the first run that we are discussing.

regards MR

On Thu, Aug 18, 2016 at 5:43 PM, Yunfei Guo < notifications@github.com> wrote:

Thanks, Mamoon. Let me test the latest freebayes and get back to you later.

On Wed, Aug 17, 2016 at 10:05 PM, rashidma < notifications@github.com

wrote:

Hi

I checked the VCF files for number of samples

VCFs number of samples NIST7035bwamem.merge.0 freebayes.multi-call.extract. vcf 1 NIST7035bwamem.merge.0 samtools.multi-call.extract. vcf 2 NIST7035bwamem.merge.realn.0 gatklite.multi-call.extract. vcf 2

NIST7086bwamem.merge.0 freebayes.multi-call.extract. vcf 1 NIST7086bwamem.merge.0 samtools.multi-call.extract. vcf 2 NIST7086bwamem.merge.realn.0 gatklite.multi-call.extract. vcf 2

I noticed frrebayes processed only one sample. Is this a known bug in freebayes??

I am trying to understand the system well so that I can use seqmule on my large number of samples. Best regards Mamoon

On Tue, Aug 16, 2016 at 5:46 PM, Yunfei Guo < notifications@github.com> wrote:

Hi MR,

I noticed that there was an error "ERROR: not all VCFs have the same number of samples!", could you check how many samples are there in each *multi-call.extract.vcf file? I don't know why there are two gatklite VCFs, though.

command to check # of samples:

for i in *multi-call.extract.vcf; do echo $i; perl -ne 'if (/^#CHROM/){print ((scalar (split /\t/,$_))-9),"\n"}' $i ; done

On Tue, Aug 16, 2016 at 4:33 AM, rashidma < notifications@github.com> wrote:

Hi, I just noticed in the *.log file of seqmule

run

the VCF " _NIST7086result/NIST7086 bwamem.merge.realn.0gatklite. multi-call.extract.vcf" has been mentioned twice. please see below colored in red. Is this creating proble?? Per sample there should be 4 VCF files (3 different aligner variant caller combinations and one consensus) BUT in case of NIST7086 why this is 5 VCF.

_2 sample(s) in this analysis: NIST7035 NIST7086All input is merged on a per-sample basis (after alignment if FASTQ).Multi-sample variant calling ENABLED.Input is exome (or captured) sequencing data.4 variant caller(s) used: GATKLiteUnifiedGenotyper SAMtools FreeBayes Consensus1 aligner(s) used: bwamemFile used for caculating coverage statistics and extracting variants: nexterarapidcapture expandedexome_targetedregions. nooverlap.bedReadgroup : READGROUPNIST7035,READGROUP NIST7086Library : LIBRARYSequencing platform: ILLUMINAReference genome build is hg19dbsnp138 is used for variant calling and recalibration (in GATK VQSR).Java memory usage is limited to 1750mjava executable path: javaMax number of processes: 12NOTICE: /tmp will be used for storing temporary files######################### ########################## FINAL OUTPUT for NIST7035:##################### ############################## NOTICE: Both consensus results and individual results are listed.WARNING: consensus results may not be available because some variant callers or aligners may fail to generate output.NIST7035_result/ NIST7035.extract_consensus. vcfNIST7035result/NIST7035 bwamem.merge.realn.0_gatklite. multi-call.extract. vcfNIST7035result/NIST7035 bwamem.merge.0samtools.multi- call.extract.vcfNIST7035 result/NIST7035_bwamem.merge. 0_freebayes.multi-call. extract.vcf################### ##############################

FINAL

OUTPUT for NIST7086:##################### ############################## NOTICE: Both consensus results and individual results are listed.WARNING: consensus results may not be available because some variant callers or aligners may fail to generate output._

_NIST7086_result/NIST7086. extractconsensus.vcfNIST7086 result/NIST7086_bwamem.merge. realn.0_gatklite.multi-call. extract.vcfNIST7086_result/ NIST7086bwamem.merge.realn.0 gatklite.multi-call.extract. vcfNIST7086result/NIST7086 bwamem.merge.0samtools.multi- call.extract.vcfNIST7086 result/NIST7086_bwamem.merge. 0freebayes.multi-call. extract.vcf

Thanks

On Tue, Aug 16, 2016 at 8:07 AM, Mamoon Rashid < rashidmamoon@gmail.com

wrote:

Hi Yunfei, I sent you the big nohup file (~8MB) and it failed to reach you. Now I am sending you the last 1000 lines of the nohup file. I can see there are errors at step 42 and 43. I thank you so much for your time. MR

On Mon, Aug 15, 2016 at 4:05 PM, Yunfei Guo < notifications@github.com> wrote:

Hi Rashid,

Could you please provide the contents of nohup.out file? I need to know where it stopped.

Thanks.

Best, Yunfei

On Mon, Aug 15, 2016 at 12:47 AM, rashidma < notifications@github.com> wrote:

Thanks a lot Yunfei, I successfully ran the test run. Now, I was running a real sample from NIST. Out of 51 steps 48 steps ran well. I am stuck in the last few steps. The seqmule was stopped many times due to system shutdown and couple of times I killed the process. But every time I restarted it with--

nohup seqmule run NIST7035-NIST7086.script &

Please let me know any troubleshooting. I will be thankful to you. Regards MR

On Thu, Aug 11, 2016 at 1:02 AM, Yunfei Guo < notifications@github.com

wrote:

Hi Mamoon,

Please use inst/hg19_exome.legacy.bed after updating SeqMule with seqmule update --git. Thank you!

Best, Yunfei

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yunfeiguo commented 7 years ago

Hi Mamoon,

I am out of town right now. Could you please send me the files you were using (perhaps a small portion of it) along with the command and configurations you used? I would be glad to figure out where is the problem and help you get the results once I come back.

Thanks!

On Aug 28, 2016, at 9:34 PM, rashidma notifications@github.com wrote:

Hi Yunfei, Sorry for the late reply. I have run many instance of seqmule with different configurations. SOme of them run successfully for exome trio data. But i learned the following lessons-

With custom reference database the GATK_RECAL step fails.

In default mode one aligner bwamem and three variant callers (GATK, samtools, freebayes) are used by seqmule BUT freebayes FAILS on multi-sample variant calling. And the final results are not produced in this case.

One may have great precaution using custom database. You have to keep them in a separate directory and provide absolute path to the ref files in command line.

I used gatk, samtools, and varscan and i realised that varscan failed to call any variants BUT the final results are produced by seqmule.

Now, i want to know about the "average coverage in target region". Is this the width of coverage (that is prcentage of bases in target region that is covered by atleast a single base or it is the depth of coverage)?? please clarify this. I want to know that how many bases of target region not covered in the alignment.

waiting for reply Many thanks Mamoon

On Wed, Aug 24, 2016 at 6:09 AM, Yunfei Guo notifications@github.com wrote:

Hi Mamoon,

That is weird. If you could provide a small portion of your bam file so that I can reproduce the error, I can try to figure out what is going on.

SeqMule does not support MPI.

Best, Yunfei

On Tue, Aug 23, 2016 at 12:30 PM, rashidma notifications@github.com wrote:

Thanks a ton Even after installing Freebayes from github, my seqmule run could not complete. Again freebayes could not call multiple-sample variant calling. Anyway, I am running it with varscan, samtools, and gatk.

Does seqmule support parallel processing using MPI?

Thanks Mamoon

On Tue, Aug 23, 2016 at 8:42 PM, Yunfei Guo notifications@github.com wrote:

Hi Mamoon,

Please refer to http://seqmule.openbioinformatics.org/en/latest/Miscellaneous/FAQ/# which-file-is-used-as-default-argument-for-advanced

Yes. Instead of specifying -a and -b at the same time, just specify -a in seqmule pipeline. Note, however, seqmule does not support mixing single-end data with pair-end data.

