Dear all,
Samtools (both 0.1.19 and 1.0.0) make a sorting error when sorting by name.
(Minimal example dataset is here:
https://pwbc.garvan.org.au/~nenbar/samtools/test.bam)
If we look at 2 paired end reads mapping to two transcripts, the paired mates
follow each other and their mapping to the transcripts.
samtools view test.bam
>HISEQ:51:C315AACXX:4:1101:1189:97694 419 TCONS_00000145 14 3 99M = 117 136
>CCATAAGCGGAGAAAGAGGGAATGACATTGTTCTTACACGGCACAAGCAGACAAAATCAACATGGTCATTTAGAAATCGGAGGTGTGGATGCTCTCTAT
>
>CCCFFFFFHHGHHIIJGIJJIIJJJJIJJJIJJJJIJJIJGIIJJJJJIIIJGHHGHFFFEFEEECCEDEEFDDDDDDDCBDD2?>@BCDDDDDDAAA@
> NH:i:2 HI:i:1
>HISEQ:51:C315AACXX:4:1101:1189:97694 339 TCONS_00000145 117 3 33M = 14 -136
>CGGAGAAATATGGTACACCTCTTTACGTATATG ?HCA4HHEC?HHFEC?<G@ABHFDA?D?B???@ NH:i:2
>HI:i:1
>HISEQ:51:C315AACXX:4:1101:1189:97694 355 TCONS_00000146 890 3 33M = 927 136
>CATATACGTAAAGAGGTGTACCATATTTCTCCG @???B?D?ADFHBA@G<?CEFHH?CEHH4ACH? NH:i:2
>HI:i:2
>HISEQ:51:C315AACXX:4:1101:1189:97694 403 TCONS_00000146 927 3 99M = 890 -136
>ATAGAGAGCATCCACACCTCCGATTTCTAAATGACCATGTTGATTTTGTCTGCTTGTGCCGTGTAAGAACAATGTCATTCCCTCTTTCTCCGCTTATGG
>
>@AAADDDDDDCB@>?2DDBCDDDDDDDFEEDECCEEEFEFFFHGHHGJIIIJJJJJIIGJIJJIJJJJIJJJIJJJJIIJJIGJIIHHGHHFFFFFCCC
> NH:i:2 HI:i:2
However, when we sort by name, the transcripts are scrambled.
samtools sort -n test.bam test.sorted
samtools view test.sorted.bam
>HISEQ:51:C315AACXX:4:1101:1189:97694 339 TCONS_00000145 117 3 33M = 14 -136
>CGGAGAAATATGGTACACCTCTTTACGTATATG ?HCA4HHEC?HHFEC?<G@ABHFDA?D?B???@ NH:i:2
>HI:i:1
>HISEQ:51:C315AACXX:4:1101:1189:97694 355 TCONS_00000146 890 3 33M = 927 136
>CATATACGTAAAGAGGTGTACCATATTTCTCCG @???B?D?ADFHBA@G<?CEFHH?CEHH4ACH? NH:i:2
>HI:i:2
>HISEQ:51:C315AACXX:4:1101:1189:97694 419 TCONS_00000145 14 3 99M = 117 136
>CCATAAGCGGAGAAAGAGGGAATGACATTGTTCTTACACGGCACAAGCAGACAAAATCAACATGGTCATTTAGAAATCGGAGGTGTGGATGCTCTCTAT
>
>CCCFFFFFHHGHHIIJGIJJIIJJJJIJJJIJJJJIJJIJGIIJJJJJIIIJGHHGHFFFEFEEECCEDEEFDDDDDDDCBDD2?>@BCDDDDDDAAA@
> NH:i:2 HI:i:1
>HISEQ:51:C315AACXX:4:1101:1189:97694 403 TCONS_00000146 927 3 99M = 890 -136
>ATAGAGAGCATCCACACCTCCGATTTCTAAATGACCATGTTGATTTTGTCTGCTTGTGCCGTGTAAGAACAATGTCATTCCCTCTTTCTCCGCTTATGG
>
>@AAADDDDDDCB@>?2DDBCDDDDDDDFEEDECCEEEFEFFFHGHHGJIIIJJJJJIIGJIJJIJJJJIJJJIJJJJIIJJIGJIIHHGHHFFFFFCCC
> NH:i:2 HI:i:2
This causes an error in some tools (such as RSEM) that require paired read
mates to follow each other.
Is this a bug or a feature, and if a feature, how can one go around it without
using bash sort.
Cheers,
Nenad
Nenad Bartonicek, PhD
Bioinformatic Officer
Centre for Clinical Genomics
Garvan Institute of Medical Research
384 Victoria Street
Sydney NSW 2010
Australia
E: n.bartoni...@garvan.org.au<mailto:n.bartoni...@garvan.org.au>
T: +61(02)92955764
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