Hi, I’ve recently encountered an error when running the ‘align reads and estimate abundance’ tool in the Trinity de novo RNA-seq analysis pipeline.
The output is as follows:
Fatal error: Exit code 2 ()
CMD: touch /galaxy-repl/main/jobdir/028/998/28998407/working/input.fa.bowtie.started
CMD: bowtie-build /galaxy-repl/main/jobdir/028/998/28998407/working/input.fa /galaxy-repl/main/jobdir/028/998/28998407/working/input.fa.bowtie
CMD: touch /galaxy-repl/main/jobdir/028/998/28998407/working/input.fa.RSEM.rsem.prepped.started
CMD: rsem-prepare-reference --transcript-to-gene-map /galaxy-repl/main/jobdir/028/998/28998407/working/gene_to_trans.map /galaxy-repl/main/jobdir/028/998/28998407/working/input.fa /galaxy-repl/main/jobdir/028/998/28998407/working/input.fa.RSEM
$VAR1 = [
‘right’ => ‘/galaxy-repl/main/jobdir/028/998/28998407/working/paired_right.fq’,
‘left’ => ‘/galaxy-repl/main/jobdir/028/998/28998407/working/paired_left.fq’,
‘output_dir’ => ‘output’
CMD: set -o pipefail && bowtie -q --all --best --strata -m 300 --chunkmbs 512 -X 800 -S -p 6 /galaxy-repl/main/jobdir/028/998/28998407/working/input.fa.bowtie -1 /galaxy-repl/main/jobdir/028/998/28998407/working/paired_left.fq -2 /galaxy-repl/main/jobdir/028/998/28998407/working/paired_right.fq | samtools view -@ 6 -F 4 -S -b | samtools sort -@ 6 -n -o bowtie.bam
Warning: Skipping pair SRR5112684.25647332/1 HWI-D00525:102:C4NM2ANXX:2:2302:6220:36668 length=125/1 because a mate is less than 4 characters long
4NM2ANXX:2:2106:14360:26107 length=125/1 because a mate is less than 4 characters long
reads processed: 917268
reads with at least one reported alignment: 650697 (70.94%)
reads that failed to align: 266571 (29.06%)
Reported 1024386 paired-end alignments
[bam_sort_core] merging from 0 files and 6 in-memory blocks…
CMD: touch bowtie.bam.ok
CMD: convert-sam-for-rsem bowtie.bam bowtie.bam.for_rsem
Number of first and second mates in read SRR5112684.97/1’s full alignments (both mates are aligned) are not matched!
Error, cmd: convert-sam-for-rsem bowtie.bam bowtie.bam.for_rsem died with ret: 65280 at /cvmfs/main.galaxyproject.org/deps/_conda/envs/mulled-v1-3213810583b3c414a873752c2610a95351f8665124407340352879200d8f7bbf/bin/align_and_estimate_abundance.pl line 729.
I’ve found an answer to a similar question from the galaxy biostar page (https://biostar.galaxyproject.org/p/28961/) which suggested there were different numbers of reads between the pairs, but when I checked with FastQ Interlacer, there were no single reads (all reads were matched between F & R).
If anyone has any suggestions regarding this issue, that would be greatly appreciated.