Thanks.

On Mon, Aug 22, 2016 at 10:23 PM, rashidma notifications@github.com wrote:

Hi Yunfei, It is running now.

I would like to know --

which /misc/predefined_config file is used in default mode by seqmule?

Is it possible to pass singletons reads in seqmule? I have a considerable amount of reads which are unpaired.

Thanks you so much for your time. Mamoon

On Mon, Aug 22, 2016 at 7:53 PM, Yunfei Guo < notifications@github.com> wrote:

33 is the step number.

seqmule run -n 33 yourseqmule.script

On Mon, Aug 22, 2016 at 6:27 AM, rashidma < notifications@github.com> wrote:

I installed freebayes from github as you mentioned. Please let me know how to run seqmule from the step where freebayes is executed? I looked at *.script file and found ---

_[33]JOBID=0PID=14673command=/work/rashidma/software/ seqmule_by_admin/SeqMule-master/bin/secondary/../../ bin/secondary/worker /work/rashidma/Exome_data/GIAB_NIST_exome_data/ MyAnalysis/04_seqmule_qsub- ucschg19/seqmule.08162016.23639.logs 33 "/work/rashidma/software/seqmule_by_admin/SeqMule- master/bin/secondary/ runFREEBAYES -advanced NIST7035-NIST7086.config -n 9 -freebayes /work/rashidma/software/seqmule_by_admin/SeqMule- master/exe/freebayes/bin/ freebayes -vcfsorter /work/rashidma/software/seqmule_by_admin/SeqMule- master/bin/secondary/../../bin/secondary/vcfsorter -ref /work/rashidma/Databases/hg19/UCSC/ucsc.hg19.fa -samtools /work/rashidma/software/seqmule_by_admin/SeqMule- master/exe/samtools/samtools -tmpdir /tmp -vcf NIST7035_result/NIST7035_bwamem.merge.0_freebayes.multi-call.vcf -bed nexterarapidcapture_expandedexome_targetedregions.nooverlap.bed -bam NIST7035_result/NIST7035_bwamem.merge.bam -bam NIST7086_result/NIST7086_bwamem.merge.bam"dependency= 23,24message=freebayes.multi-call variant callingnCPUrequested=12status=finished

What should i do now? Thanks MR

On Mon, Aug 22, 2016 at 2:15 AM, Yunfei Guo < notifications@github.com> wrote:

Hi Mamoon,

Sorry, our servers are temporarily unavailable right now. We are still trying to fix them. Could you please try install freebayes yourself and run seqmule from the freebayes step?

Instructions:

go to seqmule directory

move exe/freebayes folder to somewhere outside seqmule folder

download freebayes by "git clone --recursive git:// github.com/ekg/freebayes.git exe/freebayes"

cd exe/freebayes

make

run seqmule from the step where freebayes is executed (look for the step number for freebayes variant calling in *.script)

Thanks.

On Sun, Aug 21, 2016 at 4:28 AM, rashidma < notifications@github.com> wrote:

Hi Yunfei, Did you test the Freebayes? In my seqmule run it failed to call SNPs for multiple samples (as we discussed in the last email). Thanks Mamoon

On Fri, Aug 19, 2016 at 7:53 PM, Yunfei Guo < notifications@github.com>

wrote:

This should not affect the results, however, it may be the reason why you see some vcf files more than once in the runtime information.

On Fri, Aug 19, 2016 at 7:56 AM, rashidma < notifications@github.com> wrote:

Yes I did run the analysis multiple times with "seqmule run *.script" in the same directory.

The analysis takes a lot of time so seqmule run saves a lot of time.

Does it affect the result after rerunning seqmule?

Thanks MR

On 19 Aug 2016 17:43, "Yunfei Guo" < notifications@github.com

wrote:

I mean for each analysis, did you run it twice (probably with 'seqmule run' after 'seqmule pipeline')?

On Thu, Aug 18, 2016 at 7:13 PM, rashidma < notifications@github.com> wrote:

I re-run the analysis in a completely new directory. So both runs should not influence each other.

Thanks MR

On 18 Aug 2016 18:41, "Yunfei Guo" < notifications@github.com

wrote:

Is it because you tried to rerun the analysis?

On Aug 18, 2016, at 8:06 AM, rashidma < notifications@github.com> wrote:

Thanks a lot for your reply. I started another run with bwa-gatk-samtools-varscan and I found that it is also incomplete.

The log file contains the *gatk-multicall-extract.vcf twice for second sample. For first sample it is ok.

I could not understand the behavior. Lets first troubleshoot the first run that we are discussing.

regards MR

On Thu, Aug 18, 2016 at 5:43 PM, Yunfei Guo < notifications@github.com> wrote:

Thanks, Mamoon. Let me test the latest freebayes and get back to you later.

On Wed, Aug 17, 2016 at 10:05 PM, rashidma < notifications@github.com

wrote:

Hi

I checked the VCF files for number of samples

VCFs number of samples NIST7035bwamem.merge.0 freebayes.multi-call.extract. vcf 1 NIST7035bwamem.merge.0 samtools.multi-call.extract. vcf 2 NIST7035bwamem.merge.realn.0 gatklite.multi-call.extract. vcf 2

NIST7086bwamem.merge.0 freebayes.multi-call.extract. vcf 1 NIST7086bwamem.merge.0 samtools.multi-call.extract. vcf 2 NIST7086bwamem.merge.realn.0 gatklite.multi-call.extract. vcf 2

I noticed frrebayes processed only one sample. Is this a known bug in freebayes??

I am trying to understand the system well so that I can use seqmule on my large number of samples. Best regards Mamoon

On Tue, Aug 16, 2016 at 5:46 PM, Yunfei Guo < notifications@github.com> wrote:

Hi MR,

I noticed that there was an error "ERROR: not all VCFs have the same number of samples!", could you check how many samples are there in each *multi-call.extract.vcf file? I don't know why there are two gatklite VCFs, though.

command to check # of samples:

for i in *multi-call.extract.vcf; do echo $i; perl -ne 'if (/^#CHROM/){print ((scalar (split /\t/,$_))-9),"\n"}' $i ; done

On Tue, Aug 16, 2016 at 4:33 AM, rashidma < notifications@github.com> wrote:

Hi, I just noticed in the *.log file of seqmule

run

the VCF " _NIST7086result/NIST7086 bwamem.merge.realn.0gatklite. multi-call.extract.vcf" has been mentioned twice. please see below colored in red. Is this creating proble?? Per sample there should be 4 VCF files (3 different aligner variant caller combinations and one consensus) BUT in case of NIST7086 why this is 5 VCF.

