Trinity v2.0.2 - Butterfly fails

Marian

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Jan 27, 2015, 2:35:24 PM1/27/15

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Hi all,

the new version of Trinity stops during butterfly. I get the following error output:

Number of  Commands: 1
Exception in thread "main" java.lang.RuntimeException: Error, detected cycles in seqvertex_graph, so not a DAG as expected!
        at TransAssembly_allProbPaths.zipper_collapse_DAG_zip_up(TransAssembly_allProbPaths.java:2098)
        at TransAssembly_allProbPaths.convert_path_DAG_to_SeqVertex_DAG(TransAssembly_allProbPaths.java:1980)
        at TransAssembly_allProbPaths.create_DAG_from_OverlapLayout(TransAssembly_allProbPaths.java:1550)
        at TransAssembly_allProbPaths.main(TransAssembly_allProbPaths.java:918)
succeeded(0), failed(1)   100% completed.    

We are sorry, commands in file: [failed_butterfly_commands.68215.txt] failed.  :-( 

Error, cmd: /mnt/various/local/genome/packages/trinity/2.0.2/trinity-plugins/parafly/bin/ParaFly -c /mnt/users/mariansc/RNA_seq_data_strandspecific/FR/Trinity_23Jan2014/read_partitions/Fb_0/CBin_0/c74123.trinity.reads.fa.out/chrysalis/butterfly_commands -shuffle -CPU 8 -failed_cmds failed_butterfly_commands.68215.txt -v  died with ret 256 at /mnt/various/local/genome/packages/trinity/2.0.2/util/support_scripts/../../Trinity line 1997.

Trinity run failed. Must investigate error above. at /mnt/various/local/genome/packages/trinity/2.0.2/util/support_scripts/../../Trinity line 1057.
succeeded(0), failed(1)   100% completed.    

We are sorry, commands in file: [FailedCommands] failed.  :-( 

Error, cmd: /mnt/various/local/genome/packages/trinity/2.0.2/trinity-plugins/parafly/bin/ParaFly -c recursive_trinity.cmds -CPU 20 -v  died with ret 256 at /local/genome/packages/trinity/2.0.2/Trinity line 1997.

Trinity run failed. Must investigate error above. at /local/genome/packages/trinity/2.0.2/Trinity line 1057.

The failed command in

[FailedCommands] :

/mnt/various/local/genome/packages/trinity/2.0.2/util/support_scripts/../../Trinity --single "/mnt/users/mariansc/RNA_seq_data_strandspecific/FR/Trinity_23Jan2014/read_partitions/Fb_0/CBin_0/c74123.trinity.reads.fa" --output "/mnt/users/mariansc/RNA_seq_data_strandspecific/FR/Trinity_23Jan2014/read_partitions/Fb_0/CBin_0/c74123.trinity.reads.fa.out" --CPU 1 --max_memory 1G --SS_lib_type F --full_cleanup --seqType fa --trinity_complete --bflyCPU 8

I have no glue what the problem could be. Since it's written something with Java I checked the version and it is Java 7 (as suggested by someone else here in the group). Could it be memory related?

Thank you for help,

marian

Brian Haas

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Jan 27, 2015, 2:38:42 PM1/27/15

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Hi Marian,

This is a software bug, so nothing you're doing wrong.

Can you please send me your

/mnt/users/mariansc/RNA_seq_data_strandspecific/FR/Trinity_23Jan2014/read_partitions/Fb_0/CBin_0/c74123.trinity.reads.fa

file?

I'll look into it right away.

thanks,

~brian

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Brian Haas

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Jan 27, 2015, 10:49:06 PM1/27/15

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Hi Marian,

I'm pretty sure I've fixed this bug... this is a very tricky area of the codebase.

There's a new version of the Butterfly.jar file here:

https://github.com/trinityrnaseq/trinityrnaseq/blob/master/Butterfly/Butterfly.jar

(click the 'raw' button to download)

that you can drop in as a replacement under TRINTY_HOME/Butterfly/

and then rerun your original Trinity command. The earlier butterfly job that failed will hopefully complete fine now, and the job should wrap up with the final assembly fasta file as usual.

