Problems at Trinity Phase 2: Assembling Clusters of Reads

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KB

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Jun 4, 2021, 5:39:40 AM6/4/21

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Hello Brian,

I'm running a RNA-assembly task using trinity.

My parameter is:

Trinity --seqType fq --max_memory 48G --CPU 16 --left ../../../rawdata/R1.clean.fq --right ../../../rawdata/R2.clean.fq --SS_lib_type RF >trinity.log 2>trinity.err.

The number of my reads is about 20-30M. The Trinity phase 1 goes well, while tons of errors happen at Trinity phase 2, like many salmon issue. Below is part of my trinity.log file. The program is still running, but at a extremely slow speed. Error is error, it must have something went wrong. Could you please help me take a look and give me some advice?

Appreciated it!

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

------------ Trinity Phase 2: Assembling Clusters of Reads ---------------------

------- (involving the Inchworm, Chrysalis, Butterfly trifecta ) ---------------

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

Thursday, June 3, 2021: 13:38:46 CMD: /project/chenzhuyifu/software/miniconda3/opt/trinity-2.12.0/trinity-plugins/BIN/ParaFly -c recursive_trinity.cmds -CPU 16 -v -shuffle

Number of Commands: 123477

succeeded(1229) 0.995327% completed. touch: setting times of '/project/chenzhuyifu/rna_seq/workspace/DMF/assembly/trinity_out_CSC1/read_partitions/Fb_0/CBin_121/c12165.trinity.reads.fa.out/chrysalis/iworm_cluster_welds_graph.txt.ok': Input/output error

touch: setting times of '.iworm.24.SR.ok': Input/output error

touch: setting times of '/project/chenzhuyifu/rna_seq/workspace/DMF/assembly/trinity_out_CSC1/read_partitions/Fb_0/CBin_535/c53594.trinity.reads.fa.out/chrysalis/readsToComponents.out.ok': Input/output error

Trinity run failed. Must investigate error above.

warning, cmd: warning, cmd: /project/chenzhuyifu/software/miniconda3/opt/trinity-2.12.0/util/support_scripts/../../Trinity --single "/project/chenzhuyifu/rna_seq/workspace/DMF/assembly/trinity_out_CSC1/read_partitions/Fb_0/CBin_121/c12165.trinity.reads.fa" --output "/project/chenzhuyifu/rna_seq/workspace/DMF/assembly/trinity_out_CSC1/read_partitions/Fb_0/CBin_121/c12165.trinity.reads.fa.out" --CPU 1 --max_memory 1G --run_as_paired --SS_lib_type F --seqType fa --trinity_complete --full_cleanup failed with ret: 7, going to retry.

/project/chenzhuyifu/software/miniconda3/opt/trinity-2.12.0/util/support_scripts/../../Trinity --single "/project/chenzhuyifu/rna_seq/workspace/DMF/assembly/trinity_out_CSC1/read_partitions/Fb_1/CBin_1226/c122729.trinity.reads.fa" --output "/project/chenzhuyifu/rna_seq/workspace/DMF/assembly/trinity_out_CSC1/read_partitions/Fb_1/CBin_1226/c122729.trinity.reads.fa.out" --CPU 1 --max_memory 1G --run_as_paired --SS_lib_type F --seqType fa --trinity_complete --full_cleanup failed with ret: warning, cmd: /project/chenzhuyifu/software/miniconda3/opt/trinity-2.12.0/util/support_scripts/../../Trinity --single "/project/chenzhuyifu/rna_seq/workspace/DMF/assembly/trinity_out_CSC1/read_partitions/Fb_0/CBin_311/c31131.trinity.reads.fa" --output "/project/chenzhuyifu/rna_seq/workspace/DMF/assembly/trinity_out_CSC1/read_partitions/Fb_0/CBin_311/c31131.trinity.reads.fa.out" --CPU 1 --max_memory 1G --run_as_paired --SS_lib_type F --seqType fa --trinity_complete --full_cleanup

Error encountered:: <!----

CMD: /project/chenzhuyifu/software/miniconda3/opt/trinity-2.12.0/util/support_scripts/partition_chrysalis_graphs_n_reads.pl --deBruijns /project/chenzhuyifu/rna_seq/workspace/DMF/assembly/trinity_out_CSC1/read_partitions/Fb_0/CBin_404/c40486.trinity.reads.fa.out/chrysalis/bundled_iworm_contigs.fasta.deBruijn --componentReads /project/chenzhuyifu/rna_seq/workspace/DMF/assembly/trinity_out_CSC1/read_partitions/Fb_0/CBin_404/c40486.trinity.reads.fa.out/chrysalis/readsToComponents.out.sort -N 1000 -L 200 2>tmp.26521.1622704895.stderr

Errmsg:

Partitioning chrysalis graphs and reads

Done partitioning graphs.