_2 sample(s) in this analysis: NIST7035 NIST7086All input is merged on a per-sample basis (after alignment if FASTQ).Multi-sample variant calling ENABLED.Input is exome (or captured) sequencing data.4 variant caller(s) used: GATKLiteUnifiedGenotyper SAMtools FreeBayes Consensus1 aligner(s) used: bwamemFile used for caculating coverage statistics and extracting variants: nexterarapidcapture expandedexome_targetedregions. nooverlap.bedReadgroup : READGROUPNIST7035,READGROUP NIST7086Library : LIBRARYSequencing platform: ILLUMINAReference genome build is hg19dbsnp138 is used for variant calling and recalibration (in GATK VQSR).Java memory usage is limited to 1750mjava executable path: javaMax number of processes: 12NOTICE: /tmp will be used for storing temporary files######################### ########################## FINAL OUTPUT for NIST7035:##################### ############################## NOTICE: Both consensus results and individual results are listed.WARNING: consensus results may not be available because some variant callers or aligners may fail to generate output.NIST7035_result/ NIST7035.extract_consensus. vcfNIST7035result/NIST7035 bwamem.merge.realn.0_gatklite. multi-call.extract. vcfNIST7035result/NIST7035 bwamem.merge.0samtools.multi- call.extract.vcfNIST7035 result/NIST7035_bwamem.merge. 0_freebayes.multi-call. extract.vcf################### ##############################

FINAL

OUTPUT for NIST7086:##################### ############################## NOTICE: Both consensus results and individual results are listed.WARNING: consensus results may not be available because some variant callers or aligners may fail to generate output._

_NIST7086_result/NIST7086. extractconsensus.vcfNIST7086 result/NIST7086_bwamem.merge. realn.0_gatklite.multi-call. extract.vcfNIST7086_result/ NIST7086bwamem.merge.realn.0 gatklite.multi-call.extract. vcfNIST7086result/NIST7086 bwamem.merge.0samtools.multi- call.extract.vcfNIST7086 result/NIST7086_bwamem.merge. 0freebayes.multi-call. extract.vcf

Thanks

On Tue, Aug 16, 2016 at 8:07 AM, Mamoon Rashid < rashidmamoon@gmail.com

wrote:

Hi Yunfei, I sent you the big nohup file (~8MB) and it failed to reach you. Now I am sending you the last 1000 lines of the nohup file. I can see there are errors at step 42 and 43. I thank you so much for your time. MR

On Mon, Aug 15, 2016 at 4:05 PM, Yunfei Guo < notifications@github.com> wrote:

Hi Rashid,

Could you please provide the contents of nohup.out file? I need to know where it stopped.

Thanks.

Best, Yunfei

On Mon, Aug 15, 2016 at 12:47 AM, rashidma < notifications@github.com> wrote:

Thanks a lot Yunfei, I successfully ran the test run. Now, I was running a real sample from NIST. Out of 51 steps 48 steps ran well. I am stuck in the last few steps. The seqmule was stopped many times due to system shutdown and couple of times I killed the process. But every time I restarted it with--

nohup seqmule run NIST7035-NIST7086.script &

Please let me know any troubleshooting. I will be thankful to you. Regards MR

rashidma commented 7 years ago

Hi Yunfei, I am using seqmule heavily. I got the several errors in several round of runs of seqmule, therefore I could not send you the proper file. I was confused. I realized that base quality recalibration was creating problem and switching it off has got the seqmule running. I noticed several such behaviors.

This time I am writing you on a separate note. I ran seqmule with BWA-backtrack and GATK haplotypecaller with my own hg19 reference genome from UCSC. It failed at step 31 Extract variants in custom regions. Please comment why it failed? I have attached the required file. Please see the error-log file. It is the stderr file from PBS run. Many thanks Mamoon

On Tue, Aug 30, 2016 at 6:20 AM, Yunfei Guo notifications@github.com wrote:

Hi Mamoon,

I am out of town right now. Could you please send me the files you were using (perhaps a small portion of it) along with the command and configurations you used? I would be glad to figure out where is the problem and help you get the results once I come back.

Thanks!

On Aug 28, 2016, at 9:34 PM, rashidma notifications@github.com wrote:

Hi Yunfei, Sorry for the late reply. I have run many instance of seqmule with different configurations. SOme of them run successfully for exome trio data. But i learned the following lessons-

With custom reference database the GATK_RECAL step fails.

In default mode one aligner bwamem and three variant callers (GATK, samtools, freebayes) are used by seqmule BUT freebayes FAILS on multi-sample variant calling. And the final results are not produced in this case.

One may have great precaution using custom database. You have to keep them in a separate directory and provide absolute path to the ref files in command line.

I used gatk, samtools, and varscan and i realised that varscan failed to call any variants BUT the final results are produced by seqmule.

Now, i want to know about the "average coverage in target region". Is this the width of coverage (that is prcentage of bases in target region that is covered by atleast a single base or it is the depth of coverage)?? please clarify this. I want to know that how many bases of target region not covered in the alignment.

waiting for reply Many thanks Mamoon

On Wed, Aug 24, 2016 at 6:09 AM, Yunfei Guo notifications@github.com wrote:

Hi Mamoon,

That is weird. If you could provide a small portion of your bam file so that I can reproduce the error, I can try to figure out what is going on.

SeqMule does not support MPI.

Best, Yunfei

On Tue, Aug 23, 2016 at 12:30 PM, rashidma notifications@github.com wrote:

Thanks a ton Even after installing Freebayes from github, my seqmule run could not complete. Again freebayes could not call multiple-sample variant calling. Anyway, I am running it with varscan, samtools, and gatk.

Does seqmule support parallel processing using MPI?

Thanks Mamoon

On Tue, Aug 23, 2016 at 8:42 PM, Yunfei Guo < notifications@github.com> wrote:

Hi Mamoon,

Please refer to http://seqmule.openbioinformatics.org/en/ latest/Miscellaneous/FAQ/# which-file-is-used-as-default-argument-for-advanced

Yes. Instead of specifying -a and -b at the same time, just specify -a in seqmule pipeline. Note, however, seqmule does not support mixing single-end data with pair-end data.

Thanks.

On Mon, Aug 22, 2016 at 10:23 PM, rashidma < notifications@github.com> wrote:

Hi Yunfei, It is running now.

I would like to know --

which /misc/predefined_config file is used in default mode by seqmule?

Is it possible to pass singletons reads in seqmule? I have a considerable amount of reads which are unpaired.

Thanks you so much for your time. Mamoon

On Mon, Aug 22, 2016 at 7:53 PM, Yunfei Guo < notifications@github.com> wrote:

33 is the step number.

seqmule run -n 33 yourseqmule.script

On Mon, Aug 22, 2016 at 6:27 AM, rashidma < notifications@github.com> wrote:

I installed freebayes from github as you mentioned. Please let me know how to run seqmule from the step where freebayes is executed? I looked at *.script file and found ---

_[33]JOBID=0PID=14673command=/work/rashidma/software/ seqmule_by_admin/SeqMule-master/bin/secondary/../../ bin/secondary/worker /work/rashidma/Exome_data/GIAB_NIST_exome_data/ MyAnalysis/04_seqmule_qsub- ucschg19/seqmule.08162016.23639.logs 33 "/work/rashidma/software/seqmule_by_admin/SeqMule- master/bin/secondary/ runFREEBAYES -advanced NIST7035-NIST7086.config -n 9 -freebayes /work/rashidma/software/seqmule_by_admin/SeqMule- master/exe/freebayes/bin/ freebayes -vcfsorter /work/rashidma/software/seqmule_by_admin/SeqMule- master/bin/secondary/../../bin/secondary/vcfsorter -ref /work/rashidma/Databases/hg19/UCSC/ucsc.hg19.fa -samtools /work/rashidma/software/seqmule_by_admin/SeqMule- master/exe/samtools/samtools -tmpdir /tmp -vcf NIST7035_result/NIST7035_bwamem.merge.0_freebayes. multi-call.vcf -bed nexterarapidcapture_expandedexome_targetedregions. nooverlap.bed -bam NIST7035_result/NIST7035_bwamem.merge.bam -bam NIST7086_result/NIST7086_bwamem.merge.bam"dependency= 23,24message=freebayes.multi-call variant callingnCPUrequested=12status=finished

What should i do now? Thanks MR

On Mon, Aug 22, 2016 at 2:15 AM, Yunfei Guo < notifications@github.com> wrote:

Hi Mamoon,

Sorry, our servers are temporarily unavailable right now. We are still trying to fix them. Could you please try install freebayes yourself and run seqmule from the freebayes step?

