No Inchworm or Butterfly output generated

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Hasnahana Chetia

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Jan 11, 2017, 5:23:18 AM1/11/17
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Dear fellow Trinity users,

I am assembling a single-end fastq file using Trinity v2.3.2.
The command is Trinity --seqType fq --max_memory 20G --single SRR1234.fastq --CPU 4
I am running into the following error-

----------------------------------------------
--------------- Inchworm ---------------------
-- (Linear contig construction from k-mers) --
----------------------------------------------

* Running CMD: /home/softwares/trinityrnaseq-Trinity-v2.3.2/Inchworm/bin//inchworm --kmers jellyfish.kmers.fa --run_inchworm -K 25 -L 25 --monitor 1   --DS  --num_threads 4  --PARALLEL_IWORM  > /media/I/SRA/trinity_out_dir/inchworm.K25.L25.DS.fa.tmp
* Running CMD: mv /media/I/SRA/trinity_out_dir/inchworm.K25.L25.DS.fa.tmp /media/I/SRA/trinity_out_dir/inchworm.K25.L25.DS.fa
Wednesday, January 11, 2017: 15:16:55    CMD: touch /media/I/SRA/trinity_out_dir/inchworm.K25.L25.DS.fa.finished
NON_FATAL_EXCEPTION: WARNING, no Inchworm output is detected at: /media/I/SRA/trinity_out_dir/inchworm.K25.L25.DS.fa at /home/softwares/trinityrnaseq-Trinity-v2.3.2/Trinity line 1539.

ERROR, no butterfly assemblies reported. Perhaps there were too few reads to assemble anything?  at /home/softwares/trinityrnaseq-Trinity-v2.3.2/Trinity line 1313.

My SRA file has 4819322 reads. Is pre-assembly normalization having this effect on the assembly?

Thanking you,

Hasnahana

Brian Haas

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Jan 11, 2017, 6:26:33 AM1/11/17
to Hasnahana Chetia, trinityrnaseq-users
This is unusual.  Can you 

   ls -ltr   trinity_out_dir/

and post results?

thx,

~b

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Hasnahana Chetia

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Jan 11, 2017, 9:44:44 AM1/11/17
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Dear Sir,
I treid running the command again. I got a new error

Wednesday, January 11, 2017: 17:28:36    CMD: touch partitioned_reads.files.list.ok
Wednesday, January 11, 2017: 17:28:36    CMD: /home/softwares/trinityrnaseq-Trinity-v2.3.2/util/support_scripts/write_partitioned_trinity_cmds.pl --reads_list_file partitioned_reads.files.list --CPU 1 --max_memory 1G  --seqType fa --trinity_complete --full_cleanup  > recursive_trinity.cmds

####################################################################################
#
#  usage: /home/softwares/trinityrnaseq-Trinity-v2.3.2/util/support_scripts/write_partitioned_trinity_cmds.pl --reads_list_file <string> [Trinity params]
#
# Required:
#
# --reads_list_file <string>      file containing list of filenames corresponding
#                                  to the reads.fasta
#
#
#####################################################################################


Error, cmd: /home/softwares/trinityrnaseq-Trinity-v2.3.2/util/support_scripts/write_partitioned_trinity_cmds.pl --reads_list_file partitioned_reads.files.list --CPU 1 --max_memory 1G  --seqType fa --trinity_complete --full_cleanup  > recursive_trinity.cmds died with ret 65280 at /home/softwares/trinityrnaseq-Trinity-v2.3.2/Trinity line 2427.

Here is the ls -ltr output of the out dir
total 34194
-rw------- 1 u u 21688320 Jan 11 17:25 single.fa
-rw------- 1  u u      173 Jan 11 17:25 Trinity.timing
-rw------- 1  u u        0 Jan 11 17:28 single.fa.ok
drwx------ 1 u u      360 Jan 11 17:28 insilico_read_normalization
-rw------- 1  u u        9 Jan 11 17:28 single.fa.read_count
-rw------- 1  u u 11859471 Jan 11 17:28 jellyfish.kmers.fa
-rw------- 1 u u     7153 Jan 11 17:28 jellyfish.kmers.fa.histo
-rw------- 1 u u       7 Jan 11 17:28 inchworm.kmer_count
-rw------- 1 u u  1448785 Jan 11 17:28 inchworm.K25.L25.DS.fa
-rw------- 1 u u        0 Jan 11 17:28 inchworm.K25.L25.DS.fa.finished
-rw------- 1 u u       0 Jan 11 17:28 partitioned_reads.files.list.ok
-rw------- 1 u u     1713 Jan 11 17:28 pipeliner.5019.cmds
drwx------ 1 u u      472 Jan 11 17:28 chrysalis
-rw------- 1 u u       0 Jan 11 17:28 partitioned_reads.files.list
-rw------- 1 u u        0 Jan 11 17:28 recursive_trinity.cmds

