Trinity for single end library distributed among different lanes.

Question

[waqaskhokhar999]

RNA-seq data is single ended and distributed among different lanes. I am trying to assembel the transcriptome using this command:

trinity/trinityrnaseq-Trinity-v2.8.3/Trinity --seqType fq --max_memory 10G --single SRR3460466.fastq,SRR3460467.fastq,SRR3460468.fastq,SRR3460469.fastq,SRR3460470.fastq,SRR3460471.fastq,SRR3460472.fastq,SRR3460473.fastq,SRR3460474.fastq,SRR3460475.fastq,SRR3460476.fastq,SRR3460477.fastq

But I am getting this error

Error, not recognizing read name formatting: [SRR3460466.1]

Command line summary:

waqas@waqas-Hybris:/media/waqas/waqas_data/19_Accessions_w/new/BUR/fastq$ trinity/trinityrnaseq-Trinity-v2.8.3/Trinity --seqType fq --max_memory 10G --single SRR3460466.fastq,SRR3460467.fastq,SRR3460468.fastq,SRR3460469.fastq,SRR3460470.fastq,SRR3460471.fastq,SRR3460472.fastq,SRR3460473.fastq,SRR3460474.fastq,SRR3460475.fastq,SRR3460476.fastq,SRR3460477.fastq


     ______  ____   ____  ____   ____  ______  __ __
    |      ||    \ |    ||    \ |    ||      ||  |  |
    |      ||  D  ) |  | |  _  | |  | |      ||  |  |
    |_|  |_||    /  |  | |  |  | |  | |_|  |_||  ~  |
      |  |  |    \  |  | |  |  | |  |   |  |  |___, |
      |  |  |  .  \ |  | |  |  | |  |   |  |  |     |
      |__|  |__|\_||____||__|__||____|  |__|  |____/

    Trinity-v2.8.3



Single read files: $VAR1 = [
          'SRR3460466.fastq',
          'SRR3460467.fastq',
          'SRR3460468.fastq',
          'SRR3460469.fastq',
          'SRR3460470.fastq',
          'SRR3460471.fastq',
          'SRR3460472.fastq',
          'SRR3460473.fastq',
          'SRR3460474.fastq',
          'SRR3460475.fastq',
          'SRR3460476.fastq',
          'SRR3460477.fastq'
        ];
Trinity version: Trinity-v2.8.3
** NOTE: Latest version of Trinity is Trinity-v2.8.4, and can be obtained at:
    https://github.com/trinityrnaseq/trinityrnaseq/releases

Wednesday, November 7, 2018: 16:00:20   CMD: java -Xmx64m -XX:ParallelGCThreads=2  -jar /media/waqas/waqas_data/19_Accessions_w/new/BUR/fastq/trinity/trinityrnaseq-Trinity-v2.8.3/util/support_scripts/ExitTester.jar 0
Wednesday, November 7, 2018: 16:00:20   CMD: java -Xmx4g -XX:ParallelGCThreads=2  -jar /media/waqas/waqas_data/19_Accessions_w/new/BUR/fastq/trinity/trinityrnaseq-Trinity-v2.8.3/util/support_scripts/ExitTester.jar 1


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

---------------------------------------------------------------
------------ In silico Read Normalization ---------------------
-- (Removing Excess Reads Beyond 200 Coverage --
---------------------------------------------------------------

# running normalization on reads: $VAR1 = [
          [
            '/media/waqas/waqas_data/19_Accessions_w/new/BUR/fastq/SRR3460466.fastq',
            '/media/waqas/waqas_data/19_Accessions_w/new/BUR/fastq/SRR3460467.fastq',
            '/media/waqas/waqas_data/19_Accessions_w/new/BUR/fastq/SRR3460468.fastq',
            '/media/waqas/waqas_data/19_Accessions_w/new/BUR/fastq/SRR3460469.fastq',
            '/media/waqas/waqas_data/19_Accessions_w/new/BUR/fastq/SRR3460470.fastq',
            '/media/waqas/waqas_data/19_Accessions_w/new/BUR/fastq/SRR3460471.fastq',
            '/media/waqas/waqas_data/19_Accessions_w/new/BUR/fastq/SRR3460472.fastq',
            '/media/waqas/waqas_data/19_Accessions_w/new/BUR/fastq/SRR3460473.fastq',
            '/media/waqas/waqas_data/19_Accessions_w/new/BUR/fastq/SRR3460474.fastq',
            '/media/waqas/waqas_data/19_Accessions_w/new/BUR/fastq/SRR3460475.fastq',
            '/media/waqas/waqas_data/19_Accessions_w/new/BUR/fastq/SRR3460476.fastq',
            '/media/waqas/waqas_data/19_Accessions_w/new/BUR/fastq/SRR3460477.fastq'
          ]
        ];