Instructions:

go to seqmule directory

move exe/freebayes folder to somewhere outside seqmule folder

download freebayes by "git clone --recursive git:// github.com/ekg/freebayes.git exe/freebayes"

cd exe/freebayes

make

run seqmule from the step where freebayes is executed (look for the step number for freebayes variant calling in *.script)

Thanks.

On Sun, Aug 21, 2016 at 4:28 AM, rashidma < notifications@github.com> wrote:

Hi Yunfei, Did you test the Freebayes? In my seqmule run it failed to call SNPs for multiple samples (as we discussed in the last email). Thanks Mamoon

On Fri, Aug 19, 2016 at 7:53 PM, Yunfei Guo < notifications@github.com>

wrote:

This should not affect the results, however, it may be the reason why you see some vcf files more than once in the runtime information.

On Fri, Aug 19, 2016 at 7:56 AM, rashidma < notifications@github.com> wrote:

Yes I did run the analysis multiple times with "seqmule run *.script" in the same directory.

The analysis takes a lot of time so seqmule run saves a lot of time.

Does it affect the result after rerunning seqmule?

Thanks MR

On 19 Aug 2016 17:43, "Yunfei Guo" < notifications@github.com

wrote:

I mean for each analysis, did you run it twice (probably with 'seqmule run' after 'seqmule pipeline')?

On Thu, Aug 18, 2016 at 7:13 PM, rashidma < notifications@github.com> wrote:

I re-run the analysis in a completely new directory. So both runs should not influence each other.

Thanks MR

On 18 Aug 2016 18:41, "Yunfei Guo" < notifications@github.com

wrote:

Is it because you tried to rerun the analysis?

On Aug 18, 2016, at 8:06 AM, rashidma < notifications@github.com> wrote:

Thanks a lot for your reply. I started another run with bwa-gatk-samtools-varscan and I found that it is also incomplete.

The log file contains the *gatk-multicall-extract.vcf twice for second sample. For first sample it is ok.

I could not understand the behavior. Lets first troubleshoot the first run that we are discussing.

regards MR

On Thu, Aug 18, 2016 at 5:43 PM, Yunfei Guo < notifications@github.com> wrote:

Thanks, Mamoon. Let me test the latest freebayes and get back to you later.

On Wed, Aug 17, 2016 at 10:05 PM, rashidma < notifications@github.com

wrote:

Hi I checked the VCF files for number of

samples

VCFs number of samples NIST7035bwamem.merge.0 freebayes.multi-call.extract. vcf 1 NIST7035bwamem.merge.0 samtools.multi-call.extract. vcf 2 NIST7035bwamem.merge.realn.0 gatklite.multi-call.extract. vcf 2

NIST7086bwamem.merge.0 freebayes.multi-call.extract. vcf 1 NIST7086bwamem.merge.0 samtools.multi-call.extract. vcf 2 NIST7086bwamem.merge.realn.0 gatklite.multi-call.extract. vcf 2

I noticed frrebayes processed only one sample. Is this a known bug in freebayes??

I am trying to understand the system well so that I can use seqmule on my large number of samples. Best regards Mamoon

On Tue, Aug 16, 2016 at 5:46 PM, Yunfei Guo < notifications@github.com> wrote:

Hi MR,

I noticed that there was an error "ERROR: not all VCFs have the same number of samples!", could you check how many samples are there in each *multi-call.extract.vcf file? I don't know why there are two gatklite VCFs, though.

command to check # of samples:

for i in *multi-call.extract.vcf; do echo $i; perl -ne 'if (/^#CHROM/){print ((scalar (split /\t/,$_))-9),"\n"}' $i ; done

On Tue, Aug 16, 2016 at 4:33 AM, rashidma < notifications@github.com> wrote:

Hi, I just noticed in the *.log file of seqmule

run

the VCF " _NIST7086result/NIST7086 bwamem.merge.realn.0gatklite. multi-call.extract.vcf" has been mentioned twice. please see below colored in red. Is this creating proble?? Per sample there should be 4 VCF files (3 different aligner variant caller combinations and one consensus) BUT in case of NIST7086 why this is 5 VCF.

_2 sample(s) in this analysis: NIST7035 NIST7086All input is merged on a per-sample basis (after alignment if FASTQ).Multi-sample variant calling ENABLED.Input is exome (or captured) sequencing data.4 variant caller(s) used: GATKLiteUnifiedGenotyper SAMtools FreeBayes Consensus1 aligner(s) used: bwamemFile used for caculating coverage statistics and extracting variants: nexterarapidcapture expandedexome_targetedregions. nooverlap.bedReadgroup : READGROUPNIST7035,READGROUP NIST7086Library : LIBRARYSequencing platform: ILLUMINAReference genome build is hg19dbsnp138 is used for variant calling and recalibration (in GATK VQSR).Java memory usage is limited to 1750mjava executable path: javaMax number of processes: 12NOTICE: /tmp will be used for storing temporary files######################### ########################## FINAL OUTPUT for NIST7035:##################### ############################## NOTICE: Both consensus results and individual results are listed.WARNING: consensus results may not be available because some variant callers or aligners may fail to generate output.NIST7035_result/ NIST7035.extract_consensus. vcfNIST7035result/NIST7035 bwamem.merge.realn.0_gatklite. multi-call.extract. vcfNIST7035result/NIST7035 bwamem.merge.0samtools.multi- call.extract.vcfNIST7035 result/NIST7035_bwamem.merge. 0_freebayes.multi-call. extract.vcf################### ##############################

FINAL

OUTPUT for NIST7086:##################### ############################## NOTICE: Both consensus results and individual results are listed.WARNING: consensus results may not be available because some variant callers or aligners may fail to generate output._

_NIST7086_result/NIST7086. extractconsensus.vcfNIST7086 result/NIST7086_bwamem.merge. realn.0_gatklite.multi-call. extract.vcfNIST7086_result/ NIST7086bwamem.merge.realn.0 gatklite.multi-call.extract. vcfNIST7086result/NIST7086 bwamem.merge.0samtools.multi- call.extract.vcfNIST7086 result/NIST7086_bwamem.merge. 0freebayes.multi-call. extract.vcf

Thanks

On Tue, Aug 16, 2016 at 8:07 AM, Mamoon Rashid < rashidmamoon@gmail.com

wrote:

Hi Yunfei, I sent you the big nohup file (~8MB) and it failed to reach you. Now I am sending you the last 1000 lines of the nohup file. I can see there are errors at step 42 and 43. I thank you so much for your time. MR

On Mon, Aug 15, 2016 at 4:05 PM, Yunfei Guo < notifications@github.com> wrote:

Hi Rashid,

Could you please provide the contents of nohup.out file? I need to know where it stopped.

Thanks.

Best, Yunfei

On Mon, Aug 15, 2016 at 12:47 AM, rashidma < notifications@github.com> wrote:

Thanks a lot Yunfei, I successfully ran the test run. Now, I was running a real sample from NIST. Out of 51 steps 48 steps ran well. I am stuck in the last few steps. The seqmule was stopped many times due to system shutdown and couple of times I killed the process. But every time I restarted it with--

nohup seqmule run NIST7035-NIST7086.script &

Please let me know any troubleshooting. I will be thankful to you. Regards MR

— You are receiving this because you authored the thread. Reply to this email directly, view it on GitHub https://github.com/WGLab/SeqMule/issues/146#issuecomment-243322086, or mute the thread https://github.com/notifications/unsubscribe-auth/ATyiOKMAgvFIStYXc4WCeBJ0XdW912j8ks5qk6GIgaJpZM4Jf64z .

