Error with trinity
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3 months ago
haniyounes • 0

Good morning,

I am new in this domain, and I want to learn about trinity denovo assembly. I tried to lunch this line command from Ubuntu Vmbox , but I always get 2 errors:

~/sharedUbuntu/S.scutata$ sudo docker run --rm -v `pwd`:`pwd` trinityrnaseq/trinityrnaseq Trinity \
>       --seqType fq \
>       --left `pwd`/FastqOutput/SRR3574594_1.fastq  \
>       --right `pwd`/FastqOutput/SRR3574594_2.fastq \
>       --max_memory 28G --min_kmer_cov 2 --CPU 4 --monitoring --output /home/sharedUbuntu/S.scutata/Trinityresults


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

    Trinity-v2.11.0



Left read files: $VAR1 = [
          '/home/sharedUbuntu/S.scutata/FastqOutput/SRR3574594_1.fastq'
        ];
Right read files: $VAR1 = [
          '/home/sharedUbuntu/S.scutata/FastqOutput/SRR3574594_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

Monday, December 21, 2020: 10:54:52 CMD: java -Xmx64m -XX:ParallelGCThreads=2  -jar /usr/local/bin/trinityrnaseq/util/support_scripts/ExitTester.jar 0
Monday, December 21, 2020: 10:54:52 CMD: java -Xmx4g -XX:ParallelGCThreads=2  -jar /usr/local/bin/trinityrnaseq/util/support_scripts/ExitTester.jar 1
Monday, December 21, 2020: 10:54:52 CMD: mkdir -p /home/sharedUbuntu/S.scutata/Trinityresults
Monday, December 21, 2020: 10:54:52 CMD: mkdir -p /home/sharedUbuntu/S.scutata/Trinityresults/chrysalis
STARTING COLLECTL
I'M THE PARENT, COLLECTL_PID=67
I'M THE CHILD RUNNING TRINITY
Running CMD: bash -c "set -eou pipefail && cd /usr/local/src/collectl && exec nohup /usr/local/bin/trinityrnaseq/trinity-plugins/COLLECTL/collectl/collectl --procopts w  --procfilt u0 --interval :60 -sZ -oD | egrep 'Trinity|trinityrnaseq|Inchworm|Chrysalis|Butterfly|jellyfish|samtools|sort|bowtie' | egrep -v 'egrep' > /usr/local/src/collectl/collectl.dat " 


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

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

# running normalization on reads: $VAR1 = [
          [
            '/home/sharedUbuntu/S.scutata/FastqOutput/SRR3574594_1.fastq'
          ],
          [
            '/home/sharedUbuntu/S.scutata/FastqOutput/SRR3574594_2.fastq'
          ]
        ];


Monday, December 21, 2020: 10:54:52 CMD: /usr/local/bin/trinityrnaseq/util/insilico_read_normalization.pl --seqType fq --JM 28G  --max_cov 200 --min_cov 2 --CPU 4 --output /home/sharedUbuntu/S.scutata/Trinityresults/insilico_read_normalization --max_CV 10000  --left /home/sharedUbuntu/S.scutata/FastqOutput/SRR3574594_1.fastq --right /home/sharedUbuntu/S.scutata/FastqOutput/SRR3574594_2.fastq --pairs_together  --PARALLEL_STATS  
-prepping seqs
Converting input files. (both directions in parallel)CMD: seqtk-trinity seq -A -R 1  /home/sharedUbuntu/S.scutata/FastqOutput/SRR3574594_1.fastq >> left.fa
CMD: seqtk-trinity seq -A -R 2  /home/sharedUbuntu/S.scutata/FastqOutput/SRR3574594_2.fastq >> right.fa
CMD finished (318 seconds)
CMD finished (319 seconds)
CMD: touch left.fa.ok
CMD finished (0 seconds)
CMD: touch right.fa.ok
CMD finished (0 seconds)
Done converting input files.CMD: cat left.fa right.fa > both.fa
CMD finished (6 seconds)
CMD: touch both.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  both.fa
CMD finished (370 seconds)
CMD: jellyfish histo -t 4 -o jellyfish.K25.min2.kmers.fa.histo mer_counts.jf
CMD finished (62 seconds)
CMD: jellyfish dump -L 2 mer_counts.jf > jellyfish.K25.min2.kmers.fa
CMD finished (122 seconds)
CMD: touch jellyfish.K25.min2.kmers.fa.success
CMD finished (0 seconds)
-generating stats files
CMD: /usr/local/bin/trinityrnaseq/util/..//Inchworm/bin/fastaToKmerCoverageStats --reads left.fa --kmers jellyfish.K25.min2.kmers.fa --kmer_size 25  --num_threads 2  --DS  > left.fa.K25.stats
-reading Kmer occurrences...
CMD: /usr/local/bin/trinityrnaseq/util/..//Inchworm/bin/fastaToKmerCoverageStats --reads right.fa --kmers jellyfish.K25.min2.kmers.fa --kmer_size 25  --num_threads 2  --DS  > right.fa.K25.stats
-reading Kmer occurrences...

 done parsing 42222591 Kmers, 42222591 added, taking 118 seconds.

