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sdparekh / zUMIs

zUMIs: A fast and flexible pipeline to process RNA sequencing data with UMIs
GNU General Public License v3.0
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Error in Rsubread #113

Closed trueth1206 closed 5 years ago

trueth1206 commented 5 years ago

Hi, Thank you for developing tools for RNA sequencing. We are using zUMI to analyze published datasets. But we've got an error. Could you help us for trouble shooting? Thank you~

The error content is as following:

Error in Rsubread::featureCounts(files = abamfile, annot.ext = saf, isGTFAnnotationFile = F, : No counts were generated. Calls: .runFeatureCount -> Execution halted During startup - Warning message: Error in gzfile(file, "rb") : cannot open the connection Calls: readRDS -> gzfile In addition: Warning message: In gzfile(file, "rb") : cannot open compressed file '/scratch/c.c1885111/analysis/an0223/output/SRR7789673/zUMIs_output/ex pression/Example.dgecounts.rds', probable reason 'No such file or directory' Execution halted

cziegenhain commented 5 years ago

Hi,

To troubleshoot, we would need a bit more information on your issue. Please send us

trueth1206 commented 5 years ago

Thank you very much for your reply.

We use data from this study : https://www.ncbi.nlm.nih.gov/sra?term=SRX4644529

The yaml file: project: Example sequence_files: file1: name: /scratch/c.c1885111/analysis/an0223//input/SRR7789673_1.fastq.gz base_definition:

The output log

You provided these parameters: YAML file: /scratch/c.c1885111/analysis/an0223/bin/../resources/SRR7789673.yaml zUMIs directory: /apps/genomics/zUMIs/2.3.0/zUMIs STAR executable STAR samtools executable samtools pigz executable pigz Rscript executable Rscript RAM limit: 20 zUMIs version 2.3.1

Filtering... Mapping... Apr 01 00:53:07 ..... started STAR run Apr 01 00:53:07 ..... loading genome Apr 01 00:53:48 ..... processing annotations GTF Counting... [1] "2019-04-01 00:54:12 BST" [1] "15324 barcodes detected." [1] "30716572 reads were assigned to barcodes that do not correspond to intact cells." [1] "9e+07 Reads per chunk" [1] "Loading reference annotation from:" [1] "/scratch/c.c1885111/analysis/an0223/resources/Mus_musculus.GRCm38.84.gtf" [1] "Annotation loaded!" [1] "Assigning reads to features (ex)"

ERROR: invalid parameter: '/scratch/c.c1885111/analysis/an0223/output/SRR7789673/Example.filtered.ta gged.Aligned.out.bam'

cziegenhain commented 5 years ago

Hey,

Thanks for the additional information. can you tell me which R version and Rsubread version you are using? Also from the log it seems like STAR did not continue further than processing GTF annotations, can you confirm that STAR actually ran through? It seems like the issue is there.

trueth1206 commented 5 years ago

Hi,

Thank you for your reply. We use Rsubread 1.32.4 and R 3.5.1. Can the version of them be an issue?

Thank you~

cziegenhain commented 5 years ago

Hey,

Those versions look good & should not produce any problems. Did you look at the log STAR produces (see above) and check for the mapping?

trueth1206 commented 5 years ago

Hi,

How can I know the STAR is actually ran through ?

I got these files/directories in my output directory: Example.BCstats.txt
Example.filtered.tagged._STARgenome Example.filtered.tagged._STARtmp
zUMIs_runlog.txt
Example.postmap.yaml Example.filtered.tagged.Log.progress.out
Rplots.pdf

And these files are empty in output directory: Example.filtered.tagged.Aligned.out.bam
Example.filtered.tagged.Log.out
zUMIs_output

And do you have any idea about why STAR did not work ? We are really confusing... Thank you very much~

cziegenhain commented 5 years ago

whats the file size of Example.filtered.tagged.Aligned.out.bam please also upload Example.filtered.tagged.Log.out for me to look at. Normally there should be a file as such Example.filtered.tagged.Log.final.out if the run completed.

trueth1206 commented 5 years ago

Hi, Thank you very much for spending your time for us.

The file size of Example.filtered.tagged.Aligned.out.bam is zero. We don't have anything in the file.

Here is the Example.filtered.tagged.Log.out.

