This is a test version of Biostars. For the public version, visit https://www.biostars.org.
Trinity analysis error
(base) ongchip@ongchip-LINUX:~/ongchip-DP/work/202408$ $TRINITY_HOME/Trinity --seqType fq --max_memory 10G --left A1_1_paired.fq.gz  --right A1_2_paired.fq.gz --CPU 2


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

    Trinity-v2.15.2



Left read files: $VAR1 = [
          'A1_1_paired.fq.gz'
        ];
Right read files: $VAR1 = [
          'A1_2_paired.fq.gz'
        ];
Trinity version: Trinity-v2.15.2
-currently using the latest production release of Trinity.

Tuesday, October 29, 2024: 16:45:19 CMD: java -Xmx64m -XX:ParallelGCThreads=2  -jar /usr/local/bin/util/support_scripts/ExitTester.jar 0
Tuesday, October 29, 2024: 16:45:19 CMD: java -Xmx4g -XX:ParallelGCThreads=2  -jar /usr/local/bin/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/ongchip/ongchip-DP/work/202408/A1_1_paired.fq.gz'
          ],
          [
            '/home/ongchip/ongchip-DP/work/202408/A1_2_paired.fq.gz'
          ]
        ];


Tuesday, October 29, 2024: 16:45:19 CMD: /usr/local/bin/util/insilico_read_normalization.pl --seqType fq --JM 10G  --max_cov 200 --min_cov 1 --CPU 2 --output /home/ongchip/ongchip-DP/work/202408/trinity_out_dir/insilico_read_normalization --max_CV 10000  --left /home/ongchip/ongchip-DP/work/202408/A1_1_paired.fq.gz --right /home/ongchip/ongchip-DP/work/202408/A1_2_paired.fq.gz --pairs_together  --PARALLEL_STATS  
-prepping seqs
Converting input files. (both directions in parallel);CMD: seqtk-trinity seq -A -R 1  <(gunzip -c /home/ongchip/ongchip-DP/work/202408/A1_1_paired.fq.gz) >> left.fa
CMD: seqtk-trinity seq -A -R 2  <(gunzip -c /home/ongchip/ongchip-DP/work/202408/A1_2_paired.fq.gz) >> right.fa
CMD finished (328 seconds)
CMD finished (328 seconds)
CMD: touch left.fa.ok
CMD finished (1 seconds)
CMD: touch right.fa.ok
CMD finished (1 seconds)
 Done converting input files. CMD: cat left.fa right.fa > both.fa
CMD finished (139 seconds)
CMD: touch both.fa.ok
CMD finished (0 seconds)
-kmer counting.
-------------------------------------------
----------- Jellyfish  --------------------
-- (building a k-mer catalog from reads) --
-------------------------------------------

CMD: jellyfish count -t 2 -m 25 -s 100000000  --canonical  both.fa
CMD finished (701 seconds)
CMD: jellyfish histo -t 2 -o jellyfish.K25.min2.kmers.fa.histo mer_counts.jf
CMD finished (23 seconds)
CMD: jellyfish dump -L 2 mer_counts.jf > jellyfish.K25.min2.kmers.fa
CMD finished (61 seconds)
CMD: touch jellyfish.K25.min2.kmers.fa.success
CMD finished (0 seconds)
-generating stats files
CMD: /usr/local/bin/util/..//Inchworm/bin/fastaToKmerCoverageStats --reads left.fa --kmers jellyfish.K25.min2.kmers.fa --kmer_size 25  --num_threads 1  --DS  > left.fa.K25.stats
CMD: /usr/local/bin/util/..//Inchworm/bin/fastaToKmerCoverageStats --reads right.fa --kmers jellyfish.K25.min2.kmers.fa --kmer_size 25  --num_threads 1  --DS  > right.fa.K25.stats
-reading Kmer occurrences...
-reading Kmer occurrences...

 done parsing 130857324 Kmers, 130857324 added, taking 175 seconds.