Let me know if it gives you any trouble.

We'll be doing some more testing before putting out another release. This bug should be a very rare occurrence for others that are currently using the 2.0.2 release.

best of luck,

~brian

Marian

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Jan 28, 2015, 8:31:24 AM1/28/15

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Hi Brian,

thank you so much for fixing. After rerunning the script with the new Butterfly.jar Trinity finished just fine.

marian

Brian Haas

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Jan 28, 2015, 8:37:12 AM1/28/15

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Excellent. Thanks for the update!

~brian

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Evan Ernst

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Jan 30, 2015, 8:55:24 PM1/30/15

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Unfortunately, the new Butterfly.jar did not work for me. After replacing it and running my original Trinity command, I get the same error:

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

-------------------- Butterfly --------------------------------

-- (Reconstruct transcripts from reads and de Bruijn graphs) --

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

Butterfly_cmds: /data/eernst/projects/xxx/trinity-dn/trinity_out_dir/read_partitions/Fb_0/CBin_0/c66203.trinity.reads.fa.out/chrysalis/butterfly_commands

Inchworm and Chrysalis complete. Butterfly commands to execute are provided here:

/data/eernst/projects/xxx/trinity-dn/trinity_out_dir/read_partitions/Fb_0/CBin_0/c66203.trinity.reads.fa.out/chrysalis/butterfly_commands

Friday, January 30, 2015: 20:49:49 CMD: /home/eernst/src/trinityrnaseq-2.0.2/trinity-plugins/parafly/bin/ParaFly -c /data/eernst/projects/xxx/trinity-dn/trinity_out_dir/read_partitions/Fb_0/CBin_0/c66203.trinity.reads.fa.out/chrysalis/butterfly_commands -shuffle -CPU 1 -failed_cmds failed_butterfly_commands.14330.txt -v

Number of Commands: 2

succeeded(1) 50% completed. Exception in thread "main" java.lang.RuntimeException: Error, detected cycles in seqvertex_graph, so not a DAG as expected!

at TransAssembly_allProbPaths.convert_path_DAG_to_SeqVertex_DAG(TransAssembly_allProbPaths.java:1981)

at TransAssembly_allProbPaths.create_DAG_from_OverlapLayout(TransAssembly_allProbPaths.java:1550)

at TransAssembly_allProbPaths.main(TransAssembly_allProbPaths.java:918)

succeeded(1), failed(1) 100% completed.

We are sorry, commands in file: [failed_butterfly_commands.14330.txt] failed. :-(

Error, cmd: /home/eernst/src/trinityrnaseq-2.0.2/trinity-plugins/parafly/bin/ParaFly -c /data/eernst/projects/xxx/trinity-dn/trinity_out_dir/read_partitions/Fb_0/CBin_0/c66203.trinity.reads.fa.out/chrysalis/butterfly_commands -shuffle -CPU 1 -failed_cmds failed_butterfly_commands.14330.txt -v died with ret 256 at /home/eernst/src/trinityrnaseq-2.0.2/util/support_scripts/../../Trinity line 1997.

Trinity run failed. Must investigate error above. at /home/eernst/src/trinityrnaseq-2.0.2/util/support_scripts/../../Trinity line 1057.

succeeded(0), failed(1) 100% completed.

We are sorry, commands in file: [FailedCommands] failed. :-(

Error, cmd: /home/eernst/src/trinityrnaseq-2.0.2/trinity-plugins/parafly/bin/ParaFly -c recursive_trinity.cmds -CPU 32 -v died with ret 256 at /home/eernst/src/trinityrnaseq-2.0.2/Trinity line 1997.

Trinity run failed. Must investigate error above. at /home/eernst/src/trinityrnaseq-2.0.2/Trinity line 1057.

On Wednesday, January 28, 2015 at 8:37:12 AM UTC-5, Brian Haas wrote:

Excellent. Thanks for the update!

