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Hwaida Mohamed

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Feb 26, 2021, 3:16:43 PM2/26/21

to trinityrnaseq-users

hello i am trying to run my fastq files on trinity using amazon cloud instance but i am facing a problem

every time i run i got this error message below

i tried to update trinity or to unzip file but nothing change, could you please help me?

Trinity --seqType fq --max_memory 32G --left $R1 --right $R2 --SS_lib_type RF --CPU 6

______ ____ ____ ____ ____ ______ __ __

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

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

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

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

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

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

Trinity-v2.8.5

Left read files: $VAR1 = [

'SRR7663002_RNA-Seq_of_Lasiopodomys_brandtii_adult_male_brain_1.pe.trim.fastq'

];

Right read files: $VAR1 = [

'SRR7663002_RNA-Seq_of_Lasiopodomys_brandtii_adult_male_brain_2.pe.trim.fastq'

];

Trinity version: Trinity-v2.8.5

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

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

Friday, February 26, 2021: 20:05:53 CMD: java -Xmx64m -XX:ParallelGCThreads=2 -jar /home/ubuntu/miniconda3/envs/ngs1/opt/trinity-2.8.5/util/support_scripts/ExitTester.jar 0

Friday, February 26, 2021: 20:05:54 CMD: java -Xmx4g -XX:ParallelGCThreads=2 -jar /home/ubuntu/miniconda3/envs/ngs1/opt/trinity-2.8.5/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 = [

[

'/home/ubuntu/SRR7663002_RNA-Seq_of_Lasiopodomys_brandtii_adult_male_brain_1.pe.trim.fastq'

[

'/home/ubuntu/SRR7663002_RNA-Seq_of_Lasiopodomys_brandtii_adult_male_brain_2.pe.trim.fastq'

]

];

Friday, February 26, 2021: 20:05:54 CMD: /home/ubuntu/miniconda3/envs/ngs1/opt/trinity-2.8.5/util/insilico_read_normalization.pl --seqType fq --JM 32G --max_cov 200 --min_cov 1 --CPU 6 --output /home/ubuntu/trinity_out_dir/insilico_read_normalization --max_CV 10000 --SS_lib_type RF --left /home/ubuntu/SRR7663002_RNA-Seq_of_Lasiopodomys_brandtii_adult_male_brain_1.pe.trim.fastq --right /home/ubuntu/SRR7663002_RNA-Seq_of_Lasiopodomys_brandtii_adult_male_brain_2.pe.trim.fastq --pairs_together --PARALLEL_STATS

-prepping seqs

Converting input files. (both directions in parallel)CMD: seqtk-trinity seq -r -A /home/ubuntu/SRR7663002_RNA-Seq_of_Lasiopodomys_brandtii_adult_male_brain_1.pe.trim.fastq >> left.fa

Error, not recognizing read name formatting: [SRR7663002.15]

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 -r -A /home/ubuntu/SRR7663002_RNA-Seq_of_Lasiopodomys_brandtii_adult_male_brain_1.pe.trim.fastq >> left.fa died with ret 512 at /home/ubuntu/miniconda3/envs/ngs1/opt/trinity-2.8.5/util/insilico_read_normalization.pl line 788.

CMD: seqtk-trinity seq -A /home/ubuntu/SRR7663002_RNA-Seq_of_Lasiopodomys_brandtii_adult_male_brain_2.pe.trim.fastq >> right.fa

Error, not recognizing read name formatting: [SRR7663002.15]

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 /home/ubuntu/SRR7663002_RNA-Seq_of_Lasiopodomys_brandtii_adult_male_brain_2.pe.trim.fastq >> right.fa died with ret 512 at /home/ubuntu/miniconda3/envs/ngs1/opt/trinity-2.8.5/util/insilico_read_normalization.pl line 788.

Error, conversion thread failed at /home/ubuntu/miniconda3/envs/ngs1/opt/trinity-2.8.5/util/insilico_read_normalization.pl line 336.

