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 align_and_estimate_abundance.pl script provided by Trinity, with the following parameters:

home/genomica/DATA/Software/Trinity/trinityrnaseq-2.8.5/util/align_and_estimate_abundance.pl
--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/
--prep_reference

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:

$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/align_and_estimate_abundance.pl 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.