Trinity run failed -- Phase 1

Question

[ddowlin]

Hi all,

I am using Trinity to de novo assemble a transcriptome.

Unfortunately my run fails very early on. i.e. in Phase 1 :Clustering of RNA-Seq Reads.

The following is printed to output:

----------------------------------------------------------------------------------
-------------- Trinity Phase 1: Clustering of RNA-Seq Reads  ---------------------
----------------------------------------------------------------------------------

Converting input files. (in parallel)Thursday, July 28, 2016: 11:25:55  
CMD: gunzip -c /home/trinity/R1.fastq.gz | /home/trinityrnaseq-2.2.0/trinity-plugins/fastool/fastool --append /1 --to-fasta >> left.fa 2> /home/trinity/R1.fastq.gz.readcount 

Thursday, July 28, 2016: 11:25:55   CMD: gunzip -c /home/trinity/R2.fastq.gz | /home/trinityrnaseq-2.2.0/trinity-plugins/fastool/fastool --append /2 --to-fasta >> right.fa 2> /home/trinity/R2.fastq.gz.readcount 

Thread 2 terminated abnormally: Error, cmd: gunzip -c /home/trinity/R2.fastq.gz | /fsimb/imbc_home/trinityrnaseq-2.2.0/trinity-plugins/fastool/fastool --append /2 --to-fasta >> right.fa 2> /home/trinity/R2.fastq.gz.readcount  died with ret 256 at ./Trinity line 2206.


Thread 1 terminated abnormally: Error, counts of reads in FQ: 41119574 (as per gunzip -c /home/trinity/R1.fastq.gz | wc -l) doesn't match fastool's report of FA records: 4031700  at /home/trinityrnaseq-2.2.0/Trinity line 3087 thread 1.

main::ensure_complete_FQtoFA_conversion("gunzip -c /home/trinity/R2.fastqc.gz"..., "/home/trinity/R2.fastqc.gz) called at /home/trinityrnaseq-2.2.0/Trinity line 2116 thread 1

main::prep_seqs(ARRAY(0xc24ce0), "fq", "left", undef) called at /home/trinityrnaseq-2.2.0/Trinity line 1314 thread 1
eval {...} called at /home/trinityrnaseq-2.2.0/Trinity line 1314 thread 1


Trinity run failed. Must investigate error above.

There seems to be two errors here:

1.) Error, cmd: gunzip -c [...] died with ret 256 and

2.) Error, counts of reads in FQ: 41119574 (as per gunzip -c /home/trinity/R1.fastq.gz | wc -l) doesn't match fastool's report of FA record

Is there perhaps a problem with my fastq files?

Any help would be greatly appreciated.

Thanks in advance.

[Farbod]

Hi,

You can ask it here: https://groups.google.com/forum/#!forum/trinityrnaseq-users

their support is great.

[GenoMax]

Before you conclude that there is a problem with your fastq files have you tried to run this a second time? Is there enough disk space available at /home/trinity to hold the uncompressed files?

[ddowlin]

Thanks for the suggestion.

I unziped both fastq.gz files and re-ran Trinity.

It now gets stuck at the Jellyfish step. The specific message I am getting is:

 ----------- Jellyfish  --------------------
-- (building a k-mer catalog from reads) --
-------------------------------------------

* Running CMD: /home/trinityrnaseq-2.2.0/trinity-plugins/jellyfish/bin/jellyfish count -t 6 -m 25 -s 5936695335  --canonical  both.fa
sh: line 1: 34469 Killed                  
/home/trinityrnaseq-2.2.0/trinity-plugins/jellyfish/bin/jellyfish count -t 6 -m 25 -s 59366953$

Trinity run failed. Must investigate error above.

[GenoMax]

Are you running this on a cluster under a job scheduler or a standalone server? How much memory do you have available?

This may indicate an available memory error since the job appears to be killed by the system now.

[ddowlin]

I am using a cluster with LSF scheduler. I requested 4 cores with 2G each.

[GenoMax]

Trinity requires large amount of RAM (see this and also this). 2G is not going to be enough.

If you don't have access to adequate amount of RAM locally, then consider using the galaxy server at Indiana. People have reported success using that resource for trinity here.

[ddowlin]

Thanks. I increased the memory and it seems to be running now.

[WouterDeCoster]

You could try to run the failing commands just in your shell to get a better idea of the error message...

[ddowlin]

I ran the first command: I get the following error

terminate called after throwing an instance of 'std::runtime_error'
what():  Can't open file 'both.fa'
Aborted

I have checked the out_directory and I can open the 'both.fa' without a problem.

Answer 1

[Buffo]

Try with cufflinks, it works better than Trinity: Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks Cole Trapnell, Adam Roberts, Loyal Goff, Geo Pertea, Daehwan Kim, David R Kelley, Harold Pimentel, Steven L Salzberg, John L Rinn & Lior Pachter.

[WouterDeCoster]

If I'm not terribly mistaken Trinity and cufflinks don't have the same functionality... cufflinks uses aligned reads.