 done parsing 42222591 Kmers, 42222591 added, taking 120 seconds.
STATS_GENERATION_TIME: 390 seconds.
STATS_GENERATION_TIME: 392 seconds.
CMD finished (516 seconds)
CMD finished (520 seconds)
CMD: touch left.fa.K25.stats.ok
CMD finished (0 seconds)
CMD: touch right.fa.K25.stats.ok
CMD finished (0 seconds)
-sorting each stats file by read name.
CMD: head -n1 left.fa.K25.stats > left.fa.K25.stats.sort && tail -n +2 left.fa.K25.stats | /usr/bin/sort --parallel=4 -k1,1 -T . -S 14G >> left.fa.K25.stats.sort
CMD: head -n1 right.fa.K25.stats > right.fa.K25.stats.sort && tail -n +2 right.fa.K25.stats | /usr/bin/sort --parallel=4 -k1,1 -T . -S 14G >> right.fa.K25.stats.sort
CMD finished (44 seconds)
CMD finished (45 seconds)
CMD: touch left.fa.K25.stats.sort.ok
CMD finished (0 seconds)
CMD: touch right.fa.K25.stats.sort.ok
CMD finished (0 seconds)
-defining normalized reads
CMD: /usr/local/bin/trinityrnaseq/util/..//util/support_scripts//nbkc_merge_left_right_stats.pl --left left.fa.K25.stats.sort --right right.fa.K25.stats.sort --sorted > pairs.K25.stats
-opening left.fa.K25.stats.sort
-opening right.fa.K25.stats.sort
-done opening files.
CMD finished (338 seconds)
CMD: touch pairs.K25.stats.ok
CMD finished (0 seconds)
CMD: /usr/local/bin/trinityrnaseq/util/..//util/support_scripts//nbkc_normalize.pl --stats_file pairs.K25.stats --max_cov 200  --min_cov 2 --max_CV 10000 > pairs.K25.stats.C200.maxCV10000.accs
2402255 / 8330662 = 28.84% reads selected during normalization.
0 / 8330662 = 0.00% reads discarded as likely aberrant based on coverage profiles.
386418 / 8330662 = 4.64% reads discarded as below minimum coverage threshold=2
CMD finished (334 seconds)
CMD: touch pairs.K25.stats.C200.maxCV10000.accs.ok
CMD finished (0 seconds)
-search and capture.
-preparing to extract selected reads from: /home/sharedUbuntu/S.scutata/FastqOutput/SRR3574594_1.fastq ... done prepping, now search and capture.
-capturing normalized reads from: /home/sharedUbuntu/S.scutata/FastqOutput/SRR3574594_1.fastq
-preparing to extract selected reads from: /home/sharedUbuntu/S.scutata/FastqOutput/SRR3574594_2.fastq ... done prepping, now search and capture.
-capturing normalized reads from: /home/sharedUbuntu/S.scutata/FastqOutput/SRR3574594_2.fastq
CMD: touch /home/sharedUbuntu/S.scutata/Trinityresults/insilico_read_normalization/SRR3574594_1.fastq.normalized_K25_maxC200_minC2_maxCV10000.fq.ok
CMD finished (0 seconds)
CMD: touch /home/sharedUbuntu/S.scutata/Trinityresults/insilico_read_normalization/SRR3574594_2.fastq.normalized_K25_maxC200_minC2_maxCV10000.fq.ok
CMD finished (0 seconds)
CMD: ln -sf /home/sharedUbuntu/S.scutata/Trinityresults/insilico_read_normalization/SRR3574594_1.fastq.normalized_K25_maxC200_minC2_maxCV10000.fq left.norm.fq
ln: failed to create symbolic link 'left.norm.fq': Operation not permitted

**Error**, cmd: ln -sf /home/sharedUbuntu/S.scutata/Trinityresults/insilico_read_normalization/SRR3574594_1.fastq.normalized_K25_maxC200_minC2_maxCV10000.fq left.norm.fq died with ret 256 at /usr/local/bin/trinityrnaseq/util/insilico_read_normalization.pl line 793.

**Error**, cmd: /usr/local/bin/trinityrnaseq/util/insilico_read_normalization.pl --seqType fq --JM 28G  --max_cov 200 --min_cov 2 --CPU 4 --output /home/sharedUbuntu/S.scutata/Trinityresults/insilico_read_normalization --max_CV 10000  --left /home/sharedUbuntu/S.scutata/FastqOutput/SRR3574594_1.fastq --right /home/sharedUbuntu/S.scutata/FastqOutput/SRR3574594_2.fastq --pairs_together  --PARALLEL_STATS   died with ret 512 at /usr/local/bin/trinityrnaseq/Trinity line 2826.
    main::process_cmd("/usr/local/bin/trinityrnaseq/util/insilico_read_normalization"...) called at /usr/local/bin/trinityrnaseq/Trinity line 3379
    main::normalize("/home/sharedUbuntu/S.scutata/Trinityresults/insilico_re"..., 200, ARRAY(0x5625fba791e0), ARRAY(0x5625fba79210)) called at /usr/local/bin/trinityrnaseq/Trinity line 3319
    main::run_normalization(200, ARRAY(0x5625fba791e0), ARRAY(0x5625fba79210)) called at /usr/local/bin/trinityrnaseq/Trinity line 1372
TERMINATING COLLECTL, PID = 67

Knowing that the command works well when I try it with the test samples provided with trinity package.

I used Omicbox (trial version), and I get also an error messages. But when I asked the application to not do normalisation step, it worked and I got a fasta file with contigs.

Can you please give me some information about this problem ?

Thanks

RNA-Seq Assembly trinity • 249 views
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