Question: Red error from RSEM Trinity -- Solution: input matched paired end reads
gravatar for j_pasook
4 months ago by
j_pasook40 wrote:


I've constructed a trinity assembly using ~30M PE read. Then I was trying to estimate abundance using "Align reads and estimate abundance on a de novo assembly of RNA-Seq data" tool in usegalaxy Europe server. I performed this exact tool before using 1M PE read sample before, and it was perfectly fine When it comes to my real 30M PE read data, the tool was run for 8 hours and it showed red error with this messages;

Fatal error: Exit code 2 ()
CMD: touch /data/dnb01/galaxy_db/job_working_directory/004/118/4118029/working/input.fa.bowtie.started
CMD: bowtie-build /data/dnb01/galaxy_db/job_working_directory/004/118/4118029/working/input.fa /data/dnb01/galaxy_db/job_working_directory/004/118/4118029/working/input.fa.bowtie
CMD: touch /data/dnb01/galaxy_db/job_working_directory/004/118/4118029/working/input.fa.RSEM.rsem.prepped.started
CMD: rsem-prepare-reference  --transcript-to-gene-map /data/dnb01/galaxy_db/job_working_directory/004/118/4118029/working/ /data/dnb01/galaxy_db/job_working_directory/004/118/4118029/working/input.fa /data/dnb01/galaxy_db/job_working_directory/004/118/4118029/working/input.fa.RSEM
$VAR1 = [
            'left' => '/data/dnb01/galaxy_db/job_working_directory/004/118/4118029/working/paired_left.fq',
            'output_dir' => 'output',
            'right' => '/data/dnb01/galaxy_db/job_working_directory/004/118/4118029/working/paired_right.fq'
CMD: set -o pipefail && bowtie -q --all --best --strata -m 300 --chunkmbs 512 -X 800 -S -p 1 /data/dnb01/galaxy_db/job_working_directory/004/118/4118029/working/input.fa.bowtie -1 /data/dnb01/galaxy_db/job_working_directory/004/118/4118029/working/paired_left.fq -2 /data/dnb01/galaxy_db/job_working_directory/004/118/4118029/working/paired_right.fq | samtools view -F 4 -S -b | samtools sort -n -o bowtie.bam 
reads processed: 54708014
reads with at least one reported alignment: 52205 (0.10%)
reads that failed to align: 54655809 (99.90%)
Reported 135391 paired-end alignments
CMD: touch bowtie.bam.ok
CMD: convert-sam-for-rsem bowtie.bam bowtie.bam.for_rsem
Number of first and second mates in read 5175620'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 /usr/local/tools/_conda/envs/mulled-v1-eea4a55dc36ed6afc8679898c026ba6276e343800300cbf167af08a314bb9f65/bin/ line 790.

I think it has to be with my data but I don't know why. Can anyone enlighten me about this ? Thanks, j_pasook

ADD COMMENTlink modified 4 months ago by Jennifer Hillman Jackson25k • written 4 months ago by j_pasook40
gravatar for Jennifer Hillman Jackson
4 months ago by
United States
Jennifer Hillman Jackson25k wrote:


The same number of reads must be entered for both forward and reverse inputs. The sequence identifiers should also be a match and be in the same order.

If you performed some QA and reads were lost, cycling the data through these two NGS: QC and manipulation tools can help you to get matched paired input:

  • FASTQ interlacer paired end reads
  • FASTQ de-interlacer on paired end reads

That said, I am not exactly certain if that is your problem or not. I asked the developers at the IUC Gitter chat (where many of the .eu server admins check-in). Please feel free to join in or just follow the conversation:

Thanks! Jen, Galaxy team

ADD COMMENTlink modified 4 months ago • written 4 months ago by Jennifer Hillman Jackson25k
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