Question: Trinity RSEM Bowtie2 align_and_estimate_abundance script error
gravatar for Light92
13 months ago by
Light9220 wrote:

Hello! I'm running a RNA-Seq differential gene expression pipeline analysis. I previously filtered my input sequences, then de-novo assembled my transcriptome through Trinity, and now, through a Trinity-provided script, I'm trying to evaluate and estimate transcript abundances through the different samples. This way, I'll be able to use the resulting data to run a differential gene expression analysis, through a R package (EBSEQ) as a part of the RSEM suite.

I got to the point I'm running the script provided by Trinity, with the following parameters:

--transcripts  /home/genomica/DATA/Lattuga/denovo_trinity_denovo_filtered_dump_completo/Trinity.fasta
--seqType fq --single --/home/genomica/DATA/Lattuga/DRR057059_1_clipped.fastq --thread_count 60 --est_method RSEM --aln_method bowtie2 --trinity_mode --debug
--output_dir /home/genomica/DATA/Lattuga/denovo_trinity_denovo_filtered_dump_completo/

where Trinity.fasta is my de-novo assembled transcriptome

and DRR057059_1_clipped.fastq one of the libraries used to assemble it (just running the script on one file for now)

Am I doing anything wrong?

I get this debug message after the script creates several files you can see below:

enter image description here

$VAR1 = [
            'single' => '//--/home/genomica/DATA/Lattuga/DRR057059_1_clipped.fastq',
            'output_dir' => '/home/genomica/DATA/Lattuga/denovo_trinity_denovo_filtered_dump_completo/'
        ]; CMD: set -o pipefail && bowtie2 --no-mixed --no-discordant --gbar 1000 --end-to-end -k 200  -q -x /home/genomica/DATA/Lattuga/denovo_trinity_denovo_filtered_dump_completo/Trinity.fasta.bowtie2
-U //--/home/genomica/DATA/Lattuga/DRR057059_1_clipped.fastq -p 60 | samtools view -@ 60 -F 4 -S -b | samtools sort -@ 60 -n -o bowtie2.bam stat: No such file or directory Warning: Could not open read file "//--/home/genomica/DATA/Lattuga/DRR057059_1_clipped.fastq" for reading; skipping... Error: No input read files were valid (ERR): bowtie2-align exited with value 1 Error, cmd: set -o pipefail && bowtie2 --no-mixed --no-discordant --gbar 1000 --end-to-end -k 200  -q
-x /home/genomica/DATA/Lattuga/denovo_trinity_denovo_filtered_dump_completo/Trinity.fasta.bowtie2
-U //--/home/genomica/DATA/Lattuga/DRR057059_1_clipped.fastq -p 60 | samtools view -@ 60 -F 4 -S -b | samtools sort -@ 60 -n -o bowtie2.bam died with ret: 256 at home/genomica/DATA/Software/Trinity/trinityrnaseq-2.8.5/util/ line 733.

Something seems to be wrong with a Trinity.fasta.bowtie2 that the script can't find (there is one in the working directly but it seems to be set in different parts)

The file DRR057059_1_clipped.fastq is a legit file, used previously to assemble the transcriptome, why shouldn't it be able to open it? Maybe it's something about the synthax?)

(I don't think it's about the path, I had set the bowtie2 path correctly previously)

Thanks in advance for your help, I'm stuck here, and it could be something basic I'm missing.

bowtie2 rna-seq rsem trinity • 421 views
ADD COMMENTlink modified 13 months ago by genomax91k • written 13 months ago by Light9220

Please use the formatting bar (especially the code option) to present your post better. I've done it for you this time.

Thank you!

ADD REPLYlink written 13 months ago by genomax91k
gravatar for predeus
13 months ago by
predeus1.4k wrote:

Dir paths look kind of funny. Did you try running the commands mentioned in the debug manually?

Try checking what "Trinity.fasta.bowtie2" actually is - is this a wrapper script?

ADD COMMENTlink written 13 months ago by predeus1.4k
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