Error report in insilico_read

YanYan

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Jan 21, 2021, 3:04:04 AM1/21/21

to trinityrnaseq-users

I got error report in the step of insilico_read_normalization.

"quals and seq lines dont match in length"

The file is download from SRA ncbi

I attached the screen here:

(base) y@y-ThinkStation-P320:~$ Trinity --seqType fq --left /home/y/SRR/SRR6767056_1.fastq --right /home/y/SRR/SRR6767056_2.fastq --CPU 4 --max_memory 10G --output /home/y/trinity_out/

______ ____ ____ ____ ____ ______ __ __

| || \ | || \ | || || | |

| || D ) | | | _ | | | | || | |

|_| |_|| / | | | | | | | |_| |_|| ~ |

| | | \ | | | | | | | | | |___, |

| | | . \ | | | | | | | | | | |

|__| |__|\_||____||__|__||____| |__| |____/

Trinity-v2.11.0

Left read files: $VAR1 = [

'/home/y/SRR/SRR6767056_1.fastq'

];

Right read files: $VAR1 = [

'/home/y/SRR/SRR6767056_2.fastq'

];

Trinity version: Trinity-v2.11.0

** NOTE: Latest version of Trinity is v2.11.0, and can be obtained at:

https://github.com/trinityrnaseq/trinityrnaseq/releases

Thursday, January 21, 2021: 15:48:18 CMD: java -Xmx64m -XX:ParallelGCThreads=2 -jar /home/y/miniconda3/opt/trinity-2.11.0/util/support_scripts/ExitTester.jar 0

Thursday, January 21, 2021: 15:48:18 CMD: java -Xmx4g -XX:ParallelGCThreads=2 -jar /home/y/miniconda3/opt/trinity-2.11.0/util/support_scripts/ExitTester.jar 1

Thursday, January 21, 2021: 15:48:18 CMD: mkdir -p /home/y/trinity_out/chrysalis

----------------------------------------------------------------------------------

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

----------------------------------------------------------------------------------

---------------------------------------------------------------

------------ In silico Read Normalization ---------------------

-- (Removing Excess Reads Beyond 200 Coverage --

---------------------------------------------------------------

# running normalization on reads: $VAR1 = [

[

'/home/y/SRR/SRR6767056_1.fastq'

],

[

'/home/y/SRR/SRR6767056_2.fastq'

]

];

Thursday, January 21, 2021: 15:48:18 CMD: /home/y/miniconda3/opt/trinity-2.11.0/util/insilico_read_normalization.pl --seqType fq --JM 10G --max_cov 200 --min_cov 1 --CPU 4 --output /home/y/trinity_out/insilico_read_normalization --max_CV 10000 --left /home/y/SRR/SRR6767056_1.fastq --right /home/y/SRR/SRR6767056_2.fastq --pairs_together --PARALLEL_STATS

-prepping seqs

Converting input files. (both directions in parallel)CMD: seqtk-trinity seq -A -R 1 /home/y/SRR/SRR6767056_1.fastq >> left.fa

CMD: seqtk-trinity seq -A -R 2 /home/y/SRR/SRR6767056_2.fastq >> right.fa

Error encountered just after sequence entry[1]: 1/1', quals and seq lines dont match in length:

... corrupt file?Thread 1 terminated abnormally: Error, cmd: seqtk-trinity seq -A -R 1 /home/y/SRR/SRR6767056_1.fastq >> left.fa died with ret 768 at /home/y/miniconda3/opt/trinity-2.11.0/util/insilico_read_normalization.pl line 793.

Error encountered just after sequence entry[1]: 1/2', quals and seq lines dont match in length:

... corrupt file?Thread 2 terminated abnormally: Error, cmd: seqtk-trinity seq -A -R 2 /home/y/SRR/SRR6767056_2.fastq >> right.fa died with ret 768 at /home/y/miniconda3/opt/trinity-2.11.0/util/insilico_read_normalization.pl line 793.