~brian

On Wed, Jan 28, 2015 at 8:31 AM, Marian <nonations...@gmail.com> wrote:

Hi Brian,

thank you so much for fixing. After rerunning the script with the new Butterfly.jar Trinity finished just fine.

marian

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Brian Haas

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Jan 30, 2015, 9:02:09 PM1/30/15

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Hi Evan,

Can you please tar up your entry:

/data/eernst/projects/xxx/trinity-dn/trinity_out_dir/read_partitions/Fb_0/CBin_0/c66203.trinity.reads.fa.out/

and send it to me?

I thought this was fixed for good, but apparently there's another edge case.

many thanks

~brian

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Brian Haas

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Jan 30, 2015, 10:27:28 PM1/30/15

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Hi Evan,

I confirmed the error, and I was able to resolve it (again). Hopefully this is fixed once and for all.

Updated code is here:

https://github.com/trinityrnaseq/trinityrnaseq/tree/master/Butterfly

fingers crossed...

~brian

Evan Ernst

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Jan 30, 2015, 10:34:58 PM1/30/15

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All commands completed successfully. :-)

Thanks, Brian!

Brian Haas

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Jan 30, 2015, 10:35:30 PM1/30/15

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excellent! :)

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Qing Li

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Aug 14, 2015, 3:24:26 PM8/14/15

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Hi all,

I am using the Trinity version 2.0.5, and still have similar errors ...

We are sorry, commands in file: [failed_butterfly_commands.25355.txt] failed. :-(

Trinity run failed. Must investigate error above.

succeeded(0), failed(6) 100% completed.

We are sorry, commands in file: [FailedCommands] failed. :-(

Trinity run failed. Must investigate error above.

my command line is : /home/qli/tool/trinityrnaseq-2.0.5/Trinity --seqType fq --max_memory 500G --left ../dup.1.fa --right ../dup.2.fa --CPU 16 --bflyHeapSpaceMax 100G --bflyCPU 14 --min_contig_length 75 --min_per_id_same_path 99 --max_diffs_same_path 1 --max_internal_gap_same_path 3 --full_cleanup --bfly_opts "--max_number_of_paths_per_node_extend 50"

Seems to be a butterfly error?

Thank you for your help!

Best,

Qing

Mark Chapman

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Aug 14, 2015, 5:40:46 PM8/14/15

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Hi Qing. One thing I notice is that you specify filetype fq but are giving fa files. Not sure this will explain it, but is something that might help. Also 2.0.6 is the newest version so if you've no reason to use an older version then maybe upgrade.
If the fq/fa change doesn't help try with default butterfly options and see if it runs.
Cheers, Mark

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Qing Li

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Aug 14, 2015, 5:48:43 PM8/14/15

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Thx, Mark! The fq/fa isn't a issue, since when I run it in default butterfly settings, no complain at all... Is there anything wrong with the bfly_opts command line? But I need to twist the parameters...

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Mark Chapman

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Aug 14, 2015, 6:09:45 PM8/14/15

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Hi Qing, That certainly suggests something is off. I would try dropping one or more of the bfly commands and see if one in particular is causing the problem.
Also, 1 if you normalise the data it will be much quicker. You can do this once and then run trinity several times
And 2 are you likely running out is space, either in terms of total number of files produced or total file size?
Trinity will try and take of from where it failed so, as you're not specifying an output folder, maybe clear up everything you've done before re running again.
Cheers, Mark

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Qing Li

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Aug 14, 2015, 6:35:51 PM8/14/15

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Thx, Mark~ I think the one caused problem is --bfly_opts "--max_number_of_paths_per_node_extend 50"

Dropping that one will no longer cause any problem, but this is the one I want to test...

The test dataset is very small, so re-running it / file size is not a big problem ,,, I did remove the dir before re-run , but no improvement ...

is my command line valid,,,?

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Mark Chapman

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Aug 14, 2015, 7:21:45 PM8/14/15

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Hi Qing, I see from older release notes that the bfly parameters have changed a few times. Maybe this was different in v2.0.2? Sorry that's all I got for the advanced bfly opts!
--Mark

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李青 Li, Qing

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Aug 17, 2015, 12:36:27 AM8/17/15

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Thx, Mark! I think it's the same since other bfly parameters passed in this way was fine,,, I guess it's just a miner bug? No worries, it may be no longer that important to me~ thx!

