Error, cmd: trinityrnaseq-v2.12.0/util/insilico_read_normalization.pl [...]died with ret 9 at ./Trinity line 2869
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Laurie Bousquet
Jun 29, 2021, 3:34:30 AM6/29/21
to trinityrnaseq-users
Hello,
I am working on de-novo transcriptome assembly, and I got this error message while trying to run Trinity (after several hours of running job).
I ran trimmomatic independently on raw files before. I'm also running the program on a local computer, so maybe the error is linked to an installation issue?
Bellow is a copy of the error output.
If you have any insight of what is going on, please let me know.
Thank you so much for your time!
Laurie
./Trinity --seqType fq --max_memory 10G --single /home/RAW_DATA/10AA1_s1.trim.fastq.gz,/home/RAW_DATA/10AA3_s2.trim.fastq.gz,/home/RAW_DATA/10AB1_s3.trim.fastq.gz,/home/RAW_DATA/1216A2_s4.trim.fastq.gz,/home/RAW_DATA/1216A3_s5.trim.fastq.gz,/home/RAW_DATA/1216B4_s6.trim.fastq.gz,/home/RAW_DATA/12AA3_s7.trim.fastq.gz,/home/RAW_DATA/12AB1_s8.trim.fastq.gz,/home/RAW_DATA/12AB4_s9.trim.fastq.gz --CPU 4 --output trinity_out --verbose
______ ____ ____ ____ ____ ______ __ __
| || \ | || \ | || || | |
| || D ) | | | _ | | | | || | |
|_| |_|| / | | | | | | | |_| |_|| ~ |
| | | \ | | | | | | | | | |___, |
| | | . \ | | | | | | | | | | |
|__| |__|\_||____||__|__||____| |__| |____/
Trinity-v2.12.0
Single read files: $VAR1 = [
'/home/RAW_DATA/10AA1_s1.trim.fastq.gz',
'/home/RAW_DATA/10AA3_s2.trim.fastq.gz',
'/home/RAW_DATA/10AB1_s3.trim.fastq.gz',
'/home/RAW_DATA/1216A2_s4.trim.fastq.gz',
'/home/RAW_DATA/1216A3_s5.trim.fastq.gz',
'/home/RAW_DATA/1216B4_s6.trim.fastq.gz',
'/home/RAW_DATA/12AA3_s7.trim.fastq.gz',
'/home/RAW_DATA/12AB1_s8.trim.fastq.gz',
'/home/RAW_DATA/12AB4_s9.trim.fastq.gz'
];
Trinity version: Trinity-v2.12.0
** NOTE: Latest version of Trinity is v2.12.0, and can be obtained at:
Found samtools at: /usr/bin/samtools
Found jellyfish at: /usr/bin/jellyfish
Paired mode requires bowtie2. Found bowtie2 at: /usr/bin/bowtie2
and bowtie-build at /usr/bin/bowtie2-build
Found salmon installed at /home/LB_W/trinity/salmon-1.5.1_linux_x86_64/bin/salmon
-since butterfly will eventually be run, lets test for proper execution of java
#######################################
Running Java Tests
Monday, June 21, 2021: 09:42:52 CMD: java -Xmx64m -XX:ParallelGCThreads=2 -jar /home/LB_W/trinity/trinityrnaseq-v2.12.0/util/support_scripts/ExitTester.jar 0
CMD finished (0 seconds)
Monday, June 21, 2021: 09:42:52 CMD: java -Xmx4g -XX:ParallelGCThreads=2 -jar /home/LB_W/trinity/trinityrnaseq-v2.12.0/util/support_scripts/ExitTester.jar 1
-we properly captured the java failure status, as needed. Looking good.
Java tests succeeded.