Error, conversion thread failed at /home/y/miniconda3/opt/trinity-2.11.0/util/insilico_read_normalization.pl line 336.

Error, cmd: /home/y/miniconda3/opt/trinity-2.11.0/util/insilico_read_normalization.pl --seqType fq --JM 10G --max_cov 200 --min_cov 1 --CPU 4 --output /home/y/trinity_out/insilico_read_normalization --max_CV 10000 --left /home/y/SRR/SRR6767056_1.fastq --right /home/y/SRR/SRR6767056_2.fastq --pairs_together --PARALLEL_STATS died with ret 7424 at /home/y/miniconda3/bin/Trinity line 2826.

main::process_cmd("/home/y/miniconda3/opt/trinity-2.11.0/util/insilico_read_norm"...) called at /home/y/miniconda3/bin/Trinity line 3379

main::normalize("/home/y/trinity_out/insilico_read_normalization", 200, ARRAY(0x559d47eb27e8), ARRAY(0x559d47eb27d0)) called at /home/y/miniconda3/bin/Trinity line 3319

main::run_normalization(200, ARRAY(0x559d47eb27e8), ARRAY(0x559d47eb27d0)) called at /home/y/miniconda3/bin/Trinity line 1372

(base) y@y-ThinkStation-P320:~$

less -S /home/y/SRR/SRR6767056_1.fastq

'@1/1'

NATATTGTGATTAGACAGGGACAGAGTCATTGTGATTTATTTAATAGGAGTCGCCAAGGAAATGAATATCCTCTTTCATAAACAAC>

'+1/1'

#07B<<<BB0<B<<B<<<'<BBBBBB<B<<B<B<<<<00<<<<<0<<<<<B<7<<B777<<BBB<<<B7<BB<<<BBB<BB<<<<<>

'@2/1'

NATCGGAAGAGCACACGTCTGAACTCCAGTCACTAATGCGCATCTCGTATGCCGTCTTCTGCTTGAAAAAAAAGAACTGCCGCCAA>

'+2/1'

#0<FFFFFFFFFBFBFFIIBFF<FFIIFFFFFIIIIIBI7FIFFBFBFFFFBFF'<<BFFFFBFFFFFBBBB##############>

'@3/1'

GTTTCTTCGAATCATAAACAAATGTATTAAATTTCATGTAAACGCAGGAAAACTGCATTTGTATTGGCATATCCTGATCCGATGCT>

'+3/1'

BBBFFFFFFFFFFIIFIIIIIIIIFFIIIIIIIIIIIFFIIFIIIFFFFFIIIIIIIIIIIIFIIIFFFFIIIFFFFFFFFFFFFF>

'@4/1'

NGGCCAAATAAACAGGGGCGCCTGCACCAACTCTTTCAGCGTAGTTGCCTTTACGCAATAGTCGATGAATACGACCAACAGGGAAT>

'+4/1'

#0<FFFFFFFFFFIFFFIIIIFFFFIIIIIIIIIFFFIFFIIIIFFFFBFFFFFFFFFFFBFFFFFFFFBBFBFFFBFFFFB<BFB>

'@5/1'

CGTTGCTACTCCACAATTATTAGTTCCATATTTCATTTTCATAAAACCACCTTCTCCCCATGTCGTTCCATATGAGTTTTTAACAA>

'+5/1'

BB<BFFFFF<FFFFFFFBIFFFFFIIFIFFFBFFFBFFFIIIIFFFFIBFB7BFFBFFFFI<FFFBFFIFFFF<BB77BFFFFB<B>

less -S /home/y/SRR/SRR6767056_2.fastq

'@1/2'

GCTCATTCTCTGTTAGCGGTGTTTGATTACCTTCTTTTGACTACGATATTGAGGATTCGTCGTTGTTTATGAAAGAGGA>

'+1/2'