WARN 00:12:44,667 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:44,667 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:44,668 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:44,670 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:44,676 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:44,678 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:44,700 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:44,701 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:44,704 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:44,707 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:44,717 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:44,748 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:44,757 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:44,757 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:44,762 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:44,778 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:44,801 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:44,805 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:44,809 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:44,816 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:44,819 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:44,854 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:44,874 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:44,885 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:44,898 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:44,919 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:44,924 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:44,930 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:44,942 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:44,956 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:44,960 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:44,961 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:44,992 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:44,994 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,016 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,026 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,032 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,035 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,045 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,046 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,080 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,081 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,087 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,087 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,101 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,105 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,123 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,144 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,147 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,157 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,160 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,171 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,193 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,200 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,230 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,230 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,233 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,233 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,234 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,253 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,263 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,266 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,266 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,266 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,271 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,274 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,274 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,275 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,280 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,282 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,282 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,284 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,284 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,285 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,291 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,291 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,296 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,296 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,299 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,300 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,313 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,313 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,331 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,332 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,337 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,353 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,359 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,363 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,370 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,372 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,401 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,406 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,412 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,418 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,423 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,428 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,429 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,433 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,445 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,447 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,462 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,483 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,494 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,500 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,508 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,523 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,599 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,601 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,602 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,615 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,615 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,616 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,620 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,624 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,633 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,676 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,690 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,692 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,693 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,697 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,702 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,709 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,711 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,712 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,727 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,737 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,739 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,739 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,768 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,775 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,780 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,790 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,801 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,801 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,809 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,810 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,816 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,828 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,828 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,830 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,830 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,834 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,839 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,842 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,853 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,858 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,924 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,925 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,925 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,951 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,954 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,959 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,977 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,982 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,999 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:45,999 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,008 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,023 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,027 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,089 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,101 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,101 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,106 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,119 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,120 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,126 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,133 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,135 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,160 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,171 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,177 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,182 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,213 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,251 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,276 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,281 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,282 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,283 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,287 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,288 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,288 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,288 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,295 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,302 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,315 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,317 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,329 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,330 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,343 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,345 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,358 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,379 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,394 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,395 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,406 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,413 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,423 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,424 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,428 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,429 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,429 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,434 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,446 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,446 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,446 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,447 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,458 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,461 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,490 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,527 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,550 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,561 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,562 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,563 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,567 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,582 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,582 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,647 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,652 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,656 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,666 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,669 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,679 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,697 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,701 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,747 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,758 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,767 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,770 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,775 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,789 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,801 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,847 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,866 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,867 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,868 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,883 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,907 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,910 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,910 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,918 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,924 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,937 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,952 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,959 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,972 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:46,978 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,021 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,028 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,037 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,043 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,044 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,086 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,089 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,096 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,128 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,140 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,142 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,147 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,148 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,149 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,160 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,161 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,169 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,192 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,193 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,202 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,203 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,213 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,243 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,250 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,253 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,272 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,274 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,277 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,282 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,284 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,295 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,311 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,311 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,311 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,313 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,326 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,329 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,340 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,349 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,351 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,354 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,361 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,380 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,389 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,441 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,461 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,467 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,468 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,468 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,474 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,475 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,480 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,485 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,487 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,487 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,489 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,498 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,498 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,503 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,544 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,554 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,560 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,561 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,561 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,562 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,583 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,592 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,615 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,626 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,636 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,638 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,646 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,646 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,675 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,676 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,699 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,719 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,724 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,736 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,741 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,762 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,762 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,762 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,763 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,765 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,769 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,770 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,776 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,782 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,783 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,784 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,790 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,797 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,842 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,850 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,858 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,859 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,866 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,876 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,902 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,904 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,920 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,922 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,933 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,942 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum WARN 00:12:47,946 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRa

yunfeiguo commented 7 years ago

Hi @rashidma,

Thanks for using SeqMule!

GATK VQSR will not work well for a number of variants (e.g. in a small region) because it requires a relatively large sample for sampling.

Regarding variant extraction, is your BED file compatible with UCSC hg19, i.e. has a subset of UCSC hg19 chromosomal names? Also I cannot find the log file.

Best, Yunfei

rashidma commented 7 years ago

Hi Yunfei, Is VQSR important? How should I disable it in order to get seqmule running?

The chromosome name is consistent in bed file and reference file. Please see the log message of seqmule below-- [

_NOTICE: checking contig(chromosome) name consistency in /work/rashidma/Databases/hg19/UCSC/ucsc.hg19.fa and /work/rashidma/Exome_data/GIAB_NIST_exome_data/nexterarapidcapture_expandedexometargetedregions.bed.NOTICE: use --no-check-chr to skip chromosome name checking] I already sent you the log file in last email. And also the last portion of stderr file. I am sending you both again.

I realised that seqmule is reading VCF.pm from another installations of VCFTOOLS outside the SEQMULE home. This might be a reason of failure. What do you think?? Please see the error-log file attached.

Many thanks for your time.

Mamoon

On Tue, Oct 25, 2016 at 10:55 PM, Yunfei Guo notifications@github.com wrote:

Hi @rashidma https://github.com/rashidma,

Thanks for using SeqMule!

GATK VQSR will not work well for a number of variants (e.g. in a small region) because it requires a relatively large sample for sampling.

Regarding variant extraction, is your BED file compatible with UCSC hg19, i.e. has a subset of UCSC hg19 chromosomal names? Also I cannot find the log file.

Best, Yunfei

— You are receiving this because you were mentioned. Reply to this email directly, view it on GitHub https://github.com/WGLab/SeqMule/issues/146#issuecomment-256157521, or mute the thread https://github.com/notifications/unsubscribe-auth/ATyiOFfbzDWFZ6_MpQUFRh1hSvkz8bVGks5q3l7HgaJpZM4Jf64z .

