Runtime error in assemble step

[lamevan]

Hi,I run the assemble step and occur a error.. Could you please explain why I am getting error here? Thank you.. [Tue Apr 9 11:32:20 2019] rule assemble_polish: input: region/Chr3-13380000-60000/asm/contigs.fasta, region/Chr3-13380000-60000/asm/contigs.fasta.fai, region/Chr3-13380000-60000/asm/contig_aligned_reads.bam, region/Chr3-13380000-60000/asm/contig_aligned_reads.bam.pbi output: region/Chr3-13380000-60000/asm/contigs_polished.fasta jobid: 690 wildcards: region_id=Chr3-13380000-60000 resources: threads=4

ESC[33mJob counts: count jobs 1 assemble_polish 1ESC[0m 'FRAMERATEHZ' Traceback (most recent call last): File "/home/linpeifan/smrtsv2/smrtsv2-master/dep/conda/build/envs/pacbio/lib/python2.7/site-packages/pbcommand/cli/core.py", line 138, in _pacbio_main_runner return_code = exe_main_func(*args, kwargs) File "/home/linpeifan/smrtsv2/smrtsv2-master/dep/conda/build/envs/pacbio/lib/python2.7/site-packages/GenomicConsensus/main.py", line 340, in args_runner return tr.main() File "/home/linpeifan/smrtsv2/smrtsv2-master/dep/conda/build/envs/pacbio/lib/python2.7/site-packages/GenomicConsensus/main.py", line 258, in main with AlignmentSet(options.inputFilename) as peekFile: File "/home/linpeifan/smrtsv2/smrtsv2-master/dep/conda/build/envs/pacbio/lib/python2.7/site-packages/pbcore/io/dataset/DataSetIO.py", line 2746, in init super(AlignmentSet, self).init(*files, *kwargs) File "/home/linpeifan/smrtsv2/smrtsv2-master/dep/conda/build/envs/pacbio/lib/python2.7/site-packages/pbcore/io/dataset/DataSetIO.py", line 1994, in init super(ReadSet, self).init(files, kwargs) File "/home/linpeifan/smrtsv2/smrtsv2-master/dep/conda/build/envs/pacbio/lib/python2.7/site-packages/pbcore/io/dataset/DataSetIO.py", line 461, in init self.updateCounts() File "/home/linpeifan/smrtsv2/smrtsv2-master/dep/conda/build/envs/pacbio/lib/python2.7/site-packages/pbcore/io/dataset/DataSetIO.py", line 2574, in updateCounts self.assertIndexed() File "/home/linpeifan/smrtsv2/smrtsv2-master/dep/conda/build/envs/pacbio/lib/python2.7/site-packages/pbcore/io/dataset/DataSetIO.py", line 2404, in assertIndexed self._assertIndexed((IndexedBamReader, CmpH5Reader)) File "/home/linpeifan/smrtsv2/smrtsv2-master/dep/conda/build/envs/pacbio/lib/python2.7/site-packages/pbcore/io/dataset/DataSetIO.py", line 1951, in _assertIndexed self._openFiles() File "/home/linpeifan/smrtsv2/smrtsv2-master/dep/conda/build/envs/pacbio/lib/python2.7/site-packages/pbcore/io/dataset/DataSetIO.py", line 2075, in _openFiles resource = IndexedBamReader(location) File "/home/linpeifan/smrtsv2/smrtsv2-master/dep/conda/build/envs/pacbio/lib/python2.7/site-packages/pbcore/io/align/BamIO.py", line 374, in init super(IndexedBamReader, self).init(fname, referenceFastaFname) File "/home/linpeifan/smrtsv2/smrtsv2-master/dep/conda/build/envs/pacbio/lib/python2.7/site-packages/pbcore/io/align/BamIO.py", line 183, in init self._loadReadGroupInfo() File "/home/linpeifan/smrtsv2/smrtsv2-master/dep/conda/build/envs/pacbio/lib/python2.7/site-packages/pbcore/io/align/BamIO.py", line 95, in _loadReadGroupInfo rgFrameRate = ds["FRAMERATEHZ"] KeyError: 'FRAMERATEHZ' [ERROR] 'FRAMERATEHZ' Traceback (most recent call last): File "/home/linpeifan/smrtsv2/smrtsv2-master/dep/conda/build/envs/pacbio/lib/python2.7/site-packages/pbcommand/cli/core.py", line 138, in _pacbio_main_runner return_code = exe_main_func(*args, kwargs) File "/home/linpeifan/smrtsv2/smrtsv2-master/dep/conda/build/envs/pacbio/lib/python2.7/site-packages/GenomicConsensus/main.py", line 340, in args_runner return tr.main() File "/home/linpeifan/smrtsv2/smrtsv2-master/dep/conda/build/envs/pacbio/lib/python2.7/site-packages/GenomicConsensus/main.py", line 