Partitioning reads...

Error, cannot write to /project/chenzhuyifu/rna_seq/workspace/DMF/assembly/trinity_out_CSC1/read_partitions/Fb_0/CBin_404/c40486.trinity.reads.fa.out/chrysalis/Component_bins/Cbin0/c0.reads.tmp at /project/chenzhuyifu/software/miniconda3/opt/trinity-2.12.0/util/support_scripts/partition_chrysalis_graphs_n_reads.pl line 109, <$fh> line 1.

--->

failed with ret: 7, going to retry.

Error encountered:: <!----

CMD: bash -c " set -o pipefail;/home/chenzhuyifu/project/software/miniconda3/bin/bowtie2 --local -k 2 --no-unal --threads 1 -f --score-min G,20,8 -x /project/chenzhuyifu/rna_seq/workspace/DMF/assembly/trinity_out_CSC1/read_partitions/Fb_0/CBin_651/c65207.trinity.reads.fa.out/chrysalis/inchworm.fa.min100 /project/chenzhuyifu/rna_seq/workspace/DMF/assembly/trinity_out_CSC1/read_partitions/Fb_0/CBin_651/c65207.trinity.reads.fa.out/single.fa | samtools view -@ 1 -F4 -Sb - | samtools sort -m 536870912 -@ 1 -no - - > /project/chenzhuyifu/rna_seq/workspace/DMF/assembly/trinity_out_CSC1/read_partitions/Fb_0/CBin_651/c65207.trinity.reads.fa.out/chrysalis/iworm.bowtie.nameSorted.bam" 2>tmp.27959.1622704896.stderr

Errmsg:

10 reads; of these:

10 (100.00%) were unpaired; of these:

0 (0.00%) aligned 0 times

10 (100.00%) aligned exactly 1 time

0 (0.00%) aligned >1 times

100.00% overall alignment rate

[E::bgzf_close] File write failed

samtools sort: failed to create "-": Input/output error

--->

, going to retry.

warning, cmd: /project/chenzhuyifu/software/miniconda3/opt/trinity-2.12.0/util/support_scripts/../../Trinity --single "/project/chenzhuyifu/rna_seq/workspace/DMF/assembly/trinity_out_CSC1/read_partitions/Fb_0/CBin_181/c18187.trinity.reads.fa" --output "/project/chenzhuyifu/rna_seq/workspace/DMF/assembly/trinity_out_CSC1/read_partitions/Fb_0/CBin_181/c18187.trinity.reads.fa.out" --CPU 1 --max_memory 1G --run_as_paired --SS_lib_type F --seqType fa --trinity_complete --full_cleanup failed with ret: warning, cmd: /project/chenzhuyifu/software/miniconda3/opt/trinity-2.12.0/util/support_scripts/../../Trinity --single "/project/chenzhuyifu/rna_seq/workspace/DMF/assembly/trinity_out_CSC1/read_partitions/Fb_1/CBin_1022/c102249.trinity.reads.fa" --output "/project/chenzhuyifu/rna_seq/workspace/DMF/assembly/trinity_out_CSC1/read_partitions/Fb_1/CBin_1022/c102249.trinity.reads.fa.out" --CPU 1 --max_memory 1G --run_as_paired --SS_lib_type F --seqType fa --trinity_complete --full_cleanup failed with ret: 7, going to retry.

7, going to retry.