STAR version=STAR_2.7.0a STAR compilation time,server,dir=Fri 8 Mar 12:03:41 GMT 2019 cl2:/tmp/software.builder/genomics/STAR /2.7.0a/el7/AVX512/gnu-8.1/STAR-2.7.0a/source

DEFAULT parameters:

versionSTAR 20201 versionGenome 20101 20200
parametersFiles -
sysShell - runMode alignReads runThreadN 1 runDirPerm User_RWX runRNGseed 777 genomeDir ./GenomeDir/ genomeLoad NoSharedMemory genomeFastaFiles -
genomeChainFiles -
genomeSAindexNbases 14 genomeChrBinNbits 18 genomeSAsparseD 1 genomeSuffixLengthMax 18446744073709551615 genomeFileSizes 0
genomeConsensusFile - readFilesType Fastx
readFilesIn Read1 Read2
readFilesPrefix - readFilesCommand -
readMatesLengthsIn NotEqual readMapNumber 18446744073709551615 readNameSeparator /
readStrand Unstranded inputBAMfile - bamRemoveDuplicatesType - bamRemoveDuplicatesMate2basesN 0 limitGenomeGenerateRAM 31000000000 limitIObufferSize 150000000 limitOutSAMoneReadBytes 100000 limitOutSJcollapsed 1000000 limitOutSJoneRead 1000 limitBAMsortRAM 0 limitSjdbInsertNsj 1000000 limitNreadsSoft 18446744073709551615 outTmpDir - outTmpKeep None outStd Log outReadsUnmapped None outQSconversionAdd 0 outMultimapperOrder Old_2.4 outSAMtype SAM
outSAMmode Full outSAMstrandField None outSAMattributes Standard
outSAMunmapped None
outSAMorder Paired outSAMprimaryFlag OneBestScore outSAMreadID Standard outSAMmapqUnique 255 outSAMflagOR 0 outSAMflagAND 65535 outSAMattrRGline -
outSAMheaderHD -
outSAMheaderPG -
outSAMheaderCommentFile - outBAMcompression 1 outBAMsortingThreadN 0 outBAMsortingBinsN 50 outSAMfilter None
outSAMmultNmax 18446744073709551615 outSAMattrIHstart 1 outSAMtlen 1 outSJfilterReads All outSJfilterCountUniqueMin 3 1 1 1
outSJfilterCountTotalMin 3 1 1 1
outSJfilterOverhangMin 30 12 12 12
outSJfilterDistToOtherSJmin 10 0 5 10
outSJfilterIntronMaxVsReadN 50000 100000 200000
outWigType None
outWigStrand Stranded
outWigReferencesPrefix - outWigNorm RPM
outFilterType Normal outFilterMultimapNmax 10 outFilterMultimapScoreRange 1 outFilterScoreMin 0 outFilterScoreMinOverLread 0.66 outFilterMatchNmin 0 outFilterMatchNminOverLread 0.66 outFilterMismatchNmax 10 outFilterMismatchNoverLmax 0.3 outFilterMismatchNoverReadLmax 1 outFilterIntronMotifs None outFilterIntronStrands RemoveInconsistentStrands clip5pNbases 0
clip3pNbases 0
clip3pAfterAdapterNbases 0
clip3pAdapterSeq -
clip3pAdapterMMp 0.1
winBinNbits 16 winAnchorDistNbins 9 winFlankNbins 4 winAnchorMultimapNmax 50 winReadCoverageRelativeMin 0.5 winReadCoverageBasesMin 0 scoreGap 0 scoreGapNoncan -8 scoreGapGCAG -4 scoreGapATAC -8 scoreStitchSJshift 1 scoreGenomicLengthLog2scale -0.25 scoreDelBase -2 scoreDelOpen -2 scoreInsOpen -2 scoreInsBase -2 seedSearchLmax 0 seedSearchStartLmax 50 seedSearchStartLmaxOverLread 1 seedPerReadNmax 1000 seedPerWindowNmax 50 seedNoneLociPerWindow 10 seedMultimapNmax 10000 seedSplitMin 12 alignIntronMin 21 alignIntronMax 0 alignMatesGapMax 0 alignTranscriptsPerReadNmax 10000 alignSJoverhangMin 5 alignSJDBoverhangMin 3 alignSJstitchMismatchNmax 0 -1 0 0
alignSplicedMateMapLmin 0 alignSplicedMateMapLminOverLmate 0.66 alignWindowsPerReadNmax 10000 alignTranscriptsPerWindowNmax 100 alignEndsType Local alignSoftClipAtReferenceEnds Yes alignEndsProtrude 0 ConcordantPair
alignInsertionFlush None peOverlapNbasesMin 0 peOverlapMMp 0.01 chimSegmentMin 0 chimScoreMin 0 chimScoreDropMax 20 chimScoreSeparation 10 chimScoreJunctionNonGTAG -1 chimMainSegmentMultNmax 10 chimJunctionOverhangMin 20 chimOutType Junctions
chimFilter banGenomicN
chimSegmentReadGapMax 0 chimMultimapNmax 0 chimMultimapScoreRange 1 chimNonchimScoreDropMin 20 chimOutJunctionFormat 0
sjdbFileChrStartEnd -
sjdbGTFfile - sjdbGTFchrPrefix - sjdbGTFfeatureExon exon sjdbGTFtagExonParentTranscript transcript_id sjdbGTFtagExonParentGene gene_id sjdbOverhang 100 sjdbScore 2 sjdbInsertSave Basic varVCFfile - waspOutputMode None quantMode -
quantTranscriptomeBAMcompression 1 quantTranscriptomeBan IndelSoftclipSingleend twopass1readsN 18446744073709551615 twopassMode None soloType None soloCBstart 1 soloUMIstart 17 soloCBlen 16 soloUMIlen 10 soloCBwhitelist - soloStrand Forward soloOutFileNames Solo.out/ genes.tsv barcodes.tsv matrix.mtx matrixSJ.mtx