Error, cmd: /home/ubuntu/miniconda3/envs/ngs1/opt/trinity-2.8.5/util/insilico_read_normalization.pl --seqType fq --JM 32G --max_cov 200 --min_cov 1 --CPU 6 --output /home/ubuntu/trinity_out_dir/insilico_read_normalization --max_CV 10000 --SS_lib_type RF --left /home/ubuntu/SRR7663002_RNA-Seq_of_Lasiopodomys_brandtii_adult_male_brain_1.pe.trim.fastq --right /home/ubuntu/SRR7663002_RNA-Seq_of_Lasiopodomys_brandtii_adult_male_brain_2.pe.trim.fastq --pairs_together --PARALLEL_STATS died with ret 7424 at /home/ubuntu/miniconda3/envs/ngs1/bin/Trinity line 2745.

main::process_cmd("/home/ubuntu/miniconda3/envs/ngs1/opt/trinity-2.8.5/util/insi"...) called at /home/ubuntu/miniconda3/envs/ngs1/bin/Trinity line 3295

main::normalize("/home/ubuntu/trinity_out_dir/insilico_read_normalization", 200, ARRAY(0x55ccf09505b8), ARRAY(0x55ccf09505a0)) called at /home/ubuntu/miniconda3/envs/ngs1/bin/Trinity line 3238

main::run_normalization(200, ARRAY(0x55ccf09505b8), ARRAY(0x55ccf09505a0)) called at /home/ubuntu/miniconda3/envs/ngs1/bin/Trinity line 1350

Mark Chapman

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Feb 26, 2021, 4:49:23 PM2/26/21

to Hwaida Mohamed, trinityrnaseq-users

Hello Hwaida,

Is the data from SRA? The read names or something aren't being recognised. You can use the suggestion in the error msg below if its from SRA, or see on the trinity webpage/Google group how to fix this, eg you can use sed to be sure the read names are correct.

Best, Mark

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Sarah Haines

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Apr 1, 2021, 12:43:54 PM4/1/21

to trinityrnaseq-users

Hello all,

I'm having a similar issue where I'm receiving this error message and Trinity ends abruptly. Trinity runs through Phase 1: Clustering of RNA-seq reads and quality trimming via trimmomatic but then ends here:

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

-- (Removing Excess Reads Beyond 200 Coverage --

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

# running normalization on reads: $VAR1 = [

[

'50_2-D_023_170_S1_L004_R1_001_rRNA.fastq.PwU.qtrim.fq',

'50_3-D_011_182_S2_L004_R1_001_rRNA.fastq.PwU.qtrim.fq',

'50_4-D_094_099_S3_L004_R1_001_rRNA.fastq.PwU.qtrim.fq',

'85_2-D_082_111_S4_L004_R1_001_rRNA.fastq.PwU.qtrim.fq',

'85_4-D_070_123_S5_L004_R1_001_rRNA.fastq.PwU.qtrim.fq',

'85_5-D_058_135_S6_L004_R1_001_rRNA.fastq.PwU.qtrim.fq',

'95_1-D_046_147_S7_L004_R1_001_rRNA.fastq.PwU.qtrim.fq',

'95_3-D_034_159_S8_L004_R1_001_rRNA.fastq.PwU.qtrim.fq',

'95_4-D_022_171_S9_L004_R1_001_rRNA.fastq.PwU.qtrim.fq'

[

'50_2-D_023_170_S1_L004_R2_001_rRNA.fastq.PwU.qtrim.fq',

'50_3-D_011_182_S2_L004_R2_001_rRNA.fastq.PwU.qtrim.fq',

'50_4-D_094_099_S3_L004_R2_001_rRNA.fastq.PwU.qtrim.fq',

'85_2-D_082_111_S4_L004_R2_001_rRNA.fastq.PwU.qtrim.fq',

'85_4-D_070_123_S5_L004_R2_001_rRNA.fastq.PwU.qtrim.fq',

'85_5-D_058_135_S6_L004_R2_001_rRNA.fastq.PwU.qtrim.fq',

'95_1-D_046_147_S7_L004_R2_001_rRNA.fastq.PwU.qtrim.fq',

'95_3-D_034_159_S8_L004_R2_001_rRNA.fastq.PwU.qtrim.fq',

'95_4-D_022_171_S9_L004_R2_001_rRNA.fastq.PwU.qtrim.fq'