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Ms. Hasnahana Chetia
Research Scholar,
Bioengineering Research Laboratory,
Dept. of Biosciences and Bioengineering ,
Indian Institute of Technology Guwahati, Assam-39

Brian Haas

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Jan 11, 2017, 9:49:39 AM1/11/17
to Hasnahana Chetia, trinityrnaseq-users
It sounds like there might be some trouble w/ the file system.

How many reads did you end up with after the normalization?

     grep '>' trinity_out_dir/single.fa | wc -l


Hasnahana Chetia

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Jan 12, 2017, 12:05:04 AM1/12/17
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I got 267676 reads.

Brian Haas

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Jan 12, 2017, 7:05:52 AM1/12/17
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I think I'll need to take a direct look at this data set.  Can you send me the SRA accession you're experimenting with? Feel free to send it direct, off-list, if you want to keep it private.

best,

~b


Mark Chapman

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Jan 12, 2017, 7:12:43 AM1/12/17
to Brian Haas, Hasnahana Chetia, trinityrnaseq-users
267k reads sounds like a very small number, is this a case where no transcripts were assembled?

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Brian Haas

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Jan 12, 2017, 8:07:50 AM1/12/17
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I'm hoping there's a good explanation like this.  Something doesn't smell right though.

-Brian
(by iPhone)

mestdag...@gmail.com

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Jan 15, 2017, 10:15:24 AM1/15/17
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Hi,

I got a similar error message when running the following command:

Trinity/trinityrnaseq-Trinity-v2.3.2/Trinity --seqType fq 

--left PC9_S5_L001_R1_001.fastq.gz,PC9_S5_L002_R1_001.fastq.gz, ... 

--right PC9_S5_L001_R2_001.fastq.gz,PC9_S5_L002_R2_001.fastq.gz, ... 

--max_memory 110G --SS_lib_type RF --no_normalize_reads --CPU 12 --output Trinity/PC9_trinity_out


These are the files that have been generated so far:

-rw-rw-r-- 1 pmestdagh pmestdagh  25G Jan 14 12:45 both.fa

-rw-rw-r-- 1 pmestdagh pmestdagh    0 Jan 14 12:45 both.fa.ok

-rw-rw-r-- 1 pmestdagh pmestdagh   10 Jan 14 12:45 both.fa.read_count

drwxrwxr-x 2 pmestdagh pmestdagh 4.0K Jan 14 16:02 chrysalis

-rw-rw-r-- 1 pmestdagh pmestdagh 9.4G Jan 14 13:58 inchworm.K25.L25.fa

-rw-rw-r-- 1 pmestdagh pmestdagh    0 Jan 14 14:03 inchworm.K25.L25.fa.finished

-rw-rw-r-- 1 pmestdagh pmestdagh   11 Jan 14 13:19 inchworm.kmer_count

-rw-rw-r-- 1 pmestdagh pmestdagh  30G Jan 14 12:58 jellyfish.kmers.fa

-rw-rw-r-- 1 pmestdagh pmestdagh  80K Jan 14 13:00 jellyfish.kmers.fa.histo

-rw-rw-r-- 1 pmestdagh pmestdagh    0 Jan 14 12:45 left.fa.ok

-rw-rw-r-- 1 pmestdagh pmestdagh    0 Jan 14 16:02 partitioned_reads.files.list

-rw-rw-r-- 1 pmestdagh pmestdagh    0 Jan 14 16:02 partitioned_reads.files.list.ok

-rw-rw-r-- 1 pmestdagh pmestdagh   26 Jan 14 12:37 PC9_S5_L001_R1_001.fastq.gz.readcount

-rw-rw-r-- 1 pmestdagh pmestdagh   27 Jan 14 12:40 PC9_S5_L001_R1_002.fastq.gz.readcount