soloFeatures Gene
soloUMIdedup 1MM_All

Command Line:

STAR --readFilesCommand samtools view -@2 --outSAMmultNmax 1 --outFilterMultimapNmax 50 --outSAMunma pped Within --outSAMtype BAM Unsorted --genomeDir /scratch/c.c1885111/analysis/an0223/resources --sj dbGTFfile /scratch/c.c1885111/analysis/an0223/resources/Mus_musculus.GRCm38.84.gtf --runThreadN 8 -- readFilesIn /scratch/c.c1885111/analysis/an0223/output/SRR7789673/zUMIs_output/.tmpMerge//Example.aa .filtered.tagged.bam,/scratch/c.c1885111/analysis/an0223/output/SRR7789673/zUMIs_output/.tmpMerge//E xample.ab.filtered.tagged.bam,/scratch/c.c1885111/analysis/an0223/output/SRR7789673/zUMIs_output/.tm pMerge//Example.ac.filtered.tagged.bam,/scratch/c.c1885111/analysis/an0223/output/SRR7789673/zUMIs_o utput/.tmpMerge//Example.ad.filtered.tagged.bam,/scratch/c.c1885111/analysis/an0223/output/SRR778967 3/zUMIs_output/.tmpMerge//Example.ae.filtered.tagged.bam,/scratch/c.c1885111/analysis/an0223/output/ SRR7789673/zUMIs_output/.tmpMerge//Example.af.filtered.tagged.bam,/scratch/c.c1885111/analysis/an022 3/output/SRR7789673/zUMIs_output/.tmpMerge//Example.ag.filtered.tagged.bam,/scratch/c.c1885111/analy sis/an0223/output/SRR7789673/zUMIs_output/.tmpMerge//Example.ah.filtered.tagged.bam --outFileNamePre fix /scratch/c.c1885111/analysis/an0223/output/SRR7789673/Example.filtered.tagged. --sjdbOverhang 97 --readFilesType SAM SE

Initial USER parameters from Command Line:

outFileNamePrefix /scratch/c.c1885111/analysis/an0223/output/SRR7789673/Example.filt ered.tagged.

All USER parameters from Command Line:

readFilesCommand samtools view -@2 ~RE-DEFINED outSAMmultNmax 1 ~RE-DEFINED outFilterMultimapNmax 50 ~RE-DEFINED outSAMunmapped Within ~RE-DEFINED outSAMtype BAM Unsorted ~RE-DEFINED genomeDir /scratch/c.c1885111/analysis/an0223/resources ~RE-DEFINED sjdbGTFfile /scratch/c.c1885111/analysis/an0223/resources/Mus_musculus.GRCm38.84.g tf ~RE-DEFINED runThreadN 8 ~RE-DEFINED readFilesIn /scratch/c.c1885111/analysis/an0223/output/SRR7789673/zUMIs_output/.tm pMerge//Example.aa.filtered.tagged.bam,/scratch/c.c1885111/analysis/an0223/output/SRR7789673/zUMIs_o utput/.tmpMerge//Example.ab.filtered.tagged.bam,/scratch/c.c1885111/analysis/an0223/output/SRR778967 3/zUMIs_output/.tmpMerge//Example.ac.filtered.tagged.bam,/scratch/c.c1885111/analysis/an0223/output/ SRR7789673/zUMIs_output/.tmpMerge//Example.ad.filtered.tagged.bam,/scratch/c.c1885111/analysis/an022 3/output/SRR7789673/zUMIs_output/.tmpMerge//Example.ae.filtered.tagged.bam,/scratch/c.c1885111/analy sis/an0223/output/SRR7789673/zUMIs_output/.tmpMerge//Example.af.filtered.tagged.bam,/scratch/c.c1885 111/analysis/an0223/output/SRR7789673/zUMIs_output/.tmpMerge//Example.ag.filtered.tagged.bam,/scratc h/c.c1885111/analysis/an0223/output/SRR7789673/zUMIs_output/.tmpMerge//Example.ah.filtered.tagged.ba m ~RE-DEFINED outFileNamePrefix /scratch/c.c1885111/analysis/an0223/output/SRR7789673/Example.filtered .tagged. ~RE-DEFINED sjdbOverhang 97 ~RE-DEFINED readFilesType SAM SE ~RE-DEFINED

