Question: Fistful of failures with Bowtie2 in Trinity
1
gravatar for akijl
7 months ago by
akijl20
akijl20 wrote:

I'm getting a bowtie error in Trinity when a run I pipeline I wrote, but only for a handful samples. Most of the reads (trimmed with Trimmomatic into paired and unpaired files) ran successfully.

The sequences that fail don't have fewer number of reads than the ones that succeed, and went through the same quality control regimen.

My trinity input for paired and unpaired sequences 'U24' to be output to folder 'trinity_U24b' is below:

./Trinity --seqType fq --max_memory 50G --CPU 6 --output ExonSeq/ExonSeq_trimmed/Exon_Seq_trimmed_assembled/trinity_U24b --left ExonSeq/ExonSeq_trimmed/U24-forward-trimmed-paired,ExonSeq/ExonSeq_trimmed/U24-forward-trimmed-unpaired --right ExonSeq/ExonSeq_trimmed/U24-reverse-trimmed-paired,ExonSeq/ExonSeq_trimmed/U24-reverse-trimmed-unpaired

I'd like to understand why it's working for some sequences and not others. Any help would be greatly appreciated.

The error message is below:

   * Running CMD: bash -c " set -o pipefail;bowtie2 --local -a --threads 6 -f -x /home/reedlab/trinityrnaseq-Trinity-v2.3.2/ExonSeq/ExonSeq_trimmed/Exon_Seq_trimmed_assembled/trinity_U24b/chrysalis/inchworm.K25.L25.DS.fa.min100 /home/reedlab/trinityrnaseq-Trinity-v2.3.2/ExonSeq/ExonSeq_trimmed/Exon_Seq_trimmed_assembled/trinity_U24b/both.fa  | samtools view -@ 6 -F4 -Sb - | samtools sort -m 4473924266 -@ 6 -no - - > /home/reedlab/trinityrnaseq-Trinity-v2.3.2/ExonSeq/ExonSeq_trimmed/Exon_Seq_trimmed_assembled/trinity_U24b/chrysalis/iworm.bowtie.nameSorted.bam" 
[bam_header_read] EOF marker is absent. The input is probably truncated.
[samopen] SAM header is present: 243613 sequences.
bowtie2-align died with signal 13 (PIPE) 
bash: line 1: 12323 Exit 1                  bowtie2 --local -a --threads 6 -f -x /home/reedlab/trinityrnaseq-Trinity-v2.3.2/ExonSeq/ExonSeq_trimmed/Exon_Seq_trimmed_assembled/trinity_U24b/chrysalis/inchworm.K25.L25.DS.fa.min100 /home/reedlab/trinityrnaseq-Trinity-v2.3.2/ExonSeq/ExonSeq_trimmed/Exon_Seq_trimmed_assembled/trinity_U24b/both.fa
     12324 Broken pipe             | samtools view -@ 6 -F4 -Sb -
     12325 Killed                  | samtools sort -m 4473924266 -@ 6 -no - - > /home/reedlab/trinityrnaseq-Trinity-v2.3.2/ExonSeq/ExonSeq_trimmed/Exon_Seq_trimmed_assembled/trinity_U24b/chrysalis/iworm.bowtie.nameSorted.bam
Error, cmd: bash -c " set -o pipefail;bowtie2 --local -a --threads 6 -f -x /home/reedlab/trinityrnaseq-Trinity-v2.3.2/ExonSeq/ExonSeq_trimmed/Exon_Seq_trimmed_assembled/trinity_U24b/chrysalis/inchworm.K25.L25.DS.fa.min100 /home/reedlab/trinityrnaseq-Trinity-v2.3.2/ExonSeq/ExonSeq_trimmed/Exon_Seq_trimmed_assembled/trinity_U24b/both.fa  | samtools view -@ 6 -F4 -Sb - | samtools sort -m 4473924266 -@ 6 -no - - > /home/reedlab/trinityrnaseq-Trinity-v2.3.2/ExonSeq/ExonSeq_trimmed/Exon_Seq_trimmed_assembled/trinity_U24b/chrysalis/iworm.bowtie.nameSorted.bam"  2>tmp.12229.stderr died with ret 35072 at /home/reedlab/trinityrnaseq-Trinity-v2.3.2/PerlLib/Pipeliner.pm line 166.
    Pipeliner::run('Pipeliner=HASH(0x2492928)') called at ./Trinity line 1745
    main::run_chrysalis('/home/reedlab/trinityrnaseq-Trinity-v2.3.2/ExonSeq/ExonSeq_tr...', '/home/reedlab/trinityrnaseq-Trinity-v2.3.2/ExonSeq/ExonSeq_tr...', 200, 500, undef, '/home/reedlab/trinityrnaseq-Trinity-v2.3.2/ExonSeq/ExonSeq_tr...', '/home/reedlab/trinityrnaseq-Trinity-v2.3.2/ExonSeq/ExonSeq_tr...') called at ./Trinity line 1590
    main::run_Trinity() called at ./Trinity line 1257
    eval {...} called at ./Trinity line 1256

Trinity run failed. Must investigate error above.enter code here
ADD COMMENTlink written 7 months ago by akijl20

Which version of bowtie and which operating system and version?

ADD REPLYlink written 7 months ago by h.mon9.6k

Version of bowtie is bowtie2-2.3.0, run on Linux Mint 17.1 Cinnamon 64-bit.

ADD REPLYlink written 7 months ago by akijl20
1

I had problems with versions 2.3.0 and 2.3.1, last one to work for me is 2.2.9 - but I use a much older OS. It is worth giving a try, though. Does bowtie2-2.3.0 works for you on a simple test?

ADD REPLYlink written 7 months ago by h.mon9.6k

It does, for everything other than about 10 sequence sets I've had no problems.

ADD REPLYlink written 7 months ago by akijl20
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