Wednesday, November 7, 2018: 16:00:20   CMD: /media/waqas/waqas_data/19_Accessions_w/new/BUR/fastq/trinity/trinityrnaseq-Trinity-v2.8.3/util/insilico_read_normalization.pl --seqType fq --JM 10G  --max_cov 200 --min_cov 1 --CPU 2 --output /media/waqas/waqas_data/19_Accessions_w/new/BUR/fastq/trinity_out_dir/insilico_read_normalization   --max_pct_stdev 10000  --single /media/waqas/waqas_data/19_Accessions_w/new/BUR/fastq/SRR3460466.fastq,/media/waqas/waqas_data/19_Accessions_w/new/BUR/fastq/SRR3460467.fastq,/media/waqas/waqas_data/19_Accessions_w/new/BUR/fastq/SRR3460468.fastq,/media/waqas/waqas_data/19_Accessions_w/new/BUR/fastq/SRR3460469.fastq,/media/waqas/waqas_data/19_Accessions_w/new/BUR/fastq/SRR3460470.fastq,/media/waqas/waqas_data/19_Accessions_w/new/BUR/fastq/SRR3460471.fastq,/media/waqas/waqas_data/19_Accessions_w/new/BUR/fastq/SRR3460472.fastq,/media/waqas/waqas_data/19_Accessions_w/new/BUR/fastq/SRR3460473.fastq,/media/waqas/waqas_data/19_Accessions_w/new/BUR/fastq/SRR3460474.fastq,/media/waqas/waqas_data/19_Accessions_w/new/BUR/fastq/SRR3460475.fastq,/media/waqas/waqas_data/19_Accessions_w/new/BUR/fastq/SRR3460476.fastq,/media/waqas/waqas_data/19_Accessions_w/new/BUR/fastq/SRR3460477.fastq
CMD: seqtk-trinity seq -A /media/waqas/waqas_data/19_Accessions_w/new/BUR/fastq/SRR3460466.fastq >> single.fa
Error, not recognizing read name formatting: [SRR3460466.1]

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 

Error, cmd: seqtk-trinity seq -A /media/waqas/waqas_data/19_Accessions_w/new/BUR/fastq/SRR3460466.fastq >> single.fa died with ret 512 at /media/waqas/waqas_data/19_Accessions_w/new/BUR/fastq/trinity/trinityrnaseq-Trinity-v2.8.3/util/insilico_read_normalization.pl line 762.
Error, cmd: /media/waqas/waqas_data/19_Accessions_w/new/BUR/fastq/trinity/trinityrnaseq-Trinity-v2.8.3/util/insilico_read_normalization.pl --seqType fq --JM 10G  --max_cov 200 --min_cov 1 --CPU 2 --output /media/waqas/waqas_data/19_Accessions_w/new/BUR/fastq/trinity_out_dir/insilico_read_normalization   --max_pct_stdev 10000  --single /media/waqas/waqas_data/19_Accessions_w/new/BUR/fastq/SRR3460466.fastq,/media/waqas/waqas_data/19_Accessions_w/new/BUR/fastq/SRR3460467.fastq,/media/waqas/waqas_data/19_Accessions_w/new/BUR/fastq/SRR3460468.fastq,/media/waqas/waqas_data/19_Accessions_w/new/BUR/fastq/SRR3460469.fastq,/media/waqas/waqas_data/19_Accessions_w/new/BUR/fastq/SRR3460470.fastq,/media/waqas/waqas_data/19_Accessions_w/new/BUR/fastq/SRR3460471.fastq,/media/waqas/waqas_data/19_Accessions_w/new/BUR/fastq/SRR3460472.fastq,/media/waqas/waqas_data/19_Accessions_w/new/BUR/fastq/SRR3460473.fastq,/media/waqas/waqas_data/19_Accessions_w/new/BUR/fastq/SRR3460474.fastq,/media/waqas/waqas_data/19_Accessions_w/new/BUR/fastq/SRR3460475.fastq,/media/waqas/waqas_data/19_Accessions_w/new/BUR/fastq/SRR3460476.fastq,/media/waqas/waqas_data/19_Accessions_w/new/BUR/fastq/SRR3460477.fastq died with ret 512 at trinity/trinityrnaseq-Trinity-v2.8.3/Trinity line 2684.
    main::process_cmd("/media/waqas/waqas_data/19_Accessions_w/new/BUR/fastq/trinity"...) called at trinity/trinityrnaseq-Trinity-v2.8.3/Trinity line 3230
    main::normalize("/media/waqas/waqas_data/19_Accessions_w/new/BUR/fastq/trinity"..., 200, ARRAY(0x56443710b4b0)) called at trinity/trinityrnaseq-Trinity-v2.8.3/Trinity line 3177
    main::run_normalization(200, ARRAY(0x56443710b4b0)) called at trinity/trinityrnaseq-Trinity-v2.8.3/Trinity line 1319

Answer 1

[h.mon]

Reading the error message carefully, you will see:

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

See also the Trinity FAQ.

[waqaskhokhar999]

I have used SRA_TOOLKIT/fastq-dump to dump the fastq files, since my data is single ended, do i still need to use --split-files as this option is used for paired ended data