Finished reading parameters from all sources
Final user re-defined parameters-----------------:

runThreadN 8 genomeDir /scratch/c.c1885111/analysis/an0223/resources readFilesType SAM SE
readFilesIn /scratch/c.c1885111/analysis/an0223/output/SRR7789673/zUMIs_output /.tmpMerge//Example.aa.filtered.tagged.bam,/scratch/c.c1885111/analysis/an0223/output/SRR7789673/zUM Is_output/.tmpMerge//Example.ab.filtered.tagged.bam,/scratch/c.c1885111/analysis/an0223/output/SRR77 89673/zUMIs_output/.tmpMerge//Example.ac.filtered.tagged.bam,/scratch/c.c1885111/analysis/an0223/out put/SRR7789673/zUMIs_output/.tmpMerge//Example.ad.filtered.tagged.bam,/scratch/c.c1885111/analysis/a n0223/output/SRR7789673/zUMIs_output/.tmpMerge//Example.ae.filtered.tagged.bam,/scratch/c.c1885111/a nalysis/an0223/output/SRR7789673/zUMIs_output/.tmpMerge//Example.af.filtered.tagged.bam,/scratch/c.c 1885111/analysis/an0223/output/SRR7789673/zUMIs_output/.tmpMerge//Example.ag.filtered.tagged.bam,/sc ratch/c.c1885111/analysis/an0223/output/SRR7789673/zUMIs_output/.tmpMerge//Example.ah.filtered.tagge d.bam
readFilesCommand samtools view -@2
outFileNamePrefix /scratch/c.c1885111/analysis/an0223/output/SRR7789673/Example.filt ered.tagged. outSAMtype BAM Unsorted
outSAMunmapped Within
outSAMmultNmax 1 outFilterMultimapNmax 50 sjdbGTFfile /scratch/c.c1885111/analysis/an0223/resources/Mus_musculus.GRCm38. 84.gtf sjdbOverhang 97

Final effective command line:

STAR --runThreadN 8 --genomeDir /scratch/c.c1885111/analysis/an0223/resources --readFilesType SAM SE --readFilesIn /scratch/c.c1885111/analysis/an0223/output/SRR7789673/zUMIs_output/.tmpM erge//Example.aa.filtered.tagged.bam,/scratch/c.c1885111/analysis/an0223/output/SRR7789673/zUMIs_out put/.tmpMerge//Example.ab.filtered.tagged.bam,/scratch/c.c1885111/analysis/an0223/output/SRR7789673/ zUMIs_output/.tmpMerge//Example.ac.filtered.tagged.bam,/scratch/c.c1885111/analysis/an0223/output/SR R7789673/zUMIs_output/.tmpMerge//Example.ad.filtered.tagged.bam,/scratch/c.c1885111/analysis/an0223/ output/SRR7789673/zUMIs_output/.tmpMerge//Example.ae.filtered.tagged.bam,/scratch/c.c1885111/analysi s/an0223/output/SRR7789673/zUMIs_output/.tmpMerge//Example.af.filtered.tagged.bam,/scratch/c.c188511 1/analysis/an0223/output/SRR7789673/zUMIs_output/.tmpMerge//Example.ag.filtered.tagged.bam,/scratch/ c.c1885111/analysis/an0223/output/SRR7789673/zUMIs_output/.tmpMerge//Example.ah.filtered.tagged.bam --readFilesCommand samtools view -@2 --outFileNamePrefix /scratch/c.c1885111/analysis/ an0223/output/SRR7789673/Example.filtered.tagged. --outSAMtype BAM Unsorted --outSAMunmappe d Within --outSAMmultNmax 1 --outFilterMultimapNmax 50 --sjdbGTFfile /scratch/c.c1885111/an alysis/an0223/resources/Mus_musculus.GRCm38.84.gtf --sjdbOverhang 97