"WARN 00:12:53,217 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum "
"WARN 00:12:53,223 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum "
"WARN 00:12:53,224 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum "
"WARN 00:12:53,230 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum "
"WARN 00:12:53,230 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum "
"WARN 00:12:53,230 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum "
"WARN 00:12:53,230 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum "
"WARN 00:12:53,231 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum "
"INFO 00:12:53,401 ProgressMeter - done 13350.0 15.0 s 19.2 m 100.0% 15.0 s 0.0 s "
"INFO 00:12:53,401 ProgressMeter - Total runtime 15.37 secs, 0.26 min, 0.00 hours "
"INFO 00:13:05,865 HelpFormatter - -------------------------------------------------------------------------------- "
"INFO 00:13:05,868 HelpFormatter - The Genome Analysis Toolkit (GATK) v3.4-0-g7e26428, Compiled 2015/05/15 03:25:41 "
"INFO 00:13:05,868 HelpFormatter - Copyright (c) 2010 The Broad Institute "
"INFO 00:13:05,868 HelpFormatter - For support and documentation go to http://www.broadinstitute.org/gatk "
"INFO 00:13:05,872 HelpFormatter - Program Args: -T CombineVariants -R /work/rashidma/Databases/hg19/UCSC/ucsc.hg19.fa --genotypemergeoption UNSORTED --variant /tmp/10260.298029492.gatklite.tmp_gatklite.snp.filter.vcf --variant /tmp/10260.298029492.gatklite.tmp_gatklite.indel.filter.vcf -o NIST7035_result/NIST7035_bwa.merge.realn.0_gatk_hc.multi-call.vcf -L nexterarapidcapture_expandedexome_targetedregions.nooverlap.bed "
"INFO 00:13:05,875 HelpFormatter - Executing as rashidma@krplpslcn005 on Linux 2.6.18-164.el5 amd64; Java HotSpot(TM) 64-Bit Server VM 1.7.0_80-b15. "
"INFO 00:13:05,875 HelpFormatter - Date/Time: 2016/10/24 00:13:05 "
"INFO 00:13:05,875 HelpFormatter - -------------------------------------------------------------------------------- "
"INFO 00:13:05,875 HelpFormatter - -------------------------------------------------------------------------------- "
"INFO 00:13:06,376 GenomeAnalysisEngine - Strictness is SILENT "
"INFO 00:13:06,534 GenomeAnalysisEngine - Downsampling Settings: Method: BY_SAMPLE, Target Coverage: 1000 "
"INFO 00:13:07,179 IntervalUtils - Processing 62085286 bp from intervals "
"INFO 00:13:07,269 GenomeAnalysisEngine - Preparing for traversal "
"INFO 00:13:07,307 GenomeAnalysisEngine - Done preparing for traversal "
"INFO 00:13:07,307 ProgressMeter - [INITIALIZATION COMPLETE; STARTING PROCESSING] "
"INFO 00:13:07,308 ProgressMeter - | processed | time | per 1M | | total | remaining "
"INFO 00:13:07,308 ProgressMeter - Location | sites | elapsed | sites | completed | runtime | runtime "
"INFO 00:13:37,341 ProgressMeter - chr19:49973784 57703.0 30.0 s 8.7 m 95.4% 31.0 s 1.0 s "
"INFO 00:13:39,369 ProgressMeter - done 61073.0 32.0 s 8.7 m 100.0% 32.0 s 0.0 s "
"INFO 00:13:39,370 ProgressMeter - Total runtime 32.06 secs, 0.53 min, 0.01 hours "

----------NOTICE----------
"[ => SeqMule Execution Status: Running 30 of 41 steps: Copy results to other samples, at Mon Oct 24 00:13:50 AST 2016, Time Elapsed: 7 hr 23 min 36 s]"
"[ => SeqMule Execution Status: step 29 is finished at Mon Oct 24 00:13:50 AST 2016, gatk_hc.multi-call variant filtering]"

----------NOTICE----------
"[ => SeqMule Execution Status: Running 31 of 41 steps: Extract variants in custom regions, at Mon Oct 24 00:13:52 AST 2016, Time Elapsed: 7 hr 23 min 38 s]"
"[ => SeqMule Execution Status: step 30 is finished at Mon Oct 24 00:13:52 AST 2016, Copy results to other samples]"

Current version: 1.2.5
Check for upgrade ...

----------NOTICE----------
"[ => SeqMule Execution Status: Running 32 of 41 steps: Extract variants in custom regions, at Mon Oct 24 00:13:53 AST 2016, Time Elapsed: 7 hr 23 min 39 s]"
Current version: 1.2.5
Check for upgrade ...

----------NOTICE----------
"[ => SeqMule Execution Status: Running 33 of 41 steps: Extract variants in custom regions, at Mon Oct 24 00:13:54 AST 2016, Time Elapsed: 7 hr 23 min 40 s]"
Current version: 1.2.5
Check for upgrade ...

----------NOTICE----------
"[ => SeqMule Execution Status: Running 35 of 41 steps: Generate alignment and coverage stat, at Mon Oct 24 00:13:55 AST 2016, Time Elapsed: 7 hr 23 min 41 s]"
Current version: 1.2.5
Check for upgrade ...

----------NOTICE----------
"[ => SeqMule Execution Status: Running 38 of 41 steps: Generate alignment and coverage stat, at Mon Oct 24 00:13:56 AST 2016, Time Elapsed: 7 hr 23 min 42 s]"
Current version: 1.2.5
Check for upgrade ...
NOTICE: unable to download '/work/rashidma/software/seqmule_by_admin/SeqMule-master/bin/secondary/../../version' from http://www.openbioinformatics.org/seqmule. Bad Internet connection?
NOTICE: unable to check for updates. Results will not be affected.
NOTICE: unable to download '/work/rashidma/software/seqmule_by_admin/SeqMule-master/bin/secondary/../../version' from http://www.openbioinformatics.org/seqmule. Bad Internet connection?
NOTICE: unable to check for updates. Results will not be affected.
Extracting variants based on specified BED file.
Extracting variants based on specified BED file.
NOTICE: unable to download '/work/rashidma/software/seqmule_by_admin/SeqMule-master/bin/secondary/../../version' from http://www.openbioinformatics.org/seqmule. Bad Internet connection?
NOTICE: unable to check for updates. Results will not be affected.
Extracting variants based on specified BED file.
NOTICE: 39130 extracted variants written to NIST7086_result/NIST7086_bwa.merge.realn.0_gatk_hc.multi-call.extract.vcf
NOTICE: 53917 extracted variants written to NIST7035_result/NIST7035_bwa.merge.realn.0_gatk_hc.multi-call.extract.vcf
"NOTICE: Multiple samples in VCF, please specify sample name (-s) if you only need stats from a specific sample (does not influence Mendelian stats)."
Generating variant stats
"NOTICE: Multiple samples in VCF, please specify sample name (-s) if you only need stats from a specific sample (does not influence Mendelian stats)."
Generating variant stats
NOTICE: unable to download '/work/rashidma/software/seqmule_by_admin/SeqMule-master/bin/secondary/../../version' from http://www.openbioinformatics.org/seqmule. Bad Internet connection?
NOTICE: unable to check for updates. Results will not be affected.
NOTICE: 53917 extracted variants written to NIST7086_result/NIST7086_bwa.merge.realn.0_gatk_hc.multi-call.extract.vcf
NOTICE: Calculating coverage for NIST7035_result/NIST7035_bwa.merge.bam. This takes a while
NOTICE: checking contig(chromosome) name consistency in nexterarapidcapture_expandedexome_targetedregions.nooverlap.bed and NIST7035_result/NIST7035_bwa.merge.bam.
"NOTICE: Multiple samples in VCF, please specify sample name (-s) if you only need stats from a specific sample (does not influence Mendelian stats)."
Generating variant stats
NOTICE: unable to download '/work/rashidma/software/seqmule_by_admin/SeqMule-master/bin/secondary/../../version' from http://www.openbioinformatics.org/seqmule. Bad Internet connection?
NOTICE: unable to check for updates. Results will not be affected.
NOTICE: Calculating coverage for NIST7086_result/NIST7086_bwa.merge.bam. This takes a while
NOTICE: checking contig(chromosome) name consistency in nexterarapidcapture_expandedexome_targetedregions.nooverlap.bed and NIST7086_result/NIST7086_bwa.merge.bam.
NOTICE: all chromosome names in BED can be found in BAM header.
NOTICE: all chromosome names in BED can be found in BAM header.
NOTICE: Calculating coverage ...
NOTICE: Start reading from SAMTools depth command ...
NOTICE: Calculating coverage ...
NOTICE: Start reading from SAMTools depth command ...
"Cannot read output from vcf-stats: Wrong number of fieldsin /tmp/14047.filtered.vcf; expected 11, got 8. The offending line was:"
[chr15 43762196 . C T 1803.44 PASS AC=2;AF=0.500;AN=4;BaseQRankSum=0.165;ClippingRankSum=0.563;DP=117;FS=2.451;MLEAC]

at /work/rashidma/software/VCFTOOLS/vcftools-vcftools-4a4e953/src/perl/Vcf.pm line 172
"Vcf::throw('VcfStats=HASH(0x80a6230)', 'Wrong number of fieldsin /tmp/14047.filtered.vcf; expected 11...') called at /work/rashidma/software/VCFTOOLS/vcftools-vcftools-4a4e953/src/perl/Vcf.pm line 506"
VcfReader::next_data_hash('VcfStats=HASH(0x80a6230)') called at /work/rashidma/software/seqmule_by_admin/SeqMule-master/exe/vcftools/bin/vcf-stats line 153
main::vcf_stats('HASH(0x7c8d430)') called at /work/rashidma/software/seqmule_by_admin/SeqMule-master/exe/vcftools/bin/vcf-stats line 12