Victor T.

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Sep 15, 2015, 11:00:33 AM9/15/15

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Hello,
I am using Trinity 2.0.6 and am still getting the same error messages even after following the advice from this thread. I am running Trinity on a server of 125GB total memory.
Also Trinity works fine with the sample data
The fasta files I am trying to assemble are about 220 Mb with 2.1M sequences each, so how likely is it to be a memory issue?

Command line: Trinity --seqType fa --max_memory 80G --left /home/stxvt/reads/argyroneta-abdomen-paired/Spider-silk-gland-RNA_S3_L001_R1_001.scythe.sickle.fasta --right /home/stxvt/reads/argyroneta-abdomen-paired/Spider-silk-gland-RNA_S3_L001_R2_001.scythe.sickle.fasta --CPU 8 --bflyHeapSpaceMax 25G --bflyCPU 6 --min_contig_length 75 --min_per_id_same_path 99 --max_diffs_same_path 1 --max_internal_gap_same_path 3 --full_cleanup --output trinity-L1paired

Error message:
Exception in thread "main" java.lang.ArrayIndexOutOfBoundsException: 1
    at TransAssembly_allProbPaths.readAndMapSingleRead(TransAssembly_allProbPaths.java:11521)
    at TransAssembly_allProbPaths.getReadStarts(TransAssembly_allProbPaths.java:11438)
    at TransAssembly_allProbPaths.main(TransAssembly_allProbPaths.java:893)
succeeded(1), failed(1)   0.00488424% completed.

We are sorry, commands in file: [failed_butterfly_commands.137915.txt] failed. :-(

Would be grateful for any advice!

Victor

Nima Rafati

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Apr 4, 2016, 3:54:43 AM4/4/16

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Dear Brian,

I am using Trinity v 2.1.0 and receive following error:

We are sorry, commands in file: [FailedCommands] failed. :-(

where there is only one failed commands:

/pica/sw/apps/bioinfo/trinity/2.1.0/milou/util/support_scripts/../../Trinity --single "/pica/v10/b2011054/private/Users/nima/tmpRabbit/Domestication/Trinity/Trinity-Domestic/trinity-BF-20160401/read_partitions/Fb_4/CBin_4401/c440168.trinity.reads.fa" --output "/pica/v10/b2011054/private/Users/nima/tmpRabbit/Domestication/Trinity/Trinity-Domestic/trinity-BF-20160401/read_partitions/Fb_4/CBin_4401/c440168.trinity.reads.fa.out" --CPU 1 --max_memory 1G --SS_lib_type F --seqType fa --trinity_complete --full_cleanup --bflyCPU 16 --min_kmer_cov 2 --min_per_id_same_path 90 --max_diffs_same_path 4

Is this related to the problem(s) that you fixed before ?

I will be grateful if you can help.

Best regards,

Nima

Brian Haas

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Apr 4, 2016, 6:21:46 AM4/4/16

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Hi Nima,

When this error has happened in the past, it had to do with cntrl-M chars in the sequences.

If you run:

cat -te /pica/v10/b2011054/private/Users/nima/tmpRabbit/Domestication/Trinity/Trinity-Domestic/trinity-BF-20160401/read_partitions/Fb_4/CBin_4401/c440168.trinity.reads.fa | less

do you see ^M at the end of each line?

~b

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Nima Rafati

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Apr 4, 2016, 6:26:17 AM4/4/16

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Dear Brian,

As always you are quick in replying (thanks!!). I ran the commands that you wrote in email and do not see any ^M. All the lines are ended with "$". One more thing that I notices is about library type. In my original command I had set --SS_lib_type RF. But in failed command it is --SS_lib_type F. Should it be like this?

Best,

Nima

Brian Haas

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Apr 4, 2016, 6:27:35 AM4/4/16

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Yes, it should be that way. OK - can you (privately) send me that file and I'll take a look?

thx,

~b

Brian Haas

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Apr 4, 2016, 6:43:03 AM4/4/16

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Hi Nima,

Can you try running:

Trinity --seqType fa --single c440168.trinity.reads.fa --CPU 2 --max_memory 1G --trinity_complete --SS_lib_type F --bflyCPU 16 --min_kmer_cov 2 --min_per_id_same_path 90 --max_diffs_same_path 4

in a clean workspace and see if it works?