Final parameters after user input--------------------------------:

versionSTAR 20201 versionGenome 20101 20200
parametersFiles -
sysShell - runMode alignReads runThreadN 8 runDirPerm User_RWX runRNGseed 777 genomeDir /scratch/c.c1885111/analysis/an0223/resources genomeLoad NoSharedMemory genomeFastaFiles -
genomeChainFiles -
genomeSAindexNbases 14 genomeChrBinNbits 18 genomeSAsparseD 1 genomeSuffixLengthMax 18446744073709551615 genomeFileSizes 0
genomeConsensusFile - readFilesType SAM SE
readFilesIn /scratch/c.c1885111/analysis/an0223/output/SRR7789673/zUMIs_output /.tmpMerge//Example.aa.filtered.tagged.bam,/scratch/c.c1885111/analysis/an0223/output/SRR7789673/zUM Is_output/.tmpMerge//Example.ab.filtered.tagged.bam,/scratch/c.c1885111/analysis/an0223/output/SRR77 89673/zUMIs_output/.tmpMerge//Example.ac.filtered.tagged.bam,/scratch/c.c1885111/analysis/an0223/out put/SRR7789673/zUMIs_output/.tmpMerge//Example.ad.filtered.tagged.bam,/scratch/c.c1885111/analysis/a n0223/output/SRR7789673/zUMIs_output/.tmpMerge//Example.ae.filtered.tagged.bam,/scratch/c.c1885111/a nalysis/an0223/output/SRR7789673/zUMIs_output/.tmpMerge//Example.af.filtered.tagged.bam,/scratch/c.c 1885111/analysis/an0223/output/SRR7789673/zUMIs_output/.tmpMerge//Example.ag.filtered.tagged.bam,/sc ratch/c.c1885111/analysis/an0223/output/SRR7789673/zUMIs_output/.tmpMerge//Example.ah.filtered.tagge d.bam
readFilesPrefix - readFilesCommand samtools view -@2
readMatesLengthsIn NotEqual readMapNumber 18446744073709551615 readNameSeparator /
readStrand Unstranded inputBAMfile - bamRemoveDuplicatesType - bamRemoveDuplicatesMate2basesN 0 limitGenomeGenerateRAM 31000000000 limitIObufferSize 150000000 limitOutSAMoneReadBytes 100000 limitOutSJcollapsed 1000000 limitOutSJoneRead 1000 limitBAMsortRAM 0 limitSjdbInsertNsj 1000000 limitNreadsSoft 18446744073709551615 outFileNamePrefix /scratch/c.c1885111/analysis/an0223/output/SRR7789673/Example.filt ered.tagged. outTmpDir - outTmpKeep None outStd Log outReadsUnmapped None outQSconversionAdd 0 outMultimapperOrder Old_2.4 outSAMtype BAM Unsorted
outSAMmode Full outSAMstrandField None outSAMattributes Standard
outSAMunmapped Within
outSAMorder Paired outSAMprimaryFlag OneBestScore outSAMreadID Standard outSAMmapqUnique 255 outSAMflagOR 0 outSAMflagAND 65535 outSAMattrRGline -
outSAMheaderHD -
outSAMheaderPG -
outSAMheaderCommentFile - outBAMcompression 1 outBAMsortingThreadN 0 outBAMsortingBinsN 50 outSAMfilter None
outSAMmultNmax 1 outSAMattrIHstart 1 outSAMtlen 1 outSJfilterReads All outSJfilterCountUniqueMin 3 1 1 1
outSJfilterCountTotalMin 3 1 1 1
outSJfilterOverhangMin 30 12 12 12
outSJfilterDistToOtherSJmin 10 0 5 10
outSJfilterIntronMaxVsReadN 50000 100000 200000
outWigType None
outWigStrand Stranded
outWigReferencesPrefix - outWigNorm RPM
outFilterType Normal outFilterMultimapNmax 50 outFilterMultimapScoreRange 1 outFilterScoreMin 0 outFilterScoreMinOverLread 0.66 outFilterMatchNmin 0 outFilterMatchNminOverLread 0.66 outFilterMismatchNmax 10 outFilterMismatchNoverLmax 0.3 outFilterMismatchNoverReadLmax 1 outFilterIntronMotifs None outFilterIntronStrands RemoveInconsistentStrands clip5pNbases 0
clip3pNbases 0
clip3pAfterAdapterNbases 0
clip3pAdapterSeq -
clip3pAdapterMMp 0.1
winBinNbits 16 winAnchorDistNbins 9 winFlankNbins 4 winAnchorMultimapNmax 50 winReadCoverageRelativeMin 0.5 winReadCoverageBasesMin 0 scoreGap 0 scoreGapNoncan -8 scoreGapGCAG -4 scoreGapATAC -8 scoreStitchSJshift 1 scoreGenomicLengthLog2scale -0.25 scoreDelBase -2 scoreDelOpen -2 scoreInsOpen -2 scoreInsBase -2 seedSearchLmax 0 seedSearchStartLmax 50 seedSearchStartLmaxOverLread 1 seedPerReadNmax 1000 seedPerWindowNmax 50 seedNoneLociPerWindow 10 seedMultimapNmax 10000 seedSplitMin 12 alignIntronMin 21 alignIntronMax 0 alignMatesGapMax 0 alignTranscriptsPerReadNmax 10000 alignSJoverhangMin 5 alignSJDBoverhangMin 3 alignSJstitchMismatchNmax 0 -1 0 0
alignSplicedMateMapLmin 0 alignSplicedMateMapLminOverLmate 0.66 alignWindowsPerReadNmax 10000 alignTranscriptsPerWindowNmax 100 alignEndsType Local alignSoftClipAtReferenceEnds Yes alignEndsProtrude 0 ConcordantPair
alignInsertionFlush None peOverlapNbasesMin 0 peOverlapMMp 0.01 chimSegmentMin 0 chimScoreMin 0 chimScoreDropMax 20 chimScoreSeparation 10 chimScoreJunctionNonGTAG -1 chimMainSegmentMultNmax 10 chimJunctionOverhangMin 20 chimOutType Junctions
chimFilter banGenomicN
chimSegmentReadGapMax 0 chimMultimapNmax 0 chimMultimapScoreRange 1 chimNonchimScoreDropMin 20 chimOutJunctionFormat 0
sjdbFileChrStartEnd -
sjdbGTFfile /scratch/c.c1885111/analysis/an0223/resources/Mus_musculus.GRCm38. 84.gtf sjdbGTFchrPrefix - sjdbGTFfeatureExon exon sjdbGTFtagExonParentTranscript transcript_id sjdbGTFtagExonParentGene gene_id sjdbOverhang 97 sjdbScore 2 sjdbInsertSave Basic varVCFfile - waspOutputMode None quantMode -
quantTranscriptomeBAMcompression 1 quantTranscriptomeBan IndelSoftclipSingleend twopass1readsN 18446744073709551615 twopassMode None soloType None soloCBstart 1 soloUMIstart 17 soloCBlen 16 soloUMIlen 10 soloCBwhitelist - soloStrand Forward soloOutFileNames Solo.out/ genes.tsv barcodes.tsv matrix.mtx matrixSJ.mtx