 done parsing 130857324 Kmers, 130857324 added, taking 175 seconds.
STATS_GENERATION_TIME: 1611 seconds.
STATS_GENERATION_TIME: 1619 seconds.
CMD finished (1807 seconds)
CMD finished (1814 seconds)
CMD: touch left.fa.K25.stats.ok
CMD finished (0 seconds)
CMD: touch right.fa.K25.stats.ok
CMD finished (1 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=2 -k1,1 -T . -S 5G >> 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=2 -k1,1 -T . -S 5G >> right.fa.K25.stats.sort
CMD finished (83 seconds)
CMD finished (96 seconds)
CMD: touch left.fa.K25.stats.sort.ok
CMD finished (0 seconds)
CMD: touch right.fa.K25.stats.sort.ok
CMD finished (1 seconds)
-defining normalized reads
CMD: /usr/local/bin/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 (711 seconds)
CMD: touch pairs.K25.stats.ok
CMD finished (0 seconds)
CMD: /usr/local/bin/util/..//util/support_scripts//nbkc_normalize.pl --stats_file pairs.K25.stats --max_cov 200  --min_cov 1 --max_CV 10000 > pairs.K25.stats.C200.maxCV10000.accs
22220886 / 37910906 = 58.61% reads selected during normalization.
0 / 37910906 = 0.00% reads discarded as likely aberrant based on coverage profiles.
0 / 37910906 = 0.00% reads discarded as below minimum coverage threshold=1
CMD finished (404 seconds)
CMD: touch pairs.K25.stats.C200.maxCV10000.accs.ok
CMD finished (0 seconds)
-search and capture.
-preparing to extract selected reads from: /home/ongchip/ongchip-DP/work/202408/A1_1_paired.fq.gz ... done prepping, now search and capture.
-capturing normalized reads from: /home/ongchip/ongchip-DP/work/202408/A1_1_paired.fq.gz
-preparing to extract selected reads from: /home/ongchip/ongchip-DP/work/202408/A1_2_paired.fq.gz ... done prepping, now search and capture.
-capturing normalized reads from: /home/ongchip/ongchip-DP/work/202408/A1_2_paired.fq.gz
CMD: touch /home/ongchip/ongchip-DP/work/202408/trinity_out_dir/insilico_read_normalization/A1_1_paired.fq.gz.normalized_K25_maxC200_minC1_maxCV10000.fq.ok
CMD finished (0 seconds)
CMD: touch /home/ongchip/ongchip-DP/work/202408/trinity_out_dir/insilico_read_normalization/A1_2_paired.fq.gz.normalized_K25_maxC200_minC1_maxCV10000.fq.ok
CMD finished (0 seconds)
CMD: ln -sf /home/ongchip/ongchip-DP/work/202408/trinity_out_dir/insilico_read_normalization/A1_1_paired.fq.gz.normalized_K25_maxC200_minC1_maxCV10000.fq left.norm.fq
ln: failed to create symbolic link 'left.norm.fq': Operation not supported
Error, cmd: ln -sf /home/ongchip/ongchip-DP/work/202408/trinity_out_dir/insilico_read_normalization/A1_1_paired.fq.gz.normalized_K25_maxC200_minC1_maxCV10000.fq left.norm.fq died with ret 256 at /usr/local/bin/util/insilico_read_normalization.pl line 807.
Error, cmd: /usr/local/bin/util/insilico_read_normalization.pl --seqType fq --JM 10G  --max_cov 200 --min_cov 1 --CPU 2 --output /home/ongchip/ongchip-DP/work/202408/trinity_out_dir/insilico_read_normalization --max_CV 10000  --left /home/ongchip/ongchip-DP/work/202408/A1_1_paired.fq.gz --right /home/ongchip/ongchip-DP/work/202408/A1_2_paired.fq.gz --pairs_together  --PARALLEL_STATS   died with ret 512 at /usr/local/bin/Trinity line 2919.
    main::process_cmd("/usr/local/bin/util/insilico_read_normalization.pl --seqType "...) called at /usr/local/bin/Trinity line 3472
    main::normalize("/home/ongchip/ongchip-DP/work/202408/trinity_out_dir/insilico"..., 200, ARRAY(0x56485ebfbe90), ARRAY(0x56485ebfbe78)) called at /usr/local/bin/Trinity line 3412
    main::run_normalization(200, ARRAY(0x56485ebfbe90), ARRAY(0x56485ebfbe78)) called at /usr/local/bin/Trinity line 1450

Hello. De novo assembly analysis was performed with Trinity, but the analysis failed with the above message. I don't know what caused the error, and how to fix it. Please help me.

trinity
• 880 views • link
written by ongchip • ongchip

1 answer

ln: failed to create symbolic link 'left.norm.fq': Operation not supported

https://github.com/trinityrnaseq/trinityrnaseq/issues/977

In the upcoming release, there's a --no_symlinks parameter. Also, you could just set env var NO_SYMLINK=TRUE and that'll work instead too.

• 0 views • link
written by Pierre Lindenbaum Pierre Lindenbaum

Thank you. Then should I commands like below?

$TRINITY_HOME/Trinity --seqType fq --max_memory 10G --left A1_1_paired.fq.gz  --right A1_2_paired.fq.gz --CPU 2 --no_symlinks
• 0 views • link
written by ongchip • ongchip

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