Error encountered:: <!----

CMD: /project/chenzhuyifu/software/miniconda3/opt/trinity-2.12.0/util/support_scripts/salmon_runner.pl /project/chenzhuyifu/rna_seq/workspace/DMF/assembly/trinity_out_CSC1/read_partitions/Fb_0/CBin_889/c88991.trinity.reads.fa.out/Trinity.tmp.fasta /project/chenzhuyifu/rna_seq/workspace/DMF/assembly/trinity_out_CSC1/read_partitions/Fb_0/CBin_889/c88991.trinity.reads.fa.out/single.fa.SR_supp.fa 2>tmp.24259.1622704806.stderr

Errmsg:

CMD: salmon --no-version-check index -t /project/chenzhuyifu/rna_seq/workspace/DMF/assembly/trinity_out_CSC1/read_partitions/Fb_0/CBin_889/c88991.trinity.reads.fa.out/Trinity.tmp.fasta -i /project/chenzhuyifu/rna_seq/workspace/DMF/assembly/trinity_out_CSC1/read_partitions/Fb_0/CBin_889/c88991.trinity.reads.fa.out/Trinity.tmp.fasta.salmon.idx -k 25 -p 1 > _salmon.27992.stderr 2>&1

sh: line 1: 28013 Segmentation fault salmon --no-version-check index -t /project/chenzhuyifu/rna_seq/workspace/DMF/assembly/trinity_out_CSC1/read_partitions/Fb_0/CBin_889/c88991.trinity.reads.fa.out/Trinity.tmp.fasta -i /project/chenzhuyifu/rna_seq/workspace/DMF/assembly/trinity_out_CSC1/read_partitions/Fb_0/CBin_889/c88991.trinity.reads.fa.out/Trinity.tmp.fasta.salmon.idx -k 25 -p 1 > _salmon.27992.stderr 2>&1

Error, cmd: salmon --no-version-check index -t /project/chenzhuyifu/rna_seq/workspace/DMF/assembly/trinity_out_CSC1/read_partitions/Fb_0/CBin_889/c88991.trinity.reads.fa.out/Trinity.tmp.fasta -i /project/chenzhuyifu/rna_seq/workspace/DMF/assembly/trinity_out_CSC1/read_partitions/Fb_0/CBin_889/c88991.trinity.reads.fa.out/Trinity.tmp.fasta.salmon.idx -k 25 -p 1 > _salmon.27992.stderr 2>&1 failed with msg: index ["/project/chenzhuyifu/rna_seq/workspace/DMF/assembly/trinity_out_CSC1/read_partitions/Fb_0/CBin_889/c88991.trinity.reads.fa.out/Trinity.tmp.fasta.salmon.idx"] did not previously exist . . . creating it

[2021-06-03 15:21:35.247] [jLog] [warning] The salmon index is being built without any decoy sequences. It is recommended that decoy sequence (either computed auxiliary decoy sequence or the genome of the organism) be provided during indexing. Further details can be found at https://salmon.readthedocs.io/en/latest/salmon.html#preparing-transcriptome-indices-mapping-based-mode.

[2021-06-03 15:21:35.248] [jLog] [info] building index

out : /project/chenzhuyifu/rna_seq/workspace/DMF/assembly/trinity_out_CSC1/read_partitions/Fb_0/CBin_889/c88991.trinity.reads.fa.out/Trinity.tmp.fasta.salmon.idx

[2021-06-03 15:21:35.250] [puff::index::jointLog] [info] Running fixFasta

[Step 1 of 4] : counting k-mers

[2021-06-03 15:21:35.255] [puff::index::jointLog] [info] Replaced 0 non-ATCG nucleotides

[2021-06-03 15:21:35.255] [puff::index::jointLog] [info] Clipped poly-A tails from 0 transcripts

wrote 1 cleaned references

[2021-06-03 15:21:35.260] [puff::index::jointLog] [info] Filter size not provided; estimating from number of distinct k-mers

[2021-06-03 15:21:35.261] [puff::index::jointLog] [info] ntHll estimated 47423 distinct k-mers, setting filter size to 2^20

Threads = 1

Vertex length = 25

Hash functions = 5

Filter size = 1048576

Capacity = 1

Files:

/project/chenzhuyifu/rna_seq/workspace/DMF/assembly/trinity_out_CSC1/read_partitions/Fb_0/CBin_889/c88991.trinity.reads.fa.out/Trinity.tmp.fasta.salmon.idx/ref_k25_fixed.fa

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

Round 0, 0:1048576

Pass Filling Filtering

1 0 0

2 0 0

True junctions count = 0

False junctions count = 2

Hash table size = 2

Candidate marks count = 2

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

Reallocating bifurcations time: 0

True marks count: 2

Edges construction time: 0

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

Distinct junctions = 0

allowedIn: 8

Max Junction ID: 43

seen.size():353 kmerInfo.size():44

approximateContigTotalLength: 420

counters for complex kmers:

(prec>1 & succ>1)=0 | (succ>1 & isStart)=0 | (prec>1 & isEnd)=0 | (isStart & isEnd)=0

contig count: 2 element count: 445 complex nodes: 0

# of ones in rank vector: 1

[2021-06-03 15:22:00.261] [puff::index::jointLog] [info] Starting the Pufferfish indexing by reading the GFA binary file.