###################################
-changing dir to output dir: /home/LB_W/trinity/trinityrnaseq-v2.12.0/trinity_out
----------------------------------------------------------------------------------
-------------- Trinity Phase 1: Clustering of RNA-Seq Reads ---------------------
----------------------------------------------------------------------------------
---------------------------------------------------------------
------------ In silico Read Normalization ---------------------
-- (Removing Excess Reads Beyond 200 Coverage --
---------------------------------------------------------------
# running normalization on reads: $VAR1 = [
[
'/home/RAW_DATA/10AA1_s1.trim.fastq.gz',
'/home/RAW_DATA/10AA3_s2.trim.fastq.gz',
'/home/RAW_DATA/10AB1_s3.trim.fastq.gz',
'/home/RAW_DATA/1216A2_s4.trim.fastq.gz',
'/home/RAW_DATA/1216A3_s5.trim.fastq.gz',
'/home/RAW_DATA/1216B4_s6.trim.fastq.gz',
'/home/RAW_DATA/12AA3_s7.trim.fastq.gz',
'/home/RAW_DATA/12AB1_s8.trim.fastq.gz',
'/home/RAW_DATA/12AB4_s9.trim.fastq.gz'
]
];
-file doesn't yet exist: /home/LB_W/trinity/trinityrnaseq-v2.12.0/trinity_out/insilico_read_normalization/single.norm.fq
Monday, June 21, 2021: 09:42:52 CMD: /home/LB_W/trinity/trinityrnaseq-v2.12.0/util/insilico_read_normalization.pl --seqType fq --JM 10G --max_cov 200 --min_cov 1 --CPU 4 --output /home/LB_W/trinity/trinityrnaseq-v2.12.0/trinity_out/insilico_read_normalization --max_CV 10000 --single /home/RAW_DATA/10AA1_s1.trim.fastq.gz,/home/RAW_DATA/10AA3_s2.trim.fastq.gz,/home/RAW_DATA/10AB1_s3.trim.fastq.gz,/home/RAW_DATA/1216A2_s4.trim.fastq.gz,/home/RAW_DATA/1216A3_s5.trim.fastq.gz,/home/RAW_DATA/1216B4_s6.trim.fastq.gz,/home/RAW_DATA/12AA3_s7.trim.fastq.gz,/home/RAW_DATA/12AB1_s8.trim.fastq.gz,/home/RAW_DATA/12AB4_s9.trim.fastq.gz
-prepping seqs
-kmer counting.
-generating stats files
CMD: /home/LB_W/trinity/trinityrnaseq-v2.12.0/util/..//Inchworm/bin/fastaToKmerCoverageStats --reads single.fa --kmers jellyfish.K25.min2.kmers.fa --kmer_size 25 --num_threads 4 --DS > single.fa.K25.stats
-reading Kmer occurrences...
done parsing 274142557 Kmers, 274142557 added, taking 171 seconds.
STATS_GENERATION_TIME: 4097 seconds.
CMD finished (4312 seconds)
CMD: touch single.fa.K25.stats.ok
CMD finished (0 seconds)
-sorting each stats file by read name.
CMD: head -n1 single.fa.K25.stats > single.fa.K25.stats.sort && tail -n +2 single.fa.K25.stats | /usr/bin/sort --parallel=4 -k1,1 -T . -S 10G >> single.fa.K25.stats.sort
CMD finished (967 seconds)
CMD: touch single.fa.K25.stats.sort.ok
CMD finished (0 seconds)
-defining normalized reads
CMD: /home/LB_W/trinity/trinityrnaseq-v2.12.0/util/..//util/support_scripts//nbkc_normalize.pl --stats_file single.fa.K25.stats.sort --max_cov 200 --min_cov 1 --max_CV 10000 > single.fa.K25.stats.sort.maxC200.minC1.maxCV10000.accs
139931428 / 517013478 = 27.07% reads selected during normalization.
0 / 517013478 = 0.00% reads discarded as likely aberrant based on coverage profiles.
0 / 517013478 = 0.00% reads discarded as below minimum coverage threshold=1
CMD finished (2182 seconds)
CMD: touch single.fa.K25.stats.sort.maxC200.minC1.maxCV10000.accs.ok
CMD finished (0 seconds)
-search and capture.
Error, cmd: /home/LB_W/trinity/trinityrnaseq-v2.12.0/util/insilico_read_normalization.pl --seqType fq --JM 10G --max_cov 200 --min_cov 1 --CPU 4 --output /home/LB_W/trinity/trinityrnaseq-v2.12.0/trinity_out/insilico_read_normalization --max_CV 10000 --single /home/RAW_DATA/10AA1_s1.trim.fastq.gz,/home/RAW_DATA/10AA3_s2.trim.fastq.gz,/home/RAW_DATA/10AB1_s3.trim.fastq.gz,/home/RAW_DATA/1216A2_s4.trim.fastq.gz,/home/RAW_DATA/1216A3_s5.trim.fastq.gz,/home/RAW_DATA/1216B4_s6.trim.fastq.gz,/home/RAW_DATA/12AA3_s7.trim.fastq.gz,/home/RAW_DATA/12AB1_s8.trim.fastq.gz,/home/RAW_DATA/12AB4_s9.trim.fastq.gz died with ret 9 at ./Trinity line 2869.
