Running rnaseq-trinity about command line

Question

[pr.khavari]

Hi everyone, I have a problem in running rnaseq_trinity, my command line is here:

/home/marziyeh/Software/trinityrnaseq-Trinity-v2.4.0/Trinity --seqType fq --max_memory 4G --left /home/marziyeh/Data/RNA-seq\ data/Main\ data/paired_32_L3_1_trimmed.fq.gz paired_33_L3_1_trimmed.fq.gz --right  paired_32_L3_2_trimmed.fq.gz paired_33_L3_2_trimmed.fq.gz --CPU 2  --output trinity_out_dir_32_32.fq.gz

, but it give this error!!!

Error, cannot locate file: paired_33_L3_1_trimmed.fq.gz at /home/marziyeh/Software/trinityrnaseq-Trinity-v2.4.0/Trinity line 2429. main::create_full_path(ARRAY(0x555a78a86df0), 1) called at /home/marziyeh/Software/trinityrnaseq-Trinity-v2.4.0/Trinity line 1159

Thanks so much for your suggestions.

[GenoMax]

You forgot to include the error!

A related note. Please do not use spaces in directory/file names in unix. Replace the space with an _ in all locations.

[pr.khavari]

Thanks for remembrance it.

[GenoMax]

I am pretty sure the problem is with the spaces you have in directory names. Please change the spaces to _ as I said above and re-try.

My bad. Missed the fact that more than one sequence file was being specified in --left and --right directives.

[pr.khavari]

I do your suggest but don't work. it is the last command line: /home/marziyeh/Software/trinityrnaseq-Trinity-v2.4.0/Trinity --seqType fq --max_memory 4G --left /home/marziyeh/Data/RNA-seqdata/Main_data/paired_32_L3_1_trimmed.fq.gz paired_33_L3_1_trimmed.fq.gz --right paired_32_L3_2_trimmed.fq.gz paired_33_L3_2_trimmed.fq.gz --CPU 2 --output trinity_out_dir_32_32.fq.gz

Answer 1

[Michael]

Trinity is behaving like most normal unix program (at least when it comes to file paths), all files need to be specified by either an absolute path, a relative path or exist in the working directory.

If you want to specify multiple input files for --right and --left they need to be comma separated, and specified with a path that exists, you specified the path only to the first file argument and not to the other files, and therefore trinity does not find them. Assume the data is in /home/marziyeh/Data/RNA-seq\ data/Main\ data/ It might look simple, but the best way of doing this is:

cd /home/marziyeh/Data/RNA-seq\ data/Main\ data/;
/home/marziyeh/Software/trinityrnaseq-Trinity-v2.4.0/Trinity --seqType fq --max_memory 4G \ 
--left paired_32_L3_1_trimmed.fq.gz,paired_33_L3_1_trimmed.fq.gz  \
--right paired_32_L3_2_trimmed.fq.gz,paired_33_L3_2_trimmed.fq.gz --CPU 2  --output trinity_out_dir_32_32.fq.gz

Despite other comments, the space in the directory name is not the problem here because it is correctly quoted, even though it is a nuisance, less so with tab completion.

A few more hints for running trinity:

• Create a little shell-script that contains these commands, to be able to reproduce the parameters afterwards, also if any of the jobs fail during a run, you need to re-run the script on the exact command to finish the failed jobs (re-entrant)
• This process will run for a very very long time for any reasonably sized transcriptome, 4G is very little RAM and 2 CPUs too, try to provide as many CPUs and RAM as possible
• If Trinity is running on a server you definitely need to run it via nohup, otherwise the process will be interrupted by any logout, and the output will flood your terminal

[pr.khavari]

yeah, I'm a verdant student, very good, that works, thanks so much.

[pr.khavari]

yeah, you right, because i run it on my laptop with 16G RAM for practice, I will run it on a server. One more question about result of it, Is it true that my results of trinity? /trinity_out_dir_32_32.fq.gz/insilico_read_normalization/tmp_normalized_reads >>>> both.fa left.fa left.fa.ok right.fa right.fa.ok