soloFeatures Gene
soloUMIdedup 1MM_All

Input read files for mate 1, from input string /scratch/c.c1885111/analysis/an0223/output/SRR7789 673/zUMIs_output/.tmpMerge//Example.aa.filtered.tagged.bam,/scratch/c.c1885111/analysis/an0223/outpu t/SRR7789673/zUMIs_output/.tmpMerge//Example.ab.filtered.tagged.bam,/scratch/c.c1885111/analysis/an0 223/output/SRR7789673/zUMIs_output/.tmpMerge//Example.ac.filtered.tagged.bam,/scratch/c.c1885111/ana lysis/an0223/output/SRR7789673/zUMIs_output/.tmpMerge//Example.ad.filtered.tagged.bam,/scratch/c.c18 85111/analysis/an0223/output/SRR7789673/zUMIs_output/.tmpMerge//Example.ae.filtered.tagged.bam,/scra tch/c.c1885111/analysis/an0223/output/SRR7789673/zUMIs_output/.tmpMerge//Example.af.filtered.tagged. bam,/scratch/c.c1885111/analysis/an0223/output/SRR7789673/zUMIs_output/.tmpMerge//Example.ag.filtere d.tagged.bam,/scratch/c.c1885111/analysis/an0223/output/SRR7789673/zUMIs_output/.tmpMerge//Example.a h.filtered.tagged.bam -rw-rw-r-- 1 c.c1885111 c.c1885111 819755336 4月 1 10:58 /scratch/c.c1885111/analysis/an0223/outpu t/SRR7789673/zUMIs_output/.tmpMerge//Example.aa.filtered.tagged.bam -rw-rw-r-- 1 c.c1885111 c.c1885111 817636215 4月 1 10:58 /scratch/c.c1885111/analysis/an0223/outpu t/SRR7789673/zUMIs_output/.tmpMerge//Example.ab.filtered.tagged.bam -rw-rw-r-- 1 c.c1885111 c.c1885111 811795611 4月 1 10:58 /scratch/c.c1885111/analysis/an0223/outpu t/SRR7789673/zUMIs_output/.tmpMerge//Example.ac.filtered.tagged.bam -rw-rw-r-- 1 c.c1885111 c.c1885111 818098768 4月 1 10:58 /scratch/c.c1885111/analysis/an0223/outpu t/SRR7789673/zUMIs_output/.tmpMerge//Example.ad.filtered.tagged.bam -rw-rw-r-- 1 c.c1885111 c.c1885111 823167123 4月 1 10:58 /scratch/c.c1885111/analysis/an0223/outpu t/SRR7789673/zUMIs_output/.tmpMerge//Example.ae.filtered.tagged.bam -rw-rw-r-- 1 c.c1885111 c.c1885111 829456958 4月 1 10:58 /scratch/c.c1885111/analysis/an0223/outpu t/SRR7789673/zUMIs_output/.tmpMerge//Example.af.filtered.tagged.bam -rw-rw-r-- 1 c.c1885111 c.c1885111 813108214 4月 1 10:58 /scratch/c.c1885111/analysis/an0223/outpu t/SRR7789673/zUMIs_output/.tmpMerge//Example.ag.filtered.tagged.bam -rw-rw-r-- 1 c.c1885111 c.c1885111 821777547 4月 1 10:58 /scratch/c.c1885111/analysis/an0223/outpu t/SRR7789673/zUMIs_output/.tmpMerge//Example.ah.filtered.tagged.bam