[2021-06-03 15:22:00.261] [puff::index::jointLog] [info] Setting the index/BinaryGfa directory /project/chenzhuyifu/rna_seq/workspace/DMF/assembly/trinity_out_CSC1/read_partitions/Fb_0/CBin_889/c88991.trinity.reads.fa.out/Trinity.tmp.fasta.salmon.idx

size = 445

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

| Loading contigs | Time = 629.46 us

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

size = 445

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

| Loading contig boundaries | Time = 614.63 us

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

Number of ones: 1

Number of ones per inventory item: 512

Inventory entries filled: 1

[2021-06-03 15:22:00.262] [puff::index::jointLog] [info] Done wrapping the rank vector with a rank9sel structure.

[2021-06-03 15:22:00.262] [puff::index::jointLog] [info] contig count for validation: 1

[2021-06-03 15:22:00.263] [puff::index::jointLog] [info] Total # of Contigs : 1

[2021-06-03 15:22:00.263] [puff::index::jointLog] [info] Total # of numerical Contigs : 1

[2021-06-03 15:22:00.263] [puff::index::jointLog] [info] Total # of contig vec entries: 0

[2021-06-03 15:22:00.263] [puff::index::jointLog] [info] bits per offset entry 0

[2021-06-03 15:22:00.263] [puff::index::jointLog] [info] Done constructing the contig vector. 2

[2021-06-03 15:22:00.267] [puff::index::jointLog] [info] # segments = 1

[2021-06-03 15:22:00.267] [puff::index::jointLog] [info] total length = 445

Error, cmd:

salmon --no-version-check index -t /project/chenzhuyifu/rna_seq/workspace/DMF/assembly/trinity_out_CSC1/read_partitions/Fb_0/CBin_889/c88991.trinity.reads.fa.out/Trinity.tmp.fasta -i /project/chenzhuyifu/rna_seq/workspace/DMF/assembly/trinity_out_CSC1/read_partitions/Fb_0/CBin_889/c88991.trinity.reads.fa.out/Trinity.tmp.fasta.salmon.idx -k 25 -p 1 > _salmon.27992.stderr 2>&1

died with ret (35584) at /project/chenzhuyifu/software/miniconda3/opt/trinity-2.12.0/util/support_scripts/../../PerlLib/Process_cmd.pm line 19.

Process_cmd::process_cmd("salmon --no-version-check index -t /project/chenzhuyifu/rna_s"...) called at /project/chenzhuyifu/software/miniconda3/opt/trinity-2.12.0/util/support_scripts/salmon_runner.pl line 41

eval {...} called at /project/chenzhuyifu/software/miniconda3/opt/trinity-2.12.0/util/support_scripts/salmon_runner.pl line 40

main::run_cmd_capture_stderr("salmon --no-version-check index -t /project/chenzhuyifu/rna_s"..., "_salmon.27992.stderr") called at /project/chenzhuyifu/software/miniconda3/opt/trinity-2.12.0/util/support_scripts/salmon_runner.pl line 24

--->

Error encountered:: <!----

CMD: /home/chenzhuyifu/project/software/miniconda3/bin/bowtie2-build --threads 1 -o 3 /project/chenzhuyifu/rna_seq/workspace/DMF/assembly/trinity_out_CSC1/read_partitions/Fb_0/CBin_157/c15753.trinity.reads.fa.out/chrysalis/inchworm.fa.min100 /project/chenzhuyifu/rna_seq/workspace/DMF/assembly/trinity_out_CSC1/read_partitions/Fb_0/CBin_157/c15753.trinity.reads.fa.out/chrysalis/inchworm.fa.min100 1>/dev/null 2>tmp.26535.1622704895.stderr

Errmsg:

Building a SMALL index

An error occurred writing the index to disk. Please check if the disk is full.