-rw-rw-r-- 1 pmestdagh pmestdagh   26 Jan 14 12:38 PC9_S5_L001_R2_001.fastq.gz.readcount

-rw-rw-r-- 1 pmestdagh pmestdagh   27 Jan 14 12:40 PC9_S5_L001_R2_002.fastq.gz.readcount

-rw-rw-r-- 1 pmestdagh pmestdagh   26 Jan 14 12:38 PC9_S5_L002_R1_001.fastq.gz.readcount

-rw-rw-r-- 1 pmestdagh pmestdagh   27 Jan 14 12:41 PC9_S5_L002_R1_002.fastq.gz.readcount

-rw-rw-r-- 1 pmestdagh pmestdagh   26 Jan 14 12:38 PC9_S5_L002_R2_001.fastq.gz.readcount

-rw-rw-r-- 1 pmestdagh pmestdagh   27 Jan 14 12:41 PC9_S5_L002_R2_002.fastq.gz.readcount

-rw-rw-r-- 1 pmestdagh pmestdagh   26 Jan 14 12:38 PC9_S5_L003_R1_001.fastq.gz.readcount

-rw-rw-r-- 1 pmestdagh pmestdagh   27 Jan 14 12:42 PC9_S5_L003_R1_002.fastq.gz.readcount

-rw-rw-r-- 1 pmestdagh pmestdagh   26 Jan 14 12:38 PC9_S5_L003_R2_001.fastq.gz.readcount

-rw-rw-r-- 1 pmestdagh pmestdagh   27 Jan 14 12:43 PC9_S5_L003_R2_002.fastq.gz.readcount

-rw-rw-r-- 1 pmestdagh pmestdagh   26 Jan 14 12:39 PC9_S5_L004_R1_001.fastq.gz.readcount

-rw-rw-r-- 1 pmestdagh pmestdagh   27 Jan 14 12:44 PC9_S5_L004_R1_002.fastq.gz.readcount

-rw-rw-r-- 1 pmestdagh pmestdagh   26 Jan 14 12:39 PC9_S5_L004_R2_001.fastq.gz.readcount

-rw-rw-r-- 1 pmestdagh pmestdagh   27 Jan 14 12:44 PC9_S5_L004_R2_002.fastq.gz.readcount

-rw-rw-r-- 1 pmestdagh pmestdagh 1.6K Jan 14 16:02 pipeliner.2121.cmds

-rw-rw-r-- 1 pmestdagh pmestdagh    0 Jan 14 16:02 recursive_trinity.cmds

-rw-rw-r-- 1 pmestdagh pmestdagh    0 Jan 14 12:45 right.fa.ok

-rw-rw-r-- 1 pmestdagh pmestdagh 1.5K Jan 14 12:37 Trinity.timing



####################################################################################

#

#  usage: /store/home/pmestdagh/TxDx/Trinity/trinityrnaseq-Trinity-v2.3.2/util/support_scripts/write_partitioned_trinity_cmds.pl --reads_list_file <string> [Trinity params]

#

# Required:

#

# --reads_list_file <string>      file containing list of filenames corresponding 

#                                  to the reads.fasta

#

#

#####################################################################################



Error, cmd: /store/home/pmestdagh/TxDx/Trinity/trinityrnaseq-Trinity-v2.3.2/util/support_scripts/write_partitioned_trinity_cmds.pl --reads_list_file partitioned_reads.files.list --CPU 1 --max_memory 1G  --run_as_paired  --SS_lib_type F  --seqType fa --trinity_complete --full_cleanup  > recursive_trinity.cmds died with ret 65280 at /home/pmestdagh/TxDx/Trinity/trinityrnaseq-Trinity-v2.3.2/Trinity line 2427.

 

Brian Haas

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Jan 15, 2017, 11:16:12 AM1/15/17
to mestdag...@gmail.com, trinityrnaseq-users, Mark Chapman, Hasnahana Chetia
Hi,

sorry to see it crashed.  Let's look at what's in the chrysalis subfolder

    ls -ltr  trinity_out_dir/chrysalis/

thanks!