----------ERROR----------
"[ => SeqMule Execution Status: step 31 FAILED at Mon Oct 24 00:14:17 AST 2016, Extract variants in custom regions]"
ERROR: command failed
"/work/rashidma/software/seqmule_by_admin/SeqMule-master/bin/secondary/../../bin/secondary/worker /work/rashidma/Exome_data/GIAB_NIST_exome_data/MyAnalysis/04_seqmule_23-10-2016/seqmule.10232016.18305.logs 31 ""/work/rashidma/software/seqmule_by_admin/SeqMule-master/bin/secondary/../../bin/seqmule stats -tmpdir /tmp -capture nexterarapidcapture_expandedexome_targetedregions.nooverlap.bed -vcf NIST7086_result/NIST7086_bwa.merge.realn.0_gatk_hc.multi-call.vcf -p NIST7086_result/NIST7086_bwa.merge.realn.0_gatk_hc.multi-call"""
!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
"After fixing the problem, please execute 'cd /work/rashidma/Exome_data/GIAB_NIST_exome_data/MyAnalysis/04_seqmule_23-10-2016' and 'seqmule run NIST7035-NIST7086.script' to resume analysis."
!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!

real 444m46.571s
user 6m54.889s
sys 5m14.296s
cut: write error: Broken pipe
cut: write error: Broken pipe

yunfeiguo commented 7 years ago

VQSR is a better filtering method than hard filters. You can disable it in advanced_config file. Look for forceSNPHardFilter and forceINDELHardFilter.

You are right about vcf module loading. I will fix it asap when I got time. For now you can try hard code the seqmule's vcf module into seqmule script.

On Wed, Oct 26, 2016 at 8:51 AM, rashidma notifications@github.com wrote:

Hi Yunfei, Is VQSR important? How should I disable it in order to get seqmule running?

The chromosome name is consistent in bed file and reference file. Please see the log message of seqmule below-- [

_NOTICE: checking contig(chromosome) name consistency in /work/rashidma/Databases/hg19/UCSC/ucsc.hg19.fa and /work/rashidma/Exome_data/GIAB_NIST_exomedata/nexterarapidcapture expandedexometargetedregions.bed.NOTICE: use --no-check-chr to skip chromosome name checking] I already sent you the log file in last email. And also the last portion of stderr file. I am sending you both again.

I realised that seqmule is reading VCF.pm from another installations of VCFTOOLS outside the SEQMULE home. This might be a reason of failure. What do you think?? Please see the error-log file attached.

Many thanks for your time.

Mamoon

On Tue, Oct 25, 2016 at 10:55 PM, Yunfei Guo notifications@github.com wrote:

Hi @rashidma https://github.com/rashidma,

Thanks for using SeqMule!

GATK VQSR will not work well for a number of variants (e.g. in a small region) because it requires a relatively large sample for sampling.

Regarding variant extraction, is your BED file compatible with UCSC hg19, i.e. has a subset of UCSC hg19 chromosomal names? Also I cannot find the log file.

Best, Yunfei

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Mamoon Rashid, PhD *Post-Doctoral Fellow Marine Microbial Ecology Lab Red Sea Research Center 4700 King Abdullah University of Science and Technology (KAUST) Room 2217-WS03, Ibn Al-Haytham Building (2) 4700 King Abdullah University of Science and Technology* Thuwal 23955-6900, Kingdom of Saudi Arabia Office: +966 (0) 2 808-2671

"WARN 00:12:53,217 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum " "WARN 00:12:53,223 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum " "WARN 00:12:53,224 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum " "WARN 00:12:53,230 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum " "WARN 00:12:53,230 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum " "WARN 00:12:53,230 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum " "WARN 00:12:53,230 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum " "WARN 00:12:53,231 Interpreter - ![0,14]: 'ReadPosRankSum < -20.0;' undefined variable ReadPosRankSum " "INFO 00:12:53,401 ProgressMeter - done 13350.0 15.0 s 19.2 m 100.0% 15.0 s 0.0 s " "INFO 00:12:53,401 ProgressMeter - Total runtime 15.37 secs, 0.26 min, 0.00 hours " "INFO 00:13:05,865 HelpFormatter - ------------------------------ -------------------------------------------------- " "INFO 00:13:05,868 HelpFormatter - The Genome Analysis Toolkit (GATK) v3.4-0-g7e26428, Compiled 2015/05/15 03:25:41 " "INFO 00:13:05,868 HelpFormatter - Copyright (c) 2010 The Broad Institute " "INFO 00:13:05,868 HelpFormatter - For support and documentation go to http://www.broadinstitute.org/gatk " "INFO 00:13:05,872 HelpFormatter - Program Args: -T CombineVariants -R /work/rashidma/Databases/hg19/UCSC/ucsc.hg19.fa --genotypemergeoption UNSORTED --variant /tmp/10260.298029492.gatklite.tmp_gatklite.snp.filter.vcf --variant /tmp/10260.298029492.gatklite.tmp_gatklite.indel.filter.vcf -o NIST7035_result/NIST7035_bwa.merge.realn.0_gatk_hc.multi-call.vcf -L nexterarapidcapture_expandedexome_targetedregions.nooverlap.bed " "INFO 00:13:05,875 HelpFormatter - Executing as rashidma@krplpslcn005 on Linux 2.6.18-164.el5 amd64; Java HotSpot(TM) 64-Bit Server VM 1.7.0_80-b15. " "INFO 00:13:05,875 HelpFormatter - Date/Time: 2016/10/24 00:13:05 " "INFO 00:13:05,875 HelpFormatter - ------------------------------ -------------------------------------------------- " "INFO 00:13:05,875 HelpFormatter - ------------------------------ -------------------------------------------------- " "INFO 00:13:06,376 GenomeAnalysisEngine - Strictness is SILENT " "INFO 00:13:06,534 GenomeAnalysisEngine - Downsampling Settings: Method: BY_SAMPLE, Target Coverage: 1000 " "INFO 00:13:07,179 IntervalUtils - Processing 62085286 bp from intervals " "INFO 00:13:07,269 GenomeAnalysisEngine - Preparing for traversal " "INFO 00:13:07,307 GenomeAnalysisEngine - Done preparing for traversal " "INFO 00:13:07,307 ProgressMeter - [INITIALIZATION COMPLETE; STARTING PROCESSING] " "INFO 00:13:07,308 ProgressMeter - | processed | time | per 1M | | total | remaining " "INFO 00:13:07,308 ProgressMeter - Location | sites | elapsed | sites | completed | runtime | runtime " "INFO 00:13:37,341 ProgressMeter - chr19:49973784 57703.0 30.0 s 8.7 m 95.4% 31.0 s 1.0 s " "INFO 00:13:39,369 ProgressMeter - done 61073.0 32.0 s 8.7 m 100.0% 32.0 s 0.0 s " "INFO 00:13:39,370 ProgressMeter - Total runtime 32.06 secs, 0.53 min, 0.01 hours "

----------NOTICE---------- "[ => SeqMule Execution Status: Running 30 of 41 steps: Copy results to other samples, at Mon Oct 24 00:13:50 AST 2016, Time Elapsed: 7 hr 23 min 36 s]" "[ => SeqMule Execution Status: step 29 is finished at Mon Oct 24 00:13:50 AST 2016, gatk_hc.multi-call variant filtering]"

----------NOTICE---------- "[ => SeqMule Execution Status: Running 31 of 41 steps: Extract variants in custom regions, at Mon Oct 24 00:13:52 AST 2016, Time Elapsed: 7 hr 23 min 38 s]" "[ => SeqMule Execution Status: step 30 is finished at Mon Oct 24 00:13:52 AST 2016, Copy results to other samples]"

Current version: 1.2.5 Check for upgrade ...