258, in main with AlignmentSet(options.inputFilename) as peekFile: File "/home/linpeifan/smrtsv2/smrtsv2-master/dep/conda/build/envs/pacbio/lib/python2.7/site-packages/pbcore/io/dataset/DataSetIO.py", line 2746, in init super(AlignmentSet, self).init(*files, *kwargs) File "/home/linpeifan/smrtsv2/smrtsv2-master/dep/conda/build/envs/pacbio/lib/python2.7/site-packages/pbcore/io/dataset/DataSetIO.py", line 1994, in init super(ReadSet, self).init(files, kwargs) File "/home/linpeifan/smrtsv2/smrtsv2-master/dep/conda/build/envs/pacbio/lib/python2.7/site-packages/pbcore/io/dataset/DataSetIO.py", line 461, in init self.updateCounts() File "/home/linpeifan/smrtsv2/smrtsv2-master/dep/conda/build/envs/pacbio/lib/python2.7/site-packages/pbcore/io/dataset/DataSetIO.py", line 2574, in updateCounts self.assertIndexed() File "/home/linpeifan/smrtsv2/smrtsv2-master/dep/conda/build/envs/pacbio/lib/python2.7/site-packages/pbcore/io/dataset/DataSetIO.py", line 2404, in assertIndexed self._assertIndexed((IndexedBamReader, CmpH5Reader)) File "/home/linpeifan/smrtsv2/smrtsv2-master/dep/conda/build/envs/pacbio/lib/python2.7/site-packages/pbcore/io/dataset/DataSetIO.py", line 1951, in _assertIndexed self._openFiles() File "/home/linpeifan/smrtsv2/smrtsv2-master/dep/conda/build/envs/pacbio/lib/python2.7/site-packages/pbcore/io/dataset/DataSetIO.py", line 2075, in _openFiles resource = IndexedBamReader(location) File "/home/linpeifan/smrtsv2/smrtsv2-master/dep/conda/build/envs/pacbio/lib/python2.7/site-packages/pbcore/io/align/BamIO.py", line 374, in init super(IndexedBamReader, self).init(fname, referenceFastaFname) File "/home/linpeifan/smrtsv2/smrtsv2-master/dep/conda/build/envs/pacbio/lib/python2.7/site-packages/pbcore/io/align/BamIO.py", line 183, in init self._loadReadGroupInfo() File "/home/linpeifan/smrtsv2/smrtsv2-master/dep/conda/build/envs/pacbio/lib/python2.7/site-packages/pbcore/io/align/BamIO.py", line 95, in _loadReadGroupInfo rgFrameRate = ds["FRAMERATEHZ"] KeyError: 'FRAMERATEHZ' ESC[32m[Tue Apr 9 11:32:21 2019]ESC[0m ESC[31mError in rule assemble_polish:ESC[0m ESC[31m jobid: 0ESC[0m ESC[31m output: region/Chr3-13380000-60000/asm/contigs_polished.fastaESC[0m ESC[31mESC[0m ESC[31mRuleException: CalledProcessError in line 250 of /home/linpeifan/smrtsv2/smrtsv2-master/rules/assemble_group.snakefile: Command ' set -euo pipefail; pb_python2 -s /home/linpeifan/smrtsv2/smrtsv2-master/dep/bin/variantCaller --referenceFilename region/Chr3-13380000-60000/asm/contigs.fasta region/Chr3-13380000-60000/asm/contig_aligned_reads.bam -o region/Chr3-13380000-60000/asm/contigs_polished.fasta -j 4 --algorithm=arrow; ' returned non-zero exit status 2. File "/home/linpeifan/smrtsv2/smrtsv2-master/rules/assemble_group.snakefile", line 250, in __rule_assemble_polish File "/home/linpeifan/smrtsv2/smrtsv2-master/dep/conda/build/envs/python3/lib/python3.6/concurrent/futures/thread.py", line 55, in runESC[0m ESC[31mExiting because a job execution failed. Look above for error messageESC[0m Shutting down, this might take some time. Exiting because a job execution failed. Look above for error message Complete log: /home/linpeifan/temp/asm_group_gr-Chr3-13000000-1000000/.snakemake/log/2019-04-09T112437.712925.snakemake.log

[paudano]

I am pretty sure this is caused by running from BAX files and not subread BAM files. When version 2 was released, we did not know how detrimental this would be to downstream tools. We just added a new section to README.md ("Creating the input FOFN file"). Pull the updated code and see that section. If it was run from BAX files, you will need to re-run SMRT-SV from the start.

[lamevan]

hi,I have input the subread bam file instead of bax file.It works but another error occur. [Wed Apr 10 16:54:12 2019] rule assemble_align_fixup: input: region/Chr11-29000000-21106/asm/contig.sam, /home/linpeifan/r1nbsv/align/alignments.fofn, /home/linpeifan/r1nbsv/reference/ref.fasta, group/reads.bam output: region/Chr11-29000000-21106/asm/contig_fixup.bam jobid: 1 wildcards: region_id=Chr11-29000000-21106 resources: threads=1

ESC[33mJob counts: count jobs 1 assemble_align_fixup 1ESC[0m bamleftalign: /home/linpeifan/smrtsv2/smrtsv2-master/dep/conda/build/envs/tools/bin/../lib/libstdc++.so.6: version GLIBCXX_3.4.20' not found (required by bamleftalign) bamleftalign: /home/linpeifan/smrtsv2/smrtsv2-master/dep/conda/build/envs/tools/bin/../lib/libstdc++.so.6: versionCXXABI_1.3.9' not found (required by bamleftalign) bamleftalign: /home/linpeifan/smrtsv2/smrtsv2-master/dep/conda/build/envs/tools/bin/../lib/libstdc++.so.6: version `GLIBCXX_3.4.21' not found (required by bamleftalign) ESC[32m[Wed Apr 10 16:54:35 2019]ESC[0m ESC[31mError in rule assemble_align_fixup:ESC[0m ESC[31m jobid: 0ESC[0m ESC[31m output: region/Chr11-29000000-21106/asm/contig_fixup.bamESC[0m ESC[31mESC[0m ESC[31mRuleException: CalledProcessError in line 108 of /home/linpeifan/smrtsv2/smrtsv2-master/rules/assemble_group.snakefile: Command ' set -euo pipefail; mkdir -p $(dirname temp/region/Chr11-29000000-21106/asm/assemble_align_fixup); alignfixup -i region/Chr11-29000000-21106/asm/contig.sam -d $(head -n 1 /home/linpeifan/r1nbsv/align/alignments.fofn) -r /home/linpeifan/r1nbsv/reference/ref.fasta -g Chr11-29000000-21106 -o temp/region/Chr11-29000000-21106/asm/assemble_align_fixup_usort.bam; tlenadd -i temp/region/Chr11-29000000-21106/asm/assemble_align_fixup_usort.bam -o temp/region/Chr11-29000000-21106/asm/assemble_align_fixup_tlen.bam -r /home/linpeifan/r1nbsv/reference/ref.fasta; rm temp/region/Chr11-29000000-21106/asm/assemble_align_fixup_usort.bam; { samtools view -H temp/region/Chr11-29000000-21106/asm/assemble_align_fixup_tlen.bam | grep -Ev '^@(RG|PG)'; samtools view temp/region/Chr11-29000000-21106/asm/assemble_align_fixup_tlen.bam; } | bamleftalign -f /home/linpeifan/r1nbsv/reference/ref.fasta | samtools sort -T temp/region/Chr11-29000000-21106/asm/assemble_align_fixup -o region/Chr11-29000000-21106/asm/contig_fixup.bam; rm temp/region/Chr11-29000000-21106/asm/assemble_align_fixup_tlen.bam ' returned non-zero exit status 1. File "/home/linpeifan/smrtsv2/smrtsv2-master/rules/assemble_group.snakefile", line 108, in __rule_assemble_align_fixup File "/home/linpeifan/smrtsv2/smrtsv2-master/dep/conda/build/envs/python3/lib/python3.6/concurrent/futures/thread.py", line 55, in runESC[0m ESC[31mExiting because a job execution failed. Look above for error messageESC[0m Shutting down, this might take some time. Exiting because a job execution failed. Look above for error message Complete log: /home/linpeifan/tmp/asm_group_gr-Chr11-29000000-21106/.snakemake/log/2019-04-10T164153.540316.snakemake.log Could you explain why this error occured?Thanks!

[paudano]

It must be finding bamleftalign outside of SMRT-SV. The bamleftalign executable in SMRT-SV is compiled with gcc 4.9.0, and it doesn't have GLIBCXX_3.4.21 (latest supported C++ interface is GLIBCXX_3.4.19).

Two questions: 1) Is bamleftalign in dep/bin? 2) Are you running smrtsv.py or smrtsv?

