Question: Trinity assembly error
0
gravatar for MAPK
17 months ago by
MAPK1.4k
United States
MAPK1.4k wrote:

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
rnaseq • 1.3k views
ADD COMMENTlink modified 17 months ago • written 17 months ago by MAPK1.4k
1

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?

ADD REPLYlink modified 17 months ago • written 17 months ago by Kevin Blighe41k

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.

ADD REPLYlink modified 17 months ago • written 17 months ago by MAPK1.4k

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.

ADD REPLYlink modified 17 months ago • written 17 months ago by MAPK1.4k
1

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

ADD REPLYlink written 17 months ago by Kevin Blighe41k

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

ADD REPLYlink written 17 months ago by Kevin Blighe41k
1
gravatar for MAPK
17 months ago by
MAPK1.4k
United States
MAPK1.4k wrote:

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

ADD COMMENTlink modified 17 months ago • written 17 months ago by MAPK1.4k
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