]

];

Thursday, April 1, 2021: 11:42:24 CMD: /apps/trinityrnaseq/2.11.0/util/insilico_read_normalization.pl --seqType fq --JM 2500G --max_cov 200 --min_cov 1 --CPU 75 --output /tmp/slurmtmp.3675441/trinity_output_files_Haines_Dust/insilico_read_normalization --max_CV 10000 --left 50_2-D_023_170_S1_L004_R1_001_rRNA.fastq.PwU.qtrim.fq,50_3-D_011_182_S2_L004_R1_001_rRNA.fastq.PwU.qtrim.fq,50_4-D_094_099_S3_L004_R1_001_rRNA.fastq.PwU.qtrim.fq,85_2-D_082_111_S4_L004_R1_001_rRNA.fastq.PwU.qtrim.fq,85_4-D_070_123_S5_L004_R1_001_rRNA.fastq.PwU.qtrim.fq,85_5-D_058_135_S6_L004_R1_001_rRNA.fastq.PwU.qtrim.fq,95_1-D_046_147_S7_L004_R1_001_rRNA.fastq.PwU.qtrim.fq,95_3-D_034_159_S8_L004_R1_001_rRNA.fastq.PwU.qtrim.fq,95_4-D_022_171_S9_L004_R1_001_rRNA.fastq.PwU.qtrim.fq --right 50_2-D_023_170_S1_L004_R2_001_rRNA.fastq.PwU.qtrim.fq,50_3-D_011_182_S2_L004_R2_001_rRNA.fastq.PwU.qtrim.fq,50_4-D_094_099_S3_L004_R2_001_rRNA.fastq.PwU.qtrim.fq,85_2-D_082_111_S4_L004_R2_001_rRNA.fastq.PwU.qtrim.fq,85_4-D_070_123_S5_L004_R2_001_rRNA.fastq.PwU.qtrim.fq,85_5-D_058_135_S6_L004_R2_001_rRNA.fastq.PwU.qtrim.fq,95_1-D_046_147_S7_L004_R2_001_rRNA.fastq.PwU.qtrim.fq,95_3-D_034_159_S8_L004_R2_001_rRNA.fastq.PwU.qtrim.fq,95_4-D_022_171_S9_L004_R2_001_rRNA.fastq.PwU.qtrim.fq --pairs_together --PARALLEL_STATS

-prepping seqs

Converting input files. (both directions in parallel)CMD: seqtk-trinity seq -A -R 1 /tmp/slurmtmp.3675441/trinity_output_files_Haines_Dust/50_2-D_023_170_S1_L004_R1_001_rRNA.fastq.PwU.qtrim.fq >> left.fa

CMD: seqtk-trinity seq -A -R 2 /tmp/slurmtmp.3675441/trinity_output_files_Haines_Dust/50_2-D_023_170_S1_L004_R2_001_rRNA.fastq.PwU.qtrim.fq >> right.fa

bash: line 1: 265467 Segmentation fault (core dumped) seqtk-trinity seq -A -R 1 /tmp/slurmtmp.3675441/trinity_output_files_Haines_Dust/50_2-D_023_170_S1_L004_R1_001_rRNA.fastq.PwU.qtrim.fq >> left.fa

Thread 1 terminated abnormally: Error, cmd: seqtk-trinity seq -A -R 1 /tmp/slurmtmp.3675441/trinity_output_files_Haines_Dust/50_2-D_023_170_S1_L004_R1_001_rRNA.fastq.PwU.qtrim.fq >> left.fa died with ret 35584 at /apps/trinityrnaseq/2.11.0/util/insilico_read_normalization.pl line 793.

bash: line 1: 265468 Segmentation fault (core dumped) seqtk-trinity seq -A -R 2 /tmp/slurmtmp.3675441/trinity_output_files_Haines_Dust/50_2-D_023_170_S1_L004_R2_001_rRNA.fastq.PwU.qtrim.fq >> right.fa

Thread 2 terminated abnormally: Error, cmd: seqtk-trinity seq -A -R 2 /tmp/slurmtmp.3675441/trinity_output_files_Haines_Dust/50_2-D_023_170_S1_L004_R2_001_rRNA.fastq.PwU.qtrim.fq >> right.fa died with ret 35584 at /apps/trinityrnaseq/2.11.0/util/insilico_read_normalization.pl line 793.