~brian


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Pieter Mestdagh

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Jan 15, 2017, 12:03:39 PM1/15/17
to Brian Haas, trinityrnaseq-users, Mark Chapman, Hasnahana Chetia
Hi Brian,

Thanks for your quick response. Here you go:

-rw-rw-r-- 1 pmestdagh pmestdagh 0 Jan 14 14:03 inchworm.K25.L25.fa.min100
-rw-rw-r-- 1 pmestdagh pmestdagh 0 Jan 14 14:42 iworm_cluster_welds_graph.txt
-rw-rw-r-- 1 pmestdagh pmestdagh 0 Jan 14 14:42 inchworm.K25.L25.fa.min100.ok
-rw-rw-r-- 1 pmestdagh pmestdagh 0 Jan 14 15:39 iworm_cluster_welds_graph.txt.ok
-rw-rw-r-- 1 pmestdagh pmestdagh 0 Jan 14 15:39 GraphFromIwormFasta.out
-rw-rw-r-- 1 pmestdagh pmestdagh 0 Jan 14 15:56 GraphFromIwormFasta.out.ok
-rw-rw-r-- 1 pmestdagh pmestdagh 0 Jan 14 15:56 bundled_iworm_contigs.fasta.ok
-rw-rw-r-- 1 pmestdagh pmestdagh 0 Jan 14 15:56 bundled_iworm_contigs.fasta
-rw-rw-r-- 1 pmestdagh pmestdagh 0 Jan 14 15:56 readsToComponents.out
-rw-rw-r-- 1 pmestdagh pmestdagh 2 Jan 14 16:02 readsToComponents.out.rcts.out
-rw-rw-r-- 1 pmestdagh pmestdagh 0 Jan 14 16:02 readsToComponents.out.sort.ok
-rw-rw-r-- 1 pmestdagh pmestdagh 0 Jan 14 16:02 readsToComponents.out.sort
-rw-rw-r-- 1 pmestdagh pmestdagh 0 Jan 14 16:02 readsToComponents.out.ok

Thanks,
Pieter

Brian Haas

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Jan 15, 2017, 5:51:35 PM1/15/17
to Pieter Mestdagh, trinityrnaseq-users
Hi Pieter,

It looks like something strange happened at the chrysalis stage, and it's not obvious as to why.

What I'd suggest doing is to delete that chrysalis subdirectory

    rm -rf chrysalis/

and then rerun the original command.

If it crashes again, I'd need to look at a couple of input files to try to debug it:  

     both.fa
    inchworm.K25.L25.fa

which you could hopefully share with me privately.   Let's see if it comes to that though.

best of luck!

~brian
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Pieter Mestdagh

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Jan 16, 2017, 5:06:08 AM1/16/17
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Hi Brain,

I ran the sequence of commands you suggested but got exactly the same error. Let me know if I can privately share the input files.

Thanks,
Pieter

Brian Haas

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Jan 22, 2017, 11:08:24 AM1/22/17
to Mark Chapman, Hasnahana Chetia, trinityrnaseq-users
It turns out that the issue here is due to just the complexity being very high, the number of reads relatively small (~5M total reads in the SRA sample I examined), single-end instead of paired-end, and the reads being short (46 base length).

best,

~brian


On Thu, Jan 12, 2017 at 8:07 AM, Brian Haas <bh...@broadinstitute.org> wrote:
I'm hoping there's a good explanation like this.  Something doesn't smell right though.

-Brian
(by iPhone)


On Jan 12, 2017, at 7:12 AM, Mark Chapman <markcha...@gmail.com> wrote:

267k reads sounds like a very small number, is this a case where no transcripts were assembled?
On 12 January 2017 at 12:05, Brian Haas <bh...@broadinstitute.org> wrote:

I think I'll need to take a direct look at this data set.  Can you send me the SRA accession you're experimenting with? Feel free to send it direct, off-list, if you want to keep it private.

best,

~b


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------------------------------------
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Life Sciences Building 85
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Southampton
SO17 1BJ

Brian Haas

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Jan 22, 2017, 11:09:22 AM1/22/17
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.... and so it really wasn't able to assemble anything here given the minimum default contig length of 200 bases.  

~b

taua...@gmail.com

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May 19, 2017, 1:45:10 PM5/19/17
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Hello Brian,

I have the exact same issue as in the first post. It is a large SRA dataset (>100 million read pairs). I am using v2.3.2. My job ran out of time, so I just relaunched it. It died very shortly with the error below. Before it ran out of time it seemed to be going just fine (tail of output below).

I didn't understand from your last post if this is a general problem and what we can do about it.

Thanks!