main::process_cmd("/home/LB_W/trinity/trinityrnaseq-v2.12.0"...) called at ./Trinity line 3422
main::normalize("/home/LB_W/trinity/trinityrnaseq-v2.12.0"..., 200, ARRAY(0x55aff9aece68)) called at ./Trinity line 3362
main::run_normalization(200, ARRAY(0x55aff9aece68)) called at ./Trinity line 1389
Brian Haas
Jul 2, 2021, 10:27:35 AM7/2/21
to Laurie Bousquet, trinityrnaseq-users
hi,
It looks like it's having trouble working with your fastq input files here.
Can you try running this command directly:
/home/LB_W/trinity/trinityrnaseq-v2.12.0/util/insilico_read_normalization.pl --seqType fq --JM 10G --max_cov 200 --min_cov 1 --CPU 4 --output /home/LB_W/trinity/trinityrnaseq-v2.12.0/trinity_out/insilico_read_normalization --max_CV 10000 --single /home/RAW_DATA/10AA1_s1.trim.fastq.gz,/home/RAW_DATA/10AA3_s2.trim.fastq.gz,/home/RAW_DATA/10AB1_s3.trim.fastq.gz,/home/RAW_DATA/1216A2_s4.trim.fastq.gz,/home/RAW_DATA/1216A3_s5.trim.fastq.gz,/home/RAW_DATA/1216B4_s6.trim.fastq.gz,/home/RAW_DATA/12AA3_s7.trim.fastq.gz,/home/RAW_DATA/12AB1_s8.trim.fastq.gz,/home/RAW_DATA/12AB4_s9.trim.fastq.gz
and see if it reports any more helpful error message?
best,
~b
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Laurie Bousquet
Jul 4, 2021, 11:37:24 AM7/4/21
to trinityrnaseq-users
Hi Brian,
Thank you very much for your answer.
I tried what you indicated me and now, according to the output message, the process is killed (without more information, but at think at the same step). Below is the new output.
In the trinity_out/insilico_read_normalization/ directory, I have as an output only one fq file (11AA1_s1.trim.fastq.gz_ext_all_reads.normalized_K25_maxC200_minC1_maxCV10000.fq), and the 'tmp_normalized_reads directory'
What should I look for in fastq files that may prevent the program to run? ( the test during the installation ran normaly)>
Thank you so much for your time and answer,
Laurie
output:
/home/lab-miguel/Documents/LB_W/trinity/trinityrnaseq-v2.12.0/util/insilico_read_normalization.pl --seqType fq --JM 10G --max_cov 200 --min_cov 1 --CPU 4 --output /home/lab-miguel/Documents/LB_W/trinity/trinityrnaseq-v2.12.0/trinity_out/insilico_read_normalization --max_CV 10000 --single /home/lab-miguel/Documents/LB_W/TRIM/11AA1_s1.trim.fastq.gz,/home/lab-miguel/Documents/LB_W/TRIM/11AA3_s2.trim.fastq.gz,/home/lab-miguel/Documents/LB_W/TRIM/11AB1_s3.trim.fastq.gz,/home/lab-miguel/Documents/LB_W/TRIM/1216A2_s4.trim.fastq.gz,/home/lab-miguel/Documents/LB_W/TRIM/1216A3_s5.trim.fastq.gz,/home/lab-miguel/Documents/LB_W/TRIM/1216B4_s6.trim.fastq.gz,/home/lab-miguel/Documents/LB_W/TRIM/12AA3_s7.trim.fastq.gz,/home/lab-miguel/Documents/LB_W/TRIM/12AB1_s8.trim.fastq.gz,/home/lab-miguel/Documents/LB_W/TRIM/12AB4_s9.trim.fastq.gz
-prepping seqs
CMD: seqtk-trinity seq -A -R 1 <(gunzip -c /home/lab-miguel/Documents/LB_W/TRIM/11AA1_s1.trim.fastq.gz) >> single.fa
CMD finished (109 seconds)
CMD: seqtk-trinity seq -A -R 1 <(gunzip -c /home/lab-miguel/Documents/LB_W/TRIM/11AA3_s2.trim.fastq.gz) >> single.fa
CMD finished (124 seconds)
CMD: seqtk-trinity seq -A -R 1 <(gunzip -c /home/lab-miguel/Documents/LB_W/TRIM/11AB1_s3.trim.fastq.gz) >> single.fa
CMD finished (139 seconds)
CMD: seqtk-trinity seq -A -R 1 <(gunzip -c /home/lab-miguel/Documents/LB_W/TRIM/1216A2_s4.trim.fastq.gz) >> single.fa