readsCommandsFile: exec > "/scratch/c.c1885111/analysis/an0223/output/SRR7789673/Example.filtered.tagged._STARtmp/tmp.f ifo.read1" echo FILE 0 samtools view -@2 "/scratch/c.c1885111/analysis/an0223/output/SRR7789673/zUMIs_output/.tmpM erge//Example.aa.filtered.tagged.bam" echo FILE 1 samtools view -@2 "/scratch/c.c1885111/analysis/an0223/output/SRR7789673/zUMIs_output/.tmpM erge//Example.ab.filtered.tagged.bam" echo FILE 2 samtools view -@2 "/scratch/c.c1885111/analysis/an0223/output/SRR7789673/zUMIs_output/.tmpM erge//Example.ac.filtered.tagged.bam" echo FILE 3 samtools view -@2 "/scratch/c.c1885111/analysis/an0223/output/SRR7789673/zUMIs_output/.tmpM erge//Example.ad.filtered.tagged.bam" echo FILE 4 samtools view -@2 "/scratch/c.c1885111/analysis/an0223/output/SRR7789673/zUMIs_output/.tmpM erge//Example.ae.filtered.tagged.bam" echo FILE 5 samtools view -@2 "/scratch/c.c1885111/analysis/an0223/output/SRR7789673/zUMIs_output/.tmpM erge//Example.af.filtered.tagged.bam" echo FILE 6 samtools view -@2 "/scratch/c.c1885111/analysis/an0223/output/SRR7789673/zUMIs_output/.tmpM erge//Example.ag.filtered.tagged.bam" echo FILE 7 samtools view -@2 "/scratch/c.c1885111/analysis/an0223/output/SRR7789673/zUMIs_output/.tmpM erge//Example.ah.filtered.tagged.bam"

Finished loading and checking parameters Reading genome generation parameters:

STAR --runMode genomeGenerate --runThreadN 4 --genomeDir /scratch/c.c1885111/analysis/an02

23/bin/../resources/ --genomeFastaFiles /scratch/c.c1885111/analysis/an0223/bin/../resources/Mus_m usculus.GRCm38.dna_sm.primary_assembly.fa --sjdbGTFfile /scratch/c.c1885111/analysis/an0223/bin /../resources/Mus_musculus.GRCm38.84.gtf --sjdbOverhang 97