Error:

Friday, May 19, 2017: 12:28:01  CMD: touch /n/regal/Giribet_lab/tauanajc/prep_for_assemblies/Nautilus_pompilius/trinity/TEMP_trinity/inchworm.K25.L25.fa.finished

NON_FATAL_EXCEPTION: WARNING, no Inchworm output is detected at: /n/regal/Giribet_lab/tauanajc/prep_for_assemblies/Nautilus_pompilius/trinity/TEMP_trinity/inchworm.K25.L25.fa at /n/sw/fasrcsw/apps/Core/trinityrnaseq/2.3.2-fasrc01/Trinity line 1539.


Previous output:

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

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

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


Thursday, May 18, 2017: 13:31:49 CMD: /n/sw/fasrcsw/apps/Core/trinityrnaseq/2.3.2-fasrc01/trinity-plugins/parafly/bin/ParaFly -c recursive_trinity.cmds -CPU 12 -v 

Number of Commands: 252101

succeeded(27898), failed(1)   11.0666% completed.




On Sunday, January 22, 2017 at 11:09:22 AM UTC-5, Brian Haas wrote:
.... and so it really wasn't able to assemble anything here given the minimum default contig length of 200 bases.  

~b
On Sun, Jan 22, 2017 at 11:08 AM, Brian Haas <bh...@broadinstitute.org> wrote:
It turns out that the issue here is due to just the complexity being very high, the number of reads relatively small (~5M total reads in the SRA sample I examined), single-end instead of paired-end, and the reads being short (46 base length).

best,

~brian

On Thu, Jan 12, 2017 at 8:07 AM, Brian Haas <bh...@broadinstitute.org> wrote:
I'm hoping there's a good explanation like this.  Something doesn't smell right though.

-Brian
(by iPhone)


On Jan 12, 2017, at 7:12 AM, Mark Chapman <markcha...@gmail.com> wrote:

267k reads sounds like a very small number, is this a case where no transcripts were assembled?
On 12 January 2017 at 12:05, Brian Haas <bh...@broadinstitute.org> wrote:

I think I'll need to take a direct look at this data set.  Can you send me the SRA accession you're experimenting with? Feel free to send it direct, off-list, if you want to keep it private.

best,

~b


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Dr. Mark A. Chapman
------------------------------------
Centre for Biological Sciences
University of Southampton
Life Sciences Building 85
Highfield Campus
Southampton
SO17 1BJ
--
--
Brian J. Haas
The Broad Institute
http://broadinstitute.org/~bhaas

 

Brian Haas

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May 19, 2017, 1:50:08 PM5/19/17
to Tauana Junqueira da Cunha, trinityrnaseq-users
Hi Tauana,

Let's try this.  Try running your original trinity command, and send me the full log from the run including the final error message.

Then, send me the results of running

    ls -altr  /path/to/your/trinity_out_dir/

so we can see what files were generated.

best,

~b

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taua...@gmail.com

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May 19, 2017, 2:33:16 PM5/19/17
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Thanks for the fast response, Brian!

Here are my output attached. The larger out file is from the first run that was going ok until it ran out of time. The shorter is from when I tried to relaunch it. And then the content of the output folder.

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Trinity.123.87522336.out
Trinity.123.88098426.out
ls -altr :path:to:your:trinity_out_dir:.txt

Brian Haas

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May 19, 2017, 3:23:06 PM5/19/17
to Tauana Junqueira da Cunha, trinityrnaseq-users
Thanks!  This was very helpful.

I'm still trying to figure out how it went awry here, but to get you moving again, let's try this.

In the trinity_out_dir/

  try

    ln -s inchworm.K25.L25.DS.fa  inchworm.K25.L25.fa

and then rerun your original trinity command.  Hopefully, it picks up where it left off earlier.  If there's another (or different) error, we'll take it from there.

best,

~brian
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taua...@gmail.com

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May 19, 2017, 3:29:41 PM5/19/17
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Thanks, Brian, I just relaunched it. It might be sitting in our cluster queue for a while, so I will come back later if something goes wrong at the end of the run.

Tauana
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Brian Haas

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May 19, 2017, 3:30:43 PM5/19/17
to Tauana Junqueira da Cunha, trinityrnaseq-users

The other thing that comes to mind....   If you ran Trinity originally w/o the --SS_lib_type parameter, but then included it on the re-run, that would cause this kind of trouble (I think).   There could still be a bug, though...  I'm digging. ;-)

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Brian J. Haas
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