----------NOTICE---------- "[ => SeqMule Execution Status: Running 32 of 41 steps: Extract variants in custom regions, at Mon Oct 24 00:13:53 AST 2016, Time Elapsed: 7 hr 23 min 39 s]" Current version: 1.2.5 Check for upgrade ...

----------NOTICE---------- "[ => SeqMule Execution Status: Running 33 of 41 steps: Extract variants in custom regions, at Mon Oct 24 00:13:54 AST 2016, Time Elapsed: 7 hr 23 min 40 s]" Current version: 1.2.5 Check for upgrade ...

----------NOTICE---------- "[ => SeqMule Execution Status: Running 35 of 41 steps: Generate alignment and coverage stat, at Mon Oct 24 00:13:55 AST 2016, Time Elapsed: 7 hr 23 min 41 s]" Current version: 1.2.5 Check for upgrade ...

----------NOTICE---------- "[ => SeqMule Execution Status: Running 38 of 41 steps: Generate alignment and coverage stat, at Mon Oct 24 00:13:56 AST 2016, Time Elapsed: 7 hr 23 min 42 s]" Current version: 1.2.5 Check for upgrade ... NOTICE: unable to download '/work/rashidma/software/ seqmule_by_admin/SeqMule-master/bin/secondary/../../version' from http://www.openbioinformatics.org/seqmule. Bad Internet connection? NOTICE: unable to check for updates. Results will not be affected. NOTICE: unable to download '/work/rashidma/software/ seqmule_by_admin/SeqMule-master/bin/secondary/../../version' from http://www.openbioinformatics.org/seqmule. Bad Internet connection? NOTICE: unable to check for updates. Results will not be affected. Extracting variants based on specified BED file. Extracting variants based on specified BED file. NOTICE: unable to download '/work/rashidma/software/ seqmule_by_admin/SeqMule-master/bin/secondary/../../version' from http://www.openbioinformatics.org/seqmule. Bad Internet connection? NOTICE: unable to check for updates. Results will not be affected. Extracting variants based on specified BED file. NOTICE: 39130 extracted variants written to NIST7086_result/NIST7086_bwa. merge.realn.0_gatk_hc.multi-call.extract.vcf NOTICE: 53917 extracted variants written to NIST7035_result/NIST7035_bwa. merge.realn.0_gatk_hc.multi-call.extract.vcf "NOTICE: Multiple samples in VCF, please specify sample name (-s) if you only need stats from a specific sample (does not influence Mendelian stats)." Generating variant stats "NOTICE: Multiple samples in VCF, please specify sample name (-s) if you only need stats from a specific sample (does not influence Mendelian stats)." Generating variant stats NOTICE: unable to download '/work/rashidma/software/ seqmule_by_admin/SeqMule-master/bin/secondary/../../version' from http://www.openbioinformatics.org/seqmule. Bad Internet connection? NOTICE: unable to check for updates. Results will not be affected. NOTICE: 53917 extracted variants written to NIST7086_result/NIST7086_bwa. merge.realn.0_gatk_hc.multi-call.extract.vcf NOTICE: Calculating coverage for NIST7035_result/NIST7035_bwa.merge.bam. This takes a while NOTICE: checking contig(chromosome) name consistency in nexterarapidcapture_expandedexome_targetedregions.nooverlap.bed and NIST7035_result/NIST7035_bwa.merge.bam. "NOTICE: Multiple samples in VCF, please specify sample name (-s) if you only need stats from a specific sample (does not influence Mendelian stats)." Generating variant stats NOTICE: unable to download '/work/rashidma/software/ seqmule_by_admin/SeqMule-master/bin/secondary/../../version' from http://www.openbioinformatics.org/seqmule. Bad Internet connection? NOTICE: unable to check for updates. Results will not be affected. NOTICE: Calculating coverage for NIST7086_result/NIST7086_bwa.merge.bam. This takes a while NOTICE: checking contig(chromosome) name consistency in nexterarapidcapture_expandedexome_targetedregions.nooverlap.bed and NIST7086_result/NIST7086_bwa.merge.bam. NOTICE: all chromosome names in BED can be found in BAM header. NOTICE: all chromosome names in BED can be found in BAM header. NOTICE: Calculating coverage ... NOTICE: Start reading from SAMTools depth command ... NOTICE: Calculating coverage ... NOTICE: Start reading from SAMTools depth command ... "Cannot read output from vcf-stats: Wrong number of fieldsin /tmp/14047.filtered.vcf; expected 11, got 8. The offending line was:" [chr15 43762196 . C T 1803.44 PASS AC=2;AF=0.500;AN=4;BaseQRankSum=0.165; ClippingRankSum=0.563;DP=117;FS=2.451;MLEAC]

at /work/rashidma/software/VCFTOOLS/vcftools-vcftools-4a4e953/src/perl/Vcf.pm line 172 "Vcf::throw('VcfStats=HASH(0x80a6230)', 'Wrong number of fieldsin /tmp/14047.filtered.vcf; expected 11...') called at /work/rashidma/software/ VCFTOOLS/vcftools-vcftools-4a4e953/src/perl/Vcf.pm line 506" VcfReader::next_data_hash('VcfStats=HASH(0x80a6230)') called at /work/rashidma/software/seqmule_by_admin/SeqMule- master/exe/vcftools/bin/vcf-stats line 153 main::vcf_stats('HASH(0x7c8d430)') called at /work/rashidma/software/ seqmule_by_admin/SeqMule-master/exe/vcftools/bin/vcf-stats line 12

----------ERROR---------- "[ => SeqMule Execution Status: step 31 FAILED at Mon Oct 24 00:14:17 AST 2016, Extract variants in custom regions]" ERROR: command failed "/work/rashidma/software/seqmule_by_admin/SeqMule- master/bin/secondary/../../bin/secondary/worker /work/rashidma/Exome_data/ GIAB_NIST_exome_data/MyAnalysis/04_seqmule_23-10- 2016/seqmule.10232016.18305.logs 31 ""/work/rashidma/software/ seqmule_by_admin/SeqMule-master/bin/secondary/../../bin/seqmule stats -tmpdir /tmp -capture nexterarapidcapture_expandedexome_targetedregions.nooverlap.bed -vcf NIST7086_result/NIST7086_bwa.merge.realn.0_gatk_hc.multi-call.vcf -p NIST7086_result/NIST7086_bwa.merge.realn.0_gatk_hc.multi-call""" !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! "After fixing the problem, please execute 'cd /work/rashidma/Exome_data/ GIAB_NIST_exome_data/MyAnalysis/04_seqmule_23-10-2016' and 'seqmule run NIST7035-NIST7086.script' to resume analysis." !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!

real 444m46.571s user 6m54.889s sys 5m14.296s cut: write error: Broken pipe cut: write error: Broken pipe

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