Error, conversion thread failed at /apps/trinityrnaseq/2.11.0/util/insilico_read_normalization.pl line 336.

Error, cmd: /apps/trinityrnaseq/2.11.0/util/insilico_read_normalization.pl --seqType fq --JM 2500G --max_cov 200 --min_cov 1 --CPU 75 --output /tmp/slurmtmp.3675441/trinity_output_files_Haines_Dust/insilico_read_normalization --max_CV 10000 --left 50_2-D_023_170_S1_L004_R1_001_rRNA.fastq.PwU.qtrim.fq,50_3-D_011_182_S2_L004_R1_001_rRNA.fastq.PwU.qtrim.fq,50_4-D_094_099_S3_L004_R1_001_rRNA.fastq.PwU.qtrim.fq,85_2-D_082_111_S4_L004_R1_001_rRNA.fastq.PwU.qtrim.fq,85_4-D_070_123_S5_L004_R1_001_rRNA.fastq.PwU.qtrim.fq,85_5-D_058_135_S6_L004_R1_001_rRNA.fastq.PwU.qtrim.fq,95_1-D_046_147_S7_L004_R1_001_rRNA.fastq.PwU.qtrim.fq,95_3-D_034_159_S8_L004_R1_001_rRNA.fastq.PwU.qtrim.fq,95_4-D_022_171_S9_L004_R1_001_rRNA.fastq.PwU.qtrim.fq --right 50_2-D_023_170_S1_L004_R2_001_rRNA.fastq.PwU.qtrim.fq,50_3-D_011_182_S2_L004_R2_001_rRNA.fastq.PwU.qtrim.fq,50_4-D_094_099_S3_L004_R2_001_rRNA.fastq.PwU.qtrim.fq,85_2-D_082_111_S4_L004_R2_001_rRNA.fastq.PwU.qtrim.fq,85_4-D_070_123_S5_L004_R2_001_rRNA.fastq.PwU.qtrim.fq,85_5-D_058_135_S6_L004_R2_001_rRNA.fastq.PwU.qtrim.fq,95_1-D_046_147_S7_L004_R2_001_rRNA.fastq.PwU.qtrim.fq,95_3-D_034_159_S8_L004_R2_001_rRNA.fastq.PwU.qtrim.fq,95_4-D_022_171_S9_L004_R2_001_rRNA.fastq.PwU.qtrim.fq --pairs_together --PARALLEL_STATS died with ret 6400 at /apps/trinityrnaseq/2.11.0/Trinity line 2826.

main::process_cmd('/apps/trinityrnaseq/2.11.0/util/insilico_read_normalization.p...') called at /apps/trinityrnaseq/2.11.0/Trinity line 3379

main::normalize('/tmp/slurmtmp.3675441/trinity_output_files_Haines_Dust/insili...', 200, 'ARRAY(0x9b8c20)', 'ARRAY(0x9b8c50)') called at /apps/trinityrnaseq/2.11.0/Trinity line 3319

main::run_normalization(200, 'ARRAY(0x9b8c20)', 'ARRAY(0x9b8c50)') called at /apps/trinityrnaseq/2.11.0/Trinity line 1372

I saw a separate query where the suggestion was to lower the max-memory usage from what is called from the HPC. As my files are very large I am attempting to use a huge memory node of 79 and then memory of 2988GB. In my code however I set --max_memory 2500GB and --CPU 75.

Is there a max number of CPUs that can be requested for Trinity? Any guidance or suggestions would be appreciated!

Best,

Sarah

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