CMD finished (131 seconds)
CMD: seqtk-trinity seq -A -R 1 <(gunzip -c /home/lab-miguel/Documents/LB_W/TRIM/1216A3_s5.trim.fastq.gz) >> single.fa
CMD finished (114 seconds)
CMD: seqtk-trinity seq -A -R 1 <(gunzip -c /home/lab-miguel/Documents/LB_W/TRIM/1216B4_s6.trim.fastq.gz) >> single.fa
CMD finished (137 seconds)
CMD: seqtk-trinity seq -A -R 1 <(gunzip -c /home/lab-miguel/Documents/LB_W/TRIM/12AA3_s7.trim.fastq.gz) >> single.fa
CMD finished (164 seconds)
CMD: seqtk-trinity seq -A -R 1 <(gunzip -c /home/lab-miguel/Documents/LB_W/TRIM/12AB1_s8.trim.fastq.gz) >> single.fa
CMD finished (139 seconds)
CMD: seqtk-trinity seq -A -R 1 <(gunzip -c /home/lab-miguel/Documents/LB_W/TRIM/12AB4_s9.trim.fastq.gz) >> single.fa
CMD finished (103 seconds)
CMD: touch single.fa.ok
CMD finished (0 seconds)
-kmer counting.
-------------------------------------------
----------- Jellyfish --------------------
-- (building a k-mer catalog from reads) --
-------------------------------------------
CMD: jellyfish count -t 4 -m 25 -s 100000000 --canonical single.fa
CMD finished (1715 seconds)
CMD: jellyfish histo -t 4 -o jellyfish.K25.min2.kmers.fa.histo mer_counts.jf
CMD finished (70 seconds)
CMD: jellyfish dump -L 2 mer_counts.jf > jellyfish.K25.min2.kmers.fa
CMD finished (103 seconds)
CMD: touch jellyfish.K25.min2.kmers.fa.success
CMD finished (0 seconds)
-generating stats files
CMD: /home/lab-miguel/Documents/LB_W/trinity/trinityrnaseq-v2.12.0/util/..//Inchworm/bin/fastaToKmerCoverageStats --reads single.fa --kmers jellyfish.K25.min2.kmers.fa --kmer_size 25 --num_threads 4 --DS > single.fa.K25.stats
-reading Kmer occurrences...
done parsing 274142557 Kmers, 274142557 added, taking 170 seconds.
STATS_GENERATION_TIME: 4126 seconds.
CMD finished (4339 seconds)
CMD: touch single.fa.K25.stats.ok
CMD finished (0 seconds)
-sorting each stats file by read name.
CMD: head -n1 single.fa.K25.stats > single.fa.K25.stats.sort && tail -n +2 single.fa.K25.stats | /usr/bin/sort --parallel=4 -k1,1 -T . -S 10G >> single.fa.K25.stats.sort
CMD finished (1115 seconds)
CMD: touch single.fa.K25.stats.sort.ok
CMD finished (0 seconds)
-defining normalized reads
CMD: /home/lab-miguel/Documents/LB_W/trinity/trinityrnaseq-v2.12.0/util/..//util/support_scripts//nbkc_normalize.pl --stats_file single.fa.K25.stats.sort --max_cov 200 --min_cov 1 --max_CV 10000 > single.fa.K25.stats.sort.maxC200.minC1.maxCV10000.accs
139931428 / 517013478 = 27.07% reads selected during normalization.
0 / 517013478 = 0.00% reads discarded as likely aberrant based on coverage profiles.
0 / 517013478 = 0.00% reads discarded as below minimum coverage threshold=1
CMD finished (2273 seconds)
CMD: touch single.fa.K25.stats.sort.maxC200.minC1.maxCV10000.accs.ok
CMD finished (0 seconds)
-search and capture.
Killed
Brian Haas
Jul 4, 2021, 11:40:01 AM7/4/21
to Laurie Bousquet, trinityrnaseq-users
Hi Laurie,
It says 'killed' so it probably hit a RAM limitation (or time limitation if running on a computing grid w/ a time limit).
To view this discussion on the web visit https://groups.google.com/d/msgid/trinityrnaseq-users/f325a6bc-8ff1-4ca4-8ef3-f4ae97005f0an%40googlegroups.com.
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