bowtie2 output, sam to bam conversion error.

a_mt · 11-16-2012, 10:39 PM

Hi all,

I am trying convert a sam file (PE bowtie2 default fr output) to bam file using samtools view command. But it's throwing the following error.

Code:

$ samtools view -bS -o aln3.bam aln3.sam
[samopen] SAM header is present: 17 sequences
[sam_read1] reference '451450' is recognized as '*'
Parse error at line 2026299: sequence and quality are inconsistent

When I looked at the partcular line,

Code:

sed -n 2026299p aln3.sam
        133     chr12   451450  0       *       =       451450  0       *       *       YT:Z:UP YF:Z:LN

Its missing col1 (Qname) also CIGAR string, segment sequence and Quality scores are unavailable.

When I checked for such lines

Code:

$ sed -n '/^[^H]/=' aln3.sam | wc -l
5422

There were 5422 lines.

How do I proceed now?? Do I delete these 5422 lines from sam file?? This thread here had the same problem but it was with bwa aligner.

Also my bowtie output after aligning says (many such lines are produced before showing final alignment stats):

Code:

Warning: skipping mate #2 of read '' because length (0) <= # seed mismatches (0)
Warning: skipping mate #2 of read '' because it was < 2 characters long

I am not able interprte this. Also my alignement stats says 99.97% pairs were aligned concordantly 0 times. I am bit amused.

Code:

1018542 reads; of these:
  1018542 (100.00%) were paired; of these:
    1018249 (99.97%) aligned concordantly 0 times
    188 (0.02%) aligned concordantly exactly 1 time
    105 (0.01%) aligned concordantly >1 times

How do I proceed now??

Thank you.

Between I am new to analysis and I hope you people dont mind if my questions are too simple.

arcolombo698 · 03-21-2014, 01:27 PM

Hello. I have the same issue

Warning: skipping mate #1 of read 'SN860:381:H80WNADXX:1:1101:18171:2118 1:N:0:1' because length (0) <= # seed mismatches (0)
Warning: skipping mate #1 of read 'SN860:381:H80WNADXX:1:1101:18171:2118 1:N:0:1' because it was < 2 characters long

Brian Bushnell · 03-21-2014, 02:59 PM

It sounds like you have a 0bp read, possibly due to quality trimming. You can try removing such reads (and their mates) with a tool that allows you to specify a minimum read length.

gerbarinov · 03-29-2015, 01:23 AM

I solved this problem after trimming fastaq files with fastx_trimmer -f and fastx_trimmer -t operations with no 0bp read in output files. So the solution for me was to remove artifacts with fastx_artifacts_filter and then synchronize pairs with fastqCombinePairedEnd.py

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11-16-2012, 10:39 PM	#1
a_mt Member Location: C:/Program files/Google/Chrome Join Date: Jul 2012 Posts: 34	bowtie2 output, sam to bam conversion error. Hi all, I am trying convert a sam file (PE bowtie2 default fr output) to bam file using samtools view command. But it's throwing the following error. Code: $ samtools view -bS -o aln3.bam aln3.sam [samopen] SAM header is present: 17 sequences [sam_read1] reference '451450' is recognized as '' Parse error at line 2026299: sequence and quality are inconsistent When I looked at the partcular line, Code: sed -n 2026299p aln3.sam 133 chr12 451450 0 = 451450 0 * * YT:Z:UP YF:Z:LN Its missing col1 (Qname) also CIGAR string, segment sequence and Quality scores are unavailable. When I checked for such lines Code: $ sed -n '/^[^H]/=' aln3.sam \| wc -l 5422 There were 5422 lines. How do I proceed now?? Do I delete these 5422 lines from sam file?? This thread here had the same problem but it was with bwa aligner. Also my bowtie output after aligning says (many such lines are produced before showing final alignment stats): Code: Warning: skipping mate #2 of read '' because length (0) <= # seed mismatches (0) Warning: skipping mate #2 of read '' because it was < 2 characters long I am not able interprte this. Also my alignement stats says 99.97% pairs were aligned concordantly 0 times. I am bit amused. Code: 1018542 reads; of these: 1018542 (100.00%) were paired; of these: 1018249 (99.97%) aligned concordantly 0 times 188 (0.02%) aligned concordantly exactly 1 time 105 (0.01%) aligned concordantly >1 times How do I proceed now?? Thank you. Between I am new to analysis and I hope you people dont mind if my questions are too simple. Last edited by a_mt; 11-16-2012 at 10:41 PM.

03-21-2014, 01:27 PM	#2
arcolombo698 Senior Member Location: Los Angeles Join Date: Nov 2013 Posts: 142	Skipping Mate errors Hello. I have the same issue Warning: skipping mate #1 of read 'SN860:381:H80WNADXX:1:1101:18171:2118 1:N:0:1' because length (0) <= # seed mismatches (0) Warning: skipping mate #1 of read 'SN860:381:H80WNADXX:1:1101:18171:2118 1:N:0:1' because it was < 2 characters long

03-21-2014, 02:59 PM	#3
Brian Bushnell Super Moderator Location: Walnut Creek, CA Join Date: Jan 2014 Posts: 2,707	It sounds like you have a 0bp read, possibly due to quality trimming. You can try removing such reads (and their mates) with a tool that allows you to specify a minimum read length.

03-29-2015, 01:23 AM	#4
gerbarinov Junior Member Location: RUSSIA MOSCOW Join Date: Mar 2015 Posts: 3	I solved this problem after trimming fastaq files with fastx_trimmer -f and fastx_trimmer -t operations with no 0bp read in output files. So the solution for me was to remove artifacts with fastx_artifacts_filter and then synchronize pairs with fastqCombinePairedEnd.py