B<BFFFFFFFBFBFF<BFF70BFFFFFIIFFFBBFFIBFIIFFBFFFFFIFBFFFFIFBB<BB<B<BBBB<BBB<7<B<>

'@2/2'

GATGAGGAAAGGGGCCGGGGAGGGAAAAGGGGAGGGGGAAGGGGGGGGTCGCGGGGGTGGCCCGGTAATATAAAAAAAA>

'+2/2'

###############################################################################>

'@3/2'

GCAGTTATTTATAGAGGATTACCGATGGATAACTTAATTTTCTGTAAAAAAAATCTGATATATCCCCTTAGCATTATGA>

'+3/2'

BBBFFFFFFFFFFIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIFFFFFFFFFFFFFFFFFFFFF>

'@4/2'

GTTCGTACTTTCTTTCAAATCTTTCTTACAATAATGTCTGGTCGTGGAAAAGGCGGGAAGGTAAAGGCTAAGGCCAAGA>

'+4/2'

BBBFFFFFFFFFFIIBFIIIIIIIIIIIIIIIIIIIBFFIIIIIIFIIIIIBFIIIFBBBF'<BB<BBFBFBBBFBB7B>

'@5/2'

TACAACCCAAGCATCTGAAGCTGATTTGAAGAATAAAGTAGGAACAGTAGGCCCTATTAGTGTAGGAATAAATGGAGAT>

'+5/2'

00<BFB<FFFFFFFBBFFF7BFBFFBBBBFFBBFFFIFFFFBBFFFB7BBFFFFBF<BFF7<<B77B<FFFBFFFFBFB>

'@6/2'

GTAGGACTATCAGGCATGTCATAAGTTGATTTAACAGAAACACGTGGTGCTTCATTATCAATTTGCTGGCTAAATACTT>

'+6/2'

Brian Haas

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Jan 21, 2021, 9:21:01 AM1/21/21

to YanYan, trinityrnaseq-users

hi,

Is there really a '>' character at the end of each sequence line?

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Brian J. Haas
The Broad Institute
http://broadinstitute.org/~bhaas

YanYan

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Jan 21, 2021, 7:13:44 PM1/21/21

to trinityrnaseq-users

Sorry, there is > because I didn't fully expand the screen.

What's the problem? Is that in the head？

'@1/1'

NATATTGTGATTAGACAGGGACAGAGTCATTGTGATTTATTTAATAGGAGTCGCCAAGGAAATGAATATCCTCTTTCATAAACAACGACGAATCCTCAATATCGTAGTCA

'+1/1'

#07B<<<BB0<B<<B<<<'<BBBBBB<B<<B<B<<<<00<<<<<0<<<<<B<7<<B777<<BBB<<<B7<BB<<<BBB<BB<<<<<<<<<<7<7<0<<<7<<<<777<<<

'@2/1'

NATCGGAAGAGCACACGTCTGAACTCCAGTCACTAATGCGCATCTCGTATGCCGTCTTCTGCTTGAAAAAAAAGAACTGCCGCCAACACGGGCCACGCCAGAGGAGACCA

'+2/1'

#0<FFFFFFFFFBFBFFIIBFF<FFIIFFFFFIIIIIBI7FIFFBFBFFFFBFF'<<BFFFFBFFFFFBBBB######################################

'@3/1'

GTTTCTTCGAATCATAAACAAATGTATTAAATTTCATGTAAACGCAGGAAAACTGCATTTGTATTGGCATATCCTGATCCGATGCTATATATCAAAGCATTCCATGTCTG

'+3/1'

BBBFFFFFFFFFFIIFIIIIIIIIFFIIIIIIIIIIIFFIIFIIIFFFFFIIIIIIIIIIIIFIIIFFFFIIIFFFFFFFFFFFFFFFFFFFFFFFFFFFFBFFFFFFFF

'@4/1'

NGGCCAAATAAACAGGGGCGCCTGCACCAACTCTTTCAGCGTAGTTGCCTTTACGCAATAGTCGATGAATACGACCAACAGGGAATTGCAATCCTGCACGGGATGAACGA

'+4/1'

#0<FFFFFFFFFFIFFFIIIIFFFFIIIIIIIIIFFFIFFIIIIFFFFBFFFFFFFFFFFBFFFFFFFFBBFBFFFBFFFFB<BFBBFFBFFFFFFFFFB<BBFFFFFBB

'@5/1'

CGTTGCTACTCCACAATTATTAGTTCCATATTTCATTTTCATAAAACCACCTTCTCCCCATGTCGTTCCATATGAGTTTTTAACAATCCAGTACTTGTCCCTTGTTGTAA

'+5/1'

BB<BFFFFF<FFFFFFFBIFFFFFIIFIFFFBFFFBFFFIIIIFFFFIBFB7BFFBFFFFI<FFFBFFIFFFF<BB77BFFFFB<B<<BBB<<<<BBBBBBBBB<BB<BB

'@6/1'

CTTGGAGAAGATGAAAATGAAGTAATTGAAACTACAGATGAACATGGTGTTATTAAAAGGGTAATTCGTCATCTAATGGCACCTCCAGATATTCATTCTGTAACTTTTAC

'+6/1'

0<7B7BBB<B0B<<0BFF000B<BBFF0<BB<BBB7BFBFFF00<''B''7BF0B0BF0'''0<BBB7B7BB<'7B<7<<B<BBB0B<BBB<BB0<<<<<7<<B<B<<<<

'@7/1'

GATCGGAAGAGCACACGTCTGAACTCCAGTCACTAATGCGCATCTCGTATGCCGTCTTCTGCTTGAAAAAAAAGAGAATACATTTGAGAAGAAGGATGATTCAAGAAGGT

'+7/1'

BBBFFFFFFF0BFFFFFFFIFFFIBFFFFBFFFBBFIBFBFF0BFF<'7BBFIFBBBFF<F<<BBFBFF<B#######################################

screen.png

Brian Haas

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Jan 21, 2021, 9:17:32 PM1/21/21

to YanYan, trinityrnaseq-users

Hi,

You might try running the 'fastqc' software on your input fastqs. If there's something peculiar wrt formatting, I would hope that it would report it. If it doesn't find any problems, maybe you could give me access to the files that are cause the problem. I'll need to look at them directly to troubleshoot the issue.

best,

~b

To view this discussion on the web visit https://groups.google.com/d/msgid/trinityrnaseq-users/a4dc8929-8c14-459a-a3f9-50a3164ebf3fn%40googlegroups.com.

YanYan

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Jan 22, 2021, 3:03:43 AM1/22/21

to trinityrnaseq-users

(base) y@y-ThinkStation-P320:~$ fastqc -o /home/y/fastqc -t 4 /home/y/SRR/SRR6767056_1.fastq /home/y/SRR/SRR6767056_2.fastq

Failed to process /home/y/SRR/SRR6767056_1.fastq

uk.ac.babraham.FastQC.Sequence.SequenceFormatException: ID line didn't start with '@'

at uk.ac.babraham.FastQC.Sequence.FastQFile.readNext(FastQFile.java:158)

at uk.ac.babraham.FastQC.Sequence.FastQFile.<init>(FastQFile.java:89)

at uk.ac.babraham.FastQC.Sequence.SequenceFactory.getSequenceFile(SequenceFactory.java:106)

at uk.ac.babraham.FastQC.Sequence.SequenceFactory.getSequenceFile(SequenceFactory.java:62)

at uk.ac.babraham.FastQC.Analysis.OfflineRunner.processFile(OfflineRunner.java:129)

at uk.ac.babraham.FastQC.Analysis.OfflineRunner.<init>(OfflineRunner.java:102)

at uk.ac.babraham.FastQC.FastQCApplication.main(FastQCApplication.java:316)

Failed to process /home/y/SRR/SRR6767056_2.fastq