GstrandBit=32

versionGenome 20201 ~RE-DEFINED genomeFastaFiles /scratch/c.c1885111/analysis/an0223/bin/../resources/Mus_musculus.GRCm 38.dna_sm.primary_assembly.fa ~RE-DEFINED genomeSAindexNbases 14 ~RE-DEFINED genomeChrBinNbits 18 ~RE-DEFINED genomeSAsparseD 1 ~RE-DEFINED sjdbOverhang 97 ~RE-DEFINED sjdbFileChrStartEnd - ~RE-DEFINED sjdbGTFfile /scratch/c.c1885111/analysis/an0223/bin/../resources/Mus_musculus.GRCm 38.84.gtf ~RE-DEFINED sjdbGTFchrPrefix - ~RE-DEFINED sjdbGTFfeatureExon exon ~RE-DEFINED sjdbGTFtagExonParentTranscripttranscript_id ~RE-DEFINED sjdbGTFtagExonParentGene gene_id ~RE-DEFINED sjdbInsertSave Basic ~RE-DEFINED genomeFileSizes 2791718093 22299771940 ~RE-DEFINED Genome version is compatible with current STAR version Number of real (reference) chromosomes= 66 1 1 195471971 0 2 10 130694993 195559424 3 11 122082543 326369280 4 12 120129022 448528384 5 13 120421639 568852480 6 14 124902244 689438720 7 15 104043685 814481408 8 16 98207768 918552576 9 17 94987271 1016856576 10 18 90702639 1112014848 11 19 61431566 1202978816 12 2 182113224 1264582656 13 3 160039680 1446772736 14 4 156508116 1606942720 15 5 151834684 1763704832 16 6 149736546 1915748352 17 7 145441459 2065694720 18 8 129401213 2211184640 19 9 124595110 2340683776 20 MT 16299 2465464320 21 X 171031299 2465726464 22 Y 91744698 2636906496 23 JH584299.1 953012 2728656896 24 GL456233.1 336933 2729705472 25 JH584301.1 259875 2730229760 26 GL456211.1 241735 2730491904 27 GL456350.1 227966 2730754048 28 JH584293.1 207968 2731016192 29 GL456221.1 206961 2731278336 30 JH584297.1 205776 2731540480 31 JH584296.1 199368 2731802624 32 GL456354.1 195993 2732064768 33 JH584294.1 191905 2732326912 34 JH584298.1 184189 2732589056 35 JH584300.1 182347 2732851200 36 GL456219.1 175968 2733113344 37 GL456210.1 169725 2733375488 38 JH584303.1 158099 2733637632 39 JH584302.1 155838 2733899776 40 GL456212.1 153618 2734161920 41 JH584304.1 114452 2734424064 42 GL456379.1 72385 2734686208 43 GL456216.1 66673 2734948352 44 GL456393.1 55711 2735210496 45 GL456366.1 47073 2735472640 46 GL456367.1 42057 2735734784 47 GL456239.1 40056 2735996928 48 GL456213.1 39340 2736259072 49 GL456383.1 38659 2736521216 50 GL456385.1 35240 2736783360 51 GL456360.1 31704 2737045504 52 GL456378.1 31602 2737307648 53 GL456389.1 28772 2737569792 54 GL456372.1 28664 2737831936 55 GL456370.1 26764 2738094080 56 GL456381.1 25871 2738356224 57 GL456387.1 24685 2738618368 58 GL456390.1 24668 2738880512 59 GL456394.1 24323 2739142656 60 GL456392.1 23629 2739404800 61 GL456382.1 23158 2739666944 62 GL456359.1 22974 2739929088 63 GL456396.1 21240 2740191232 64 GL456368.1 20208 2740453376 65 JH584292.1 14945 2740715520 66 JH584295.1 1976 2740977664 Started loading the genome: Mon Apr 1 10:58:33 2019

Genome: size given as a parameter = 2791718093 SA: size given as a parameter = 22299771940 SAindex: size given as a parameter = 1 Read from SAindex: pGe.gSAindexNbases=14 nSAi=357913940 nGenome=2791718093; nSAbyte=22299771940 GstrandBit=32 SA number of indices=5406005318 Shared memory is not used for genomes. Allocated a private copy of the genome. Genome file size: 2791718093 bytes; state: good=1 eof=0 fail=0 bad=0 Loading Genome ... done! state: good=1 eof=0 fail=0 bad=0; loaded 2791718093 bytes SA file size: 22299771940 bytes; state: good=1 eof=0 fail=0 bad=0 Loading SA ... done! state: good=1 eof=0 fail=0 bad=0; loaded 22299771940 bytes Loading SAindex ... done: 1565873619 bytes Finished loading the genome: Mon Apr 1 10:59:10 2019

Processing splice junctions database sjdbN=258863, pGe.sjdbOverhang=97 alignIntronMax=alignMatesGapMax=0, the max intron size will be approximately determined by (2^winBin Nbits)*winAnchorDistNbins=589824 Apr 01 10:59:11 Loaded database junctions from the generated genome /scratch/c.c1885111/analysis/a n0223/resources/sjdbList.out.tab: 258863 total junctions

Apr 01 10:59:11 ..... processing annotations GTF

cziegenhain commented 5 years ago

It seems like your STAR run aborts when loading splice junctions from the GTF file. Thats not really a bug in zUMIs, so please try to troubleshoot this on your side. For instance make sure that you are following the instructions for STAR index generation and make sure that your machine does not run out of memory etc..

trueth1206 commented 5 years ago

Thank you very much~ We will keep try it.

trueth1206 commented 5 years ago

Hello, It works after we update the STAR version. Thank you for you kindly help.

cziegenhain commented 5 years ago

Glad to hear that! Hope that zUMIs works smooth for you in the future.