Trinity assembly error

Question

[MAPK]

Hi All, I am running this assembly using Trinity feeding the following command:

Trinity --seqType fq --max_memory 100G --right file_1.fastq
--left file_2.fastq --CPU 16 --output SRR-trinity-output

However, it keeps generating this error shown below. Can someone please help me resolve this issue here. Thanks.

Error I am getting:

Wednesday, November 8, 2017: 18:40:29   CMD: java -Xmx64m -XX:ParallelGCThreads=2  -jar /home/owner/Desktop/pan/executables/trinityrnaseq-Trinity-v2.5.1/util/support_scripts/ExitTester.jar 0
Wednesday, November 8, 2017: 18:40:29   CMD: java -Xmx64m -XX:ParallelGCThreads=2  -jar /home/owner/Desktop/pan/executables/trinityrnaseq-Trinity-v2.5.1/util/support_scripts/ExitTester.jar 1


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

---------------------------------------------------------------
------------ In silico Read Normalization ---------------------
-- (Removing Excess Reads Beyond 50 Coverage --
---------------------------------------------------------------

# running normalization on reads: $VAR1 = [
          [
            '/media/owner/b54f3251-5380-4288-9ddf-fa3357ea8294/test-plantae/pan_sra/SRX/test/file_2.fastq'
          ],
          [
            '/media/owner/b54f3251-5380-4288-9ddf-fa3357ea8294/test-plantae/pan_sra/SRX/test/file_1.fastq'
          ]
        ];


Wednesday, November 8, 2017: 18:40:29   CMD: /home/owner/Desktop/pan/executables/trinityrnaseq-Trinity-v2.5.1/util/insilico_read_normalization.pl --seqType fq --JM 100G  --max_cov 50 --CPU 16 --output /media/owner/b54f3251-5380-4288-9ddf-fa3357ea8294/test-plantae/pan_sra/SRX/test/file-trinity-output/insilico_read_normalization   --max_pct_stdev 10000  --left /media/owner/b54f3251-5380-4288-9ddf-fa3357ea8294/test-plantae/pan_sra/SRX/test/file_2.fastq --right /media/owner/b54f3251-5380-4288-9ddf-fa3357ea8294/test-plantae/pan_sra/SRX/test/file_1.fastq --pairs_together --PARALLEL_STATS  
Converting input files. (both directions in parallel)CMD: seqtk-trinity seq -A /media/owner/b54f3251-5380-4288-9ddf-fa3357ea8294/test-plantae/pan_sra/SRX/test/file_2.fastq >> left.fa
Error, not recognizing read name formatting: [file.1]

If your data come from SRA, be sure to dump the fastq file like so:

    SRA_TOOLKIT/fastq-dump --defline-seq '@$sn[_$rn]/$ri' --split-files file.sra 

Thread 1 terminated abnormally: Error, cmd: seqtk-trinity seq -A /media/owner/b54f3251-5380-4288-9ddf-fa3357ea8294/test-plantae/pan_sra/SRX/test/file_2.fastq >> left.fa died with ret 512 at /home/owner/Desktop/pan/executables/trinityrnaseq-Trinity-v2.5.1/util/insilico_read_normalization.pl line 758.
CMD: seqtk-trinity seq -A /media/owner/b54f3251-5380-4288-9ddf-fa3357ea8294/test-plantae/pan_sra/SRX/test/file_1.fastq >> right.fa
Error, not recognizing read name formatting: [file.1]

If your data come from SRA, be sure to dump the fastq file like so:

    SRA_TOOLKIT/fastq-dump --defline-seq '@$sn[_$rn]/$ri' --split-files file.sra 

Thread 2 terminated abnormally: Error, cmd: seqtk-trinity seq -A /media/owner/b54f3251-5380-4288-9ddf-fa3357ea8294/test-plantae/pan_sra/SRX/test/file_1.fastq >> right.fa died with ret 512 at /home/owner/Desktop/pan/executables/trinityrnaseq-Trinity-v2.5.1/util/insilico_read_normalization.pl line 758.
Error, conversion thread failed at /home/owner/Desktop/pan/executables/trinityrnaseq-Trinity-v2.5.1/util/insilico_read_normalization.pl line 329.
Error, cmd: /home/owner/Desktop/pan/executables/trinityrnaseq-Trinity-v2.5.1/util/insilico_read_normalization.pl --seqType fq --JM 100G  --max_cov 50 --CPU 16 --output /media/owner/b54f3251-5380-4288-9ddf-fa3357ea8294/test-plantae/pan_sra/SRX/test/file-trinity-output/insilico_read_normalization   --max_pct_stdev 10000  --left /media/owner/b54f3251-5380-4288-9ddf-fa3357ea8294/test-plantae/pan_sra/SRX/test/file_2.fastq --right /media/owner/b54f3251-5380-4288-9ddf-fa3357ea8294/test-plantae/pan_sra/SRX/test/file_1.fastq --pairs_together --PARALLEL_STATS   died with ret 7424 at /home/owner/Desktop/pan/executables/trinityrnaseq-Trinity-v2.5.1//Trinity line 2544.
    main::process_cmd('/home/owner/Desktop/pan/executables/trinityrnaseq-Trinity-v...') called at /home/owner/Desktop/pan/executables/trinityrnaseq-Trinity-v2.5.1//Trinity line 3090
    main::normalize('/media/owner/b54f3251-5380-4288-9ddf-fa3357ea8294/test...', 50, 'ARRAY(0xd58eb8)', 'ARRAY(0xd64760)') called at /home/owner/Desktop/pan/executables/trinityrnaseq-Trinity-v2.5.1//Trinity line 3037
    main::run_normalization(50, 'ARRAY(0xd58eb8)', 'ARRAY(0xd64760)') called at /home/owner/Desktop/pan/executables/trinityrnaseq-Trinity-v2.5.1//Trinity line 1297

[Kevin Blighe]

Looks like it could be a problem with the FASTQ formatting. I've seen the error reported a few times across the WWW but no solution. Take a look here in order to validate your FASTQ files: Fastq Quality Read And Score Length Check

Edit: just noticed that you're also specifying file1 as right reads and file2 as left. Did you mean to do that?

[MAPK]

Thanks. Yes, the two files are part of paired reads from fastq-dump --defline-seq '@$sn[_$rn]/$ri' --split-files *.sra option generating left and right fastq files.

[MAPK]

Additionally, it is generating the same error for the test fastq files from the Trinity package itself. I tried make test_trinity and it is generating the same error.

[Kevin Blighe]

I can't be sure but it looks like a bug in the program. There appears to be a solution here: https://groups.google.com/forum/#!topic/trinityrnaseq-users/Mo4hrTo5dSM

[Kevin Blighe]

Thanks for posting the answer below. No doubt others will encounter the same problem

Answer 1

[MAPK]

Ok. I finally figured out the problem for this: Trinity works well with Java version 7. So before running Trinity, we need to make sure we set java version 7 like this:

To get a list of your installed Java platforms, run the following command from the terminal:

sudo update-alternatives --config java

This will give you a list output similar to this:

There are 2 choices for the alternative java (providing /usr/bin/java).
   Selection    Path                                           Priority   Status
  ------------------------------------------------------------
  0            /usr/lib/jvm/java-6-oracle/jre/bin/java         1070      auto mode
  1            /usr/lib/jvm/java-7-openjdk-i386/jre/bin/java   1051      manual mode
* 2            /usr/lib/jvm/java-6-openjdk-i386/jre/bin/java   1069      manual mode
Press enter to keep the current choice[*], or type selection number:

Here select the number that represent java-7, so chose 1.

Now, Trinity should be running properly. Check by invoking this command: make test_trinity

###################################################################
Butterfly assemblies are written to /home/owner/Desktop/executables/trinityrnaseq-2.0.6/sample_data/test_Trinity_Assembly/trinity_out_dir/Trinity.fasta
###################################################################



##### Done Running Trinity #####

UPDATE*** This above solution works well for Trinity version: v2.0.6