--->

Trinity run failed. Must investigate error above.

succeeded(1230) 0.996137% completed. -salmon error reported: Error, cmd: /project/chenzhuyifu/software/miniconda3/opt/trinity-2.12.0/util/support_scripts/salmon_runner.pl /project/chenzhuyifu/rna_seq/workspace/DMF/assembly/trinity_out_CSC1/read_partitions/Fb_0/CBin_889/c88991.trinity.reads.fa.out/Trinity.tmp.fasta /project/chenzhuyifu/rna_seq/workspace/DMF/assembly/trinity_out_CSC1/read_partitions/Fb_0/CBin_889/c88991.trinity.reads.fa.out/single.fa.SR_supp.fa 2>tmp.24259.1622704806.stderr died with ret 65280 at /project/chenzhuyifu/software/miniconda3/opt/trinity-2.12.0/PerlLib/Pipeliner.pm line 187.

Pipeliner::run(Pipeliner=HASH(0x562c67c657d8)) called at /project/chenzhuyifu/software/miniconda3/opt/trinity-2.12.0/util/support_scripts/../../Trinity line 1907

eval {...} called at /project/chenzhuyifu/software/miniconda3/opt/trinity-2.12.0/util/support_scripts/../../Trinity line 1906

main::run_Trinity() called at /project/chenzhuyifu/software/miniconda3/opt/trinity-2.12.0/util/support_scripts/../../Trinity line 1416

eval {...} called at /project/chenzhuyifu/software/miniconda3/opt/trinity-2.12.0/util/support_scripts/../../Trinity line 1415

WARNING - salmon failure mode not recognized by Trinity:

Error, cmd: /project/chenzhuyifu/software/miniconda3/opt/trinity-2.12.0/util/support_scripts/salmon_runner.pl /project/chenzhuyifu/rna_seq/workspace/DMF/assembly/trinity_out_CSC1/read_partitions/Fb_0/CBin_889/c88991.trinity.reads.fa.out/Trinity.tmp.fasta /project/chenzhuyifu/rna_seq/workspace/DMF/assembly/trinity_out_CSC1/read_partitions/Fb_0/CBin_889/c88991.trinity.reads.fa.out/single.fa.SR_supp.fa 2>tmp.24259.1622704806.stderr died with ret 65280 at /project/chenzhuyifu/software/miniconda3/opt/trinity-2.12.0/PerlLib/Pipeliner.pm line 187.

Pipeliner::run(Pipeliner=HASH(0x562c67c657d8)) called at /project/chenzhuyifu/software/miniconda3/opt/trinity-2.12.0/util/support_scripts/../../Trinity line 1907

eval {...} called at /project/chenzhuyifu/software/miniconda3/opt/trinity-2.12.0/util/support_scripts/../../Trinity line 1906

main::run_Trinity() called at /project/chenzhuyifu/software/miniconda3/opt/trinity-2.12.0/util/support_scripts/../../Trinity line 1416

eval {...} called at /project/chenzhuyifu/software/miniconda3/opt/trinity-2.12.0/util/support_scripts/../../Trinity line 1415

- retaining Trinity transcripts provided as input to salmon, w/o filtering (pre-salmon mode).

trinity.log

Brian Haas

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Jun 4, 2021, 7:59:44 AM6/4/21

to KB, trinityrnaseq-users

Hi,

It's probably because you're using a newer version of salmon than we use in Trinity. You can run Trinity with --no_salmon for a 'quick fix' - it really doesn't contribute much in this context, but I prefer to use it where we can.

Here's the versions of all the software we use in Trinity:

https://github.com/trinityrnaseq/trinityrnaseq/blob/master/Docker/Dockerfile

If you can use our singularity image, it comes with everything prepackaged and is generally the easiest way to run Trinity:
https://github.com/trinityrnaseq/trinityrnaseq/wiki/Trinity-in-Docker#trinity_singularity

best,

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

KB

unread,

Jun 8, 2021, 9:11:07 PM6/8/21

to trinityrnaseq-users

Hi brian,

Thanks a lot. It seems like the issue has been solved when I turned the version of salmon back to 1.4.0 and changed the dectory of trinity_out_dir to a larger disk. Hope to contact later!