~b

Nima Rafati

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Apr 4, 2016, 7:21:05 AM4/4/16

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Hi Brian,

It worked without any problem and produced Trinity.fa (?!)

What can be the problem (path....)?

Brian Haas

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Apr 4, 2016, 7:25:02 AM4/4/16

to Nima Rafati, trinityrnaseq-users, Evan Ernst

It's weird. Try removing the output directory for this entry:

rm -r /pica/v10/b2011054/private/Users/nima/tmpRabbit/Domestication/Trinity/Trinity-Domestic/trinity-BF-20160401/read_partitions/Fb_4/CBin_4401/c440168.trinity.reads.fa.out/

and then rerunning your original Trinity command. It should pick up where it left off, ideally run this one job successfully, and then wrap up.

best,

~b

Nima Rafati

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Apr 5, 2016, 10:08:40 AM4/5/16

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Dear Brian,

It worked! It was a big assembly and took almost 15 hours to finish that's why I was late in replying.

But it was weird that only one failed command hinders the very last step.

Thanks for your help!

Best regards,

Nima

Brian Haas

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Apr 5, 2016, 11:04:49 AM4/5/16

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I'm glad to hear it finally finished ok. Thanks for the update!

~b

Mauricio Losilla

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Apr 12, 2016, 12:15:06 PM4/12/16

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Hi,

I had this same problem, my assembly completed all but 1 command. After browsing through the forum I did what Brian suggested: delete the path/to/read_partitions/cXXXXXX.trinity.reads.fa.out/ and successfully ran the failed command (Actually I didn't delete the folder, just moved it to a different location, JIC someone wants to take a look at it for debugging purposes).

I tried to look into why this happened, and maybe I found something that could be useful for other users or for the developers. This was the error message in the log file:

mv: cannot stat `path/to/read_partitions/Fb_0/CBin_581/c58145.trinity.reads.fa.out/inchworm.K25.L25.fa.tmp': No such file or directory

Error, cmd: mv path/to/read_partitions/Fb_0/CBin_581/c58145.trinity.reads.fa.out/inchworm.K25.L25.fa.tmp path/to/read_partitions/Fb_0/CBin_581/c58145.trinity.reads.fa.out/inchworm.K25.L25.fa 2>tmp.13426.stderr died with ret 256 at /opt/software/trinity/2.2.0--GCC-4.4.5/PerlLib/Pipeliner.pm line 102

Pipeliner::run('Pipeliner=HASH(0x22c4d88)') called at /opt/software/trinity/2.2.0--GCC-4.4.5/util/support_scripts/../../Trinity line 2058

eval {...} called at /opt/software/trinity/2.2.0--GCC-4.4.5/util/support_scripts/../../Trinity line 2053

main::run_inchworm('path/to...', 'single.fa', 'F', '') called at /opt/software/trinity/2.2.0--GCC-4.4.5/util/support_scripts/../../Trinity line 1428

main::run_Trinity() called at /opt/software/trinity/2.2.0--GCC-4.4.5/util/support_scripts/../../Trinity line 1209

eval {...} called at /opt/software/trinity/2.2.0--GCC-4.4.5/util/support_scripts/../../Trinity line 1208

This log says that the first error is due to a missing file: inchworm.K25.L25.fa.tmp. Upon checking the folder contents, this file was indeed absent. The file inchworm.K25.L25.fa was present, but the file inchworm.K25.L25.fa.finished was absent (I saw this file in other folders).

