Rsamtools: sortBam() and 'samtools sort' does not give the same results
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@henrik-bengtsson-4333
Last seen 4 months ago
United States
Hi, I've got a BAM file 'foo.bam' [425,850,792 bytes] that I'm trying to sort. It was generated using BWA. I fail to sort it with Rsamtools::sortBam() [Rsamtools v1.12.4] whereas with 'samtools sort' [samtools v0.1.18 (r982:295)] it works. BAM FILE: % samtools view -H foo0.bam | head @HD VN:1.3 SO:coordinate @SQ SN:1 LN:249250621 @SQ SN:2 LN:243199373 @SQ SN:3 LN:198022430 @SQ SN:4 LN:191154276 @SQ SN:5 LN:180915260 @SQ SN:6 LN:171115067 @SQ SN:7 LN:159138663 @SQ SN:8 LN:146364022 @SQ SN:9 LN:141213431 The following: > Rsamtools::sortBam("foo.bam", "foo.sort.byR") outputs file 'foo.sort.byR.bam' [425,293,387 bytes], but when trying to generate an index, both indexBam() and 'samtools index' throws an error: > Rsamtools::indexBam("foo.sort.byR.bam") Error in FUN("foo.sort.byR.bam"[[1L]], ...) : failed to build index file: foo.sort.byR.bam In addition: Warning messages: 1: In FUN("foo.sort.byR.bam"[[1L]], ...) : [bam_index_core] the alignment is not sorted: reads without coordinates prior to reads with coordinates. 2: In FUN("foo.sort.byR.bam"[[1L]], ...) : [bam_index_build2] fail to index the BAM file. and % samtools index foo.sort.byR.bam [bam_index_core] the alignment is not sorted: reads without coordinates prior to reads with coordinates. [bam_index_build2] fail to index the BAM file. Trying to call sortBam() multiple times changes nothing. However, if I use 'samtools sort' to sort the BAM file, it all works: % samtools sort foo.bam foo.sort [bam_sort_core] merging from 3 files... outputs file 'foo.sort.bam' [425,254,135 bytes]. With this file both % samtools index foo.sort.bam and > Rsamtools::indexBam("foo.sort.bam") work. They both generate identical file 'foo.sort.bam.bai' [8,274,272 bytes]. I've tried also with a toy sample BAM file 'longreads.bam' [1,923,379 bytes] and there I don't get any errors (although Rsamtools and 'samtools sort' do output differently sized *.sort.bam files). I've never had this issue before. Comments? /Henrik > sessionInfo() R version 3.0.1 (2013-05-16) Platform: x86_64-unknown-linux-gnu (64-bit) locale: [1] C attached base packages: [1] parallel stats graphics grDevices utils datasets methods [8] base other attached packages: [1] Rsamtools_1.12.4 Biostrings_2.28.0 GenomicRanges_1.12.4 [4] IRanges_1.18.2 BiocGenerics_0.6.0 loaded via a namespace (and not attached): [1] bitops_1.0-6 stats4_3.0.1 tools_3.0.1 zlibbioc_1.6.0
Alignment Rsamtools Alignment Rsamtools • 2.0k views
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@martin-morgan-1513
Last seen 3 months ago
United States
On 08/22/2013 02:13 PM, Henrik Bengtsson wrote: > Hi, > > I've got a BAM file 'foo.bam' [425,850,792 bytes] that I'm trying to > sort. It was generated using BWA. I fail to sort it with > Rsamtools::sortBam() [Rsamtools v1.12.4] whereas with 'samtools sort' > [samtools v0.1.18 (r982:295)] it works. > > BAM FILE: > % samtools view -H foo0.bam | head > @HD VN:1.3 SO:coordinate > @SQ SN:1 LN:249250621 > @SQ SN:2 LN:243199373 > @SQ SN:3 LN:198022430 > @SQ SN:4 LN:191154276 > @SQ SN:5 LN:180915260 > @SQ SN:6 LN:171115067 > @SQ SN:7 LN:159138663 > @SQ SN:8 LN:146364022 > @SQ SN:9 LN:141213431 > > The following: >> Rsamtools::sortBam("foo.bam", "foo.sort.byR") > > outputs file 'foo.sort.byR.bam' [425,293,387 bytes], but when trying > to generate an index, both indexBam() and 'samtools index' throws an > error: > >> Rsamtools::indexBam("foo.sort.byR.bam") > Error in FUN("foo.sort.byR.bam"[[1L]], ...) : failed to build index > file: foo.sort.byR.bam > In addition: Warning messages: > 1: In FUN("foo.sort.byR.bam"[[1L]], ...) : > [bam_index_core] the alignment is not sorted: reads without > coordinates prior to reads with coordinates. > 2: In FUN("foo.sort.byR.bam"[[1L]], ...) : > [bam_index_build2] fail to index the BAM file. > > and > > % samtools index foo.sort.byR.bam > [bam_index_core] the alignment is not sorted: reads without > coordinates prior to reads with coordinates. > [bam_index_build2] fail to index the BAM file. > > Trying to call sortBam() multiple times changes nothing. However, if > I use 'samtools sort' to sort the BAM file, it all works: > > % samtools sort foo.bam foo.sort > [bam_sort_core] merging from 3 files... > > outputs file 'foo.sort.bam' [425,254,135 bytes]. With this file both > > % samtools index foo.sort.bam > > and > >> Rsamtools::indexBam("foo.sort.bam") > > work. They both generate identical file 'foo.sort.bam.bai' [8,274,272 bytes]. > > I've tried also with a toy sample BAM file 'longreads.bam' [1,923,379 > bytes] and there I don't get any errors (although Rsamtools and > 'samtools sort' do output differently sized *.sort.bam files). > > I've never had this issue before. Comments? I think samtools-0.1.18 is from Sept 2, 2011 (from git) Author: Heng Li <lh3 at="" live.co.uk=""> Date: Fri Sep 2 16:57:34 2011 +0000 * Updated samtools to the latest * Added seqtk.c * Updated wgsim.c * release samtools-0.1.18 versus (not sure why this didn't get into the NEWS file) in Rsamtools in release r74547 | mtmorgan at fhcrc.org | 2013-03-18 22:45:53 -0700 (Mon, 18 Mar 2013) | 4 l ines update samtools to 0.1.19 beta so one possibility is a regression in samtools. Happy to investigate further if you've got a reproducible example. Martin > > /Henrik > >> sessionInfo() > R version 3.0.1 (2013-05-16) > Platform: x86_64-unknown-linux-gnu (64-bit) > > locale: > [1] C > > attached base packages: > [1] parallel stats graphics grDevices utils datasets methods > [8] base > > other attached packages: > [1] Rsamtools_1.12.4 Biostrings_2.28.0 GenomicRanges_1.12.4 > [4] IRanges_1.18.2 BiocGenerics_0.6.0 > > loaded via a namespace (and not attached): > [1] bitops_1.0-6 stats4_3.0.1 tools_3.0.1 zlibbioc_1.6.0 > > _______________________________________________ > Bioconductor mailing list > Bioconductor at r-project.org > https://stat.ethz.ch/mailman/listinfo/bioconductor > Search the archives: http://news.gmane.org/gmane.science.biology.informatics.conductor > -- Computational Biology / Fred Hutchinson Cancer Research Center 1100 Fairview Ave. N. PO Box 19024 Seattle, WA 98109 Location: Arnold Building M1 B861 Phone: (206) 667-2793
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On Thu, Aug 22, 2013 at 4:13 PM, Martin Morgan <mtmorgan at="" fhcrc.org=""> wrote: > On 08/22/2013 02:13 PM, Henrik Bengtsson wrote: >> >> Hi, >> >> I've got a BAM file 'foo.bam' [425,850,792 bytes] that I'm trying to >> sort. It was generated using BWA. I fail to sort it with >> Rsamtools::sortBam() [Rsamtools v1.12.4] whereas with 'samtools sort' >> [samtools v0.1.18 (r982:295)] it works. >> >> BAM FILE: >> % samtools view -H foo0.bam | head >> @HD VN:1.3 SO:coordinate >> @SQ SN:1 LN:249250621 >> @SQ SN:2 LN:243199373 >> @SQ SN:3 LN:198022430 >> @SQ SN:4 LN:191154276 >> @SQ SN:5 LN:180915260 >> @SQ SN:6 LN:171115067 >> @SQ SN:7 LN:159138663 >> @SQ SN:8 LN:146364022 >> @SQ SN:9 LN:141213431 >> >> The following: >>> >>> Rsamtools::sortBam("foo.bam", "foo.sort.byR") >> >> >> outputs file 'foo.sort.byR.bam' [425,293,387 bytes], but when trying >> to generate an index, both indexBam() and 'samtools index' throws an >> error: >> >>> Rsamtools::indexBam("foo.sort.byR.bam") >> >> Error in FUN("foo.sort.byR.bam"[[1L]], ...) : failed to build index >> file: foo.sort.byR.bam >> In addition: Warning messages: >> 1: In FUN("foo.sort.byR.bam"[[1L]], ...) : >> [bam_index_core] the alignment is not sorted: reads without >> coordinates prior to reads with coordinates. >> 2: In FUN("foo.sort.byR.bam"[[1L]], ...) : >> [bam_index_build2] fail to index the BAM file. >> >> and >> >> % samtools index foo.sort.byR.bam >> [bam_index_core] the alignment is not sorted: reads without >> coordinates prior to reads with coordinates. >> [bam_index_build2] fail to index the BAM file. >> >> Trying to call sortBam() multiple times changes nothing. However, if >> I use 'samtools sort' to sort the BAM file, it all works: >> >> % samtools sort foo.bam foo.sort >> [bam_sort_core] merging from 3 files... >> >> outputs file 'foo.sort.bam' [425,254,135 bytes]. With this file both >> >> % samtools index foo.sort.bam >> >> and >> >>> Rsamtools::indexBam("foo.sort.bam") >> >> >> work. They both generate identical file 'foo.sort.bam.bai' [8,274,272 >> bytes]. >> >> I've tried also with a toy sample BAM file 'longreads.bam' [1,923,379 >> bytes] and there I don't get any errors (although Rsamtools and >> 'samtools sort' do output differently sized *.sort.bam files). >> >> I've never had this issue before. Comments? > > > I think samtools-0.1.18 is from Sept 2, 2011 (from git) > > Author: Heng Li <lh3 at="" live.co.uk=""> > Date: Fri Sep 2 16:57:34 2011 +0000 > > * Updated samtools to the latest > * Added seqtk.c > * Updated wgsim.c > * release samtools-0.1.18 > > versus (not sure why this didn't get into the NEWS file) in Rsamtools in > release > > r74547 | mtmorgan at fhcrc.org | 2013-03-18 22:45:53 -0700 (Mon, 18 Mar 2013) | > 4 l > ines > > update samtools to 0.1.19 beta > > so one possibility is a regression in samtools. Thanks Martin, I should have known better. After using samtools 0.1.19 (stable), sortBam() of Rsamtools 1.12.4 and 'samtools sort' of samtools 0.1.19 gives identical BAM files; 425293387 Aug 22 13:42 foo.sort.Rsamtools1_12_4.bam 425293387 Aug 22 17:14 foo.sort.samtools0_1_19.bam % diff foo.sort.Rsamtools1_12_4.bam foo.sort.samtools0_1_19.bam % md5sum foo.sort.Rsamtools1_12_4.bam foo.sort.samtools0_1_19.bam 0dcd0a5b867d3e049d1edd35fe09445a foo.sort.Rsamtools1_12_4.bam 0dcd0a5b867d3e049d1edd35fe09445a foo.sort.samtools0_1_19.bam So no longer a case of BioC but samtools developers. /Henrik > Happy to investigate further if you've got a reproducible example. > > Martin > >> >> /Henrik >> >>> sessionInfo() >> >> R version 3.0.1 (2013-05-16) >> Platform: x86_64-unknown-linux-gnu (64-bit) >> >> locale: >> [1] C >> >> attached base packages: >> [1] parallel stats graphics grDevices utils datasets methods >> [8] base >> >> other attached packages: >> [1] Rsamtools_1.12.4 Biostrings_2.28.0 GenomicRanges_1.12.4 >> [4] IRanges_1.18.2 BiocGenerics_0.6.0 >> >> loaded via a namespace (and not attached): >> [1] bitops_1.0-6 stats4_3.0.1 tools_3.0.1 zlibbioc_1.6.0 >> >> _______________________________________________ >> Bioconductor mailing list >> Bioconductor at r-project.org >> https://stat.ethz.ch/mailman/listinfo/bioconductor >> Search the archives: >> http://news.gmane.org/gmane.science.biology.informatics.conductor >> > > > -- > Computational Biology / Fred Hutchinson Cancer Research Center > 1100 Fairview Ave. N. > PO Box 19024 Seattle, WA 98109 > > Location: Arnold Building M1 B861 > Phone: (206) 667-2793
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