[lamevan]

1.yes 2.smrtsv it seems works after I put another libstdc++.so.6 to the /lib directory and create an soft link.. But another error occured...[33mJob counts: count jobs 1 assemble_polish 1 Child process exited with exitcode=-7. Aborting. [ERROR] Child process exited with exitcode=-7. Aborting. Aborting [ERROR] Aborting [Thu Apr 11 18:10:23 2019] Error in rule assemble_polish:  jobid: 0  output: region/Chr7-28960000-60000/asm/contigs_polished.fasta  RuleException: CalledProcessError in line 250 of /home/linpeifan/smrtsv2/smrtsv2-master/rules/assemble_group.snakefile: Command ' set -euo pipefail; pb_python2 -s /home/linpeifan/smrtsv2/smrtsv2-master/dep/bin/variantCaller --referenceFilename region/Chr7-28960000-60000/asm/contigs.fasta region/Chr7-28960000-60000/asm/contig_aligned_reads.bam -o region/Chr7-28960000-60000/asm/contigs_polished.fasta -j 4 --algorithm=quiver; ' returned non-zero exit status 255. File "/home/linpeifan/smrtsv2/smrtsv2-master/rules/assemble_group.snakefile", line 250, in __rule_assemble_polish File "/home/linpeifan/smrtsv2/smrtsv2-master/dep/conda/build/envs/python3/lib/python3.6/concurrent/futures/thread.py", line 55, in run Exiting because a job execution failed. Look above for error message Shutting down, this might take some time. Exiting because a job execution failed. Look above for error message Complete log: /home/linpeifan/tmp/asm_group_gr-Chr7-28000000-1000000/.snakemake/log/2019-04-10T164155.812110.snakemake.log

[lamevan]

Another error occured in another job.. Job counts: count jobs 1 assemble_align_org 1 [INFO] 2019-04-11T18:09:48 [blasr] started. /usr/bin/bash: line 1: 22855 Bus error blasr region/Chr12-7920000-60000/reads/reads.bam region/Chr12-7920000-60000/asm/contigs.fasta --bam --bestn 1 --unaligned /dev/null --out temp/region/Chr12-7920000-60000/reads/align_reads.bam --nproc 4 [Thu Apr 11 18:10:13 2019] Error in rule assemble_align_org:  jobid: 0  output: region/Chr12-7920000-60000/asm/contig_aligned_reads.bam, region/Chr12-7920000-60000/asm/contig_aligned_reads.bam.pbi  RuleException: CalledProcessError in line 286 of /home/linpeifan/smrtsv2/smrtsv2-master/rules/assemble_group.snakefile: Command ' set -euo pipefail; blasr region/Chr12-7920000-60000/reads/reads.bam region/Chr12-7920000-60000/asm/contigs.fasta --bam --bestn 1 --unaligned /dev/null --out temp/region/Chr12-7920000-60000/reads/align_reads.bam --nproc 4; samtools sort -O bam -T temp/region/Chr12-7920000-60000/reads/align_reads_slice -o region/Chr12-7920000-60000/asm/contig_aligned_reads.bam temp/region/Chr12-7920000-60000/reads/align_reads.bam; pbindex region/Chr12-7920000-60000/asm/contig_aligned_reads.bam; rm temp/region/Chr12-7920000-60000/reads/align_reads.bam ' returned non-zero exit status 135. File "/home/linpeifan/smrtsv2/smrtsv2-master/rules/assemble_group.snakefile", line 286, in __rule_assemble_align_org File "/home/linpeifan/smrtsv2/smrtsv2-master/dep/conda/build/envs/python3/lib/python3.6/concurrent/futures/thread.py", line 55, in run Exiting because a job execution failed. Look above for error message Shutting down, this might take some time. Exiting because a job execution failed. Look above for error message Complete log: /home/linpeifan/tmp/asm_group_gr-Chr12-7000000-1000000/.snakemake/log/2019-04-10T164158.101764.snakemake.log

[paudano]

I think something is not right about your install environment, but I don't know what it would be. You should not need to link "libstdc++.so.6" into the lib directory, it should find that if everything in dep is installed correctly. Conda builds binaries with RPATH to find their libraries (it's like LD_LIBRARY_PATH, but built into the executable). So if you have to supplement a library, then something is not right with the install.

Also, a bus error is likely a memory error. I think a binary is trying to use a library it is not compatible with.

I would start by rebuilding everything in dep. Go into dep, run make clean, then make. If any errors occur in the build process, let's start by troubleshooting those. After that, let's continue running and troubleshooting SMRT-SV.

[paudano]

Let me know if you are able to rebuild dep and if that makes any difference.

[lamevan]

I rebuilt the environment as you said,it works now.Now it goes to assemble step and takes a long time.I will wait for the result.Thank you!

[paudano]

Great. If you run into a new issue, open a new case for that. Closing this one for now.