This suggested to me that Trinity was abruptly interrupted when working on this folder. I then checked the logs from previous runs (I often complete my assemblies in multiple steps, because of computing resources limitations. Each step is killed by the HPC system when it exceeds the running time I assign it). The last log before the failed command says:

succeeded(58133) 11.3117% completed. =>> PBS: job killed

58133 is very close to 58145, the command that eventually failed. The log of the next run reports the error, plus some extra info:

warning, command: /opt/software/trinity/2.2.0--GCC-4.4.5/util/support_scripts/../../Trinity --single "path/to/read_partitions/Fb_0/CBin_581/c58138.trinity.reads.fa" --output "/path/to/read_partitions/Fb_0/CBin_581/c58138.trinity.reads.fa.out" --CPU 1 --max_memory 1G --SS_lib_type F --seqType fa --trinity_complete --full_cleanup --min_contig_length 101 has successfully completed from a previous run. Skipping it here.

mv: cannot stat `path/to/read_partitions/Fb_0/CBin_581/c58145.trinity.reads.fa.out/inchworm.K25.L25.fa.tmp': No such file or directory

Error, cmd: mv path/to/read_partitions/Fb_0/CBin_581/c58145.trinity.reads.fa.out/inchworm.K25.L25.fa.tmp path/to/read_partitions/Fb_0/CBin_581/c58145.trinity.reads.fa.out/inchworm.K25.L25.fa 2>tmp.27088.stderr died with ret 256 at /opt/software/trinity/2.2.0--GCC-4.4.5/PerlLib/Pipeliner.pm line 102

Pipeliner::run('Pipeliner=HASH(0x1f49d88)') called at /opt/software/trinity/2.2.0--GCC-4.4.5/util/support_scripts/../../Trinity line 2058

eval {...} called at /opt/software/trinity/2.2.0--GCC-4.4.5/util/support_scripts/../../Trinity line 2053

main::run_inchworm('/mnt/ls15/scratch/users/losillam/TranscriptomeAssembly/RNAseq...', 'single.fa', 'F', '') called at /opt/software/trinity/2.2.0--GCC-4.4.5/util/support_scripts/../../Trinity line 1428

main::run_Trinity() called at /opt/software/trinity/2.2.0--GCC-4.4.5/util/support_scripts/../../Trinity line 1209

eval {...} called at /opt/software/trinity/2.2.0--GCC-4.4.5/util/support_scripts/../../Trinity line 1208

If it indicates bad_alloc(), then Inchworm ran out of memory. You'll need to either reduce the size of your data set or run Trinity on a server with more memory available.

succeeded(0), failed(1) 0.000219401% completed. WARNING, cannot remove output directory path/to/read_partitions/Fb_0/CBin_581/c58141.trinity.reads.fa.out, since not created in this run. (safety precaution)

succeeded(2), failed(1) 0.000658204% completed. WARNING, cannot remove output directory path/to/read_partitions/Fb_0/CBin_581/c58139.trinity.reads.fa.out, since not created in this run. (safety precaution)

succeeded(3), failed(1) 0.000877605% completed. WARNING, cannot remove output directory path/to/read_partitions/Fb_0/CBin_581/c58140.trinity.reads.fa.out, since not created in this run. (safety precaution)

succeeded(4), failed(1) 0.00109701% completed. WARNING, cannot remove output directory /path/to/read_partitions/Fb_0/CBin_581/c58142.trinity.reads.fa.out, since not created in this run. (safety precaution)

succeeded(12), failed(1) 0.00285222% completed. WARNING, cannot remove output directory path/to/read_partitions/Fb_0/CBin_581/c58144.trinity.reads.fa.out, since not created in this run. (safety precaution)

succeeded(19), failed(1) 0.00438802% completed. WARNING, cannot remove output directory path/to/read_partitions/Fb_0/CBin_581/c58143.trinity.reads.fa.out, since not created in this run. (safety precaution)

From this log, it looks like the previous Trinity execution completed 58138 commands, and not 58133 as that log reported. In addition, the folders cXXXXX.trinity.reads.fa.out had been created up until the one that failed. Hence, I think that what caused the failure was one of the interruptions of a previous Trinity run. Like I said, I saved the folder (and the logs), if someone wants to take a closer look.

I hope this helps,

Mau

Walter Cocca

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May 18, 2016, 11:00:16 AM5/18/16

to trinityrnaseq-users, nimar...@gmail.com, legi...@gmail.com

Hi Brian, hi all

I'm running Trinity 2.2.0 and, as happened for others in this post, Butterfly fail. This is in the FailedCommand output: