SEQanswers

Go Back   SEQanswers > Applications Forums > RNA Sequencing



Similar Threads
Thread Thread Starter Forum Replies Last Post
Cufflinks Error: "Segmentation Fault" AsoBioInfo Bioinformatics 1 02-27-2013 03:36 AM
Maq Segmentation fault Seq84 Bioinformatics 3 07-13-2012 04:05 AM
cufflinks segmentation fault help please seq_newbie Bioinformatics 1 06-29-2011 11:52 AM
Cufflinks cufflinks v1.0.3 - segmentation fault bias correction chrNT annotations adrian Bioinformatics 0 06-08-2011 01:28 PM
Segmentation fault when running Cufflinks doggysaywhat RNA Sequencing 0 03-30-2011 10:48 AM

Reply
 
Thread Tools
Old 02-28-2013, 08:31 AM   #1
bmicro_mit1
Junior Member
 
Location: Boston

Join Date: Feb 2013
Posts: 4
Default Cufflinks 2.0.2 segmentation fault

I am using 100 bp paired end Illumina Hi-seq data with about 50M reads and trying to use tophat / cufflinks for RNA-seq analysis for human data, using Ensemble v68 gtf along with Gencode v13 lncRNA gtf annotations. These files were concatenated together to run both tophat 2.0.6 with bowtie2 2.0.6:

tophat -p 4 --solexa1.3-quals --read-realign-edit-dist 0 --no-novel-juncs --library-type fr-unstranded -G $GTF -o $OUT $GENOME $FASTQ_1 $FASTQ_2

and Cufflinks 2.0.2
cufflinks -o $OUT -p 4 -G $GTF -b $FASTA --multi-read-correct $OUT/accepted_hits.bam

A segmentation fault has continued to occur with multiple samples at similar locations as Cufflinks is re-estimating abundacnes with bias and multi-read correction. Below is the output:

[09:41:21] Learning bias parameters.
> Processed 635 loci. [*************************] 100%
[09:45:58] Re-estimating abundances with bias and multi-read correction.
> Processing Locus chr16:5289802-6826015 [******* ] 31%Segmentation fault (core dumped)

Any input would be greatly appreciated.
bmicro_mit1 is offline   Reply With Quote
Old 02-28-2013, 08:47 AM   #2
GenoMax
Senior Member
 
Location: East Coast USA

Join Date: Feb 2008
Posts: 7,049
Default

How much RAM do you have on this machine?
GenoMax is offline   Reply With Quote
Old 02-28-2013, 08:51 AM   #3
bmicro_mit1
Junior Member
 
Location: Boston

Join Date: Feb 2013
Posts: 4
Default

This was run on a couple of different machines in a SGE cluster. Some of the nodes had up to 48Gb of RAM, but in the SGE email reporting the program had died, it never reported more than 5Gb of memory usage.
bmicro_mit1 is offline   Reply With Quote
Old 02-28-2013, 08:53 AM   #4
GenoMax
Senior Member
 
Location: East Coast USA

Join Date: Feb 2008
Posts: 7,049
Default

Are there any other error messages (stderr output)?
GenoMax is offline   Reply With Quote
Old 02-28-2013, 08:55 AM   #5
bmicro_mit1
Junior Member
 
Location: Boston

Join Date: Feb 2013
Posts: 4
Default

No, just the segmentation fault. I am running with verbose mode right now to see if there is more output.
bmicro_mit1 is offline   Reply With Quote
Old 02-28-2013, 09:13 AM   #6
GenoMax
Senior Member
 
Location: East Coast USA

Join Date: Feb 2008
Posts: 7,049
Default

Are you using the -o and -e directives with your qsub (or SGE job submission script) to capture the output/stderr output? Contents of those would be useful as well.
GenoMax is offline   Reply With Quote
Old 02-28-2013, 09:14 AM   #7
bmicro_mit1
Junior Member
 
Location: Boston

Join Date: Feb 2013
Posts: 4
Default

Yes, nothing in the 'o' file, and only what I had put in the initial post from the 'e' file.
bmicro_mit1 is offline   Reply With Quote
Old 03-14-2013, 10:46 AM   #8
chrisjohn86
Junior Member
 
Location: UK

Join Date: Mar 2013
Posts: 2
Default

I have had a lot of segmentation faults with tuxedo over the last few weeks. I finally figured out it was due to bad RAM, I removed 8GB of 16GB total and it started working fine. Memoryxp is a good RAM diagnostic tool. This is one possible reason for seg faults. There are others:
http://www.cyberciti.biz/tips/segmen...inux-unix.html
chrisjohn86 is offline   Reply With Quote
Old 03-22-2013, 05:44 AM   #9
Anelda
Member
 
Location: Cape Town, South Africa

Join Date: May 2010
Posts: 30
Default

I have found that the order of the reference chromosomes in the genome.fasta file and the chromosomes in the GFF/GTF file, must be exactly the same otherwise a segmentation fault occurs. This is specifically valid in the case of Cufflinks. To demonstrate..

grep ">" genome.fasta > fasta.order
cut -f 1 genome.gff | uniq > gff.order

diff fasta.order gff.order

If the order of the chromosomes are not the same, you'll have to reshuffle. Easiest might be to reshuffle the GFF/GTF - I'm not sure if there are any scripts that can sort fasta/gff files. I just grep each chromosome from the GFF file and send it to a separate file, then cat the individual chromosome.gff files in the correct order and create new genome.gff.

Hope this helps someone!
Anelda is offline   Reply With Quote
Old 11-08-2013, 10:38 AM   #10
sterding
Member
 
Location: Boston

Join Date: Sep 2010
Posts: 36
Default

Quote:
Originally Posted by Anelda View Post
I have found that the order of the reference chromosomes in the genome.fasta file and the chromosomes in the GFF/GTF file, must be exactly the same otherwise a segmentation fault occurs.
Thanks. I am testing this. I am also curious how you found the trick
sterding is offline   Reply With Quote
Old 11-08-2013, 12:02 PM   #11
sterding
Member
 
Location: Boston

Join Date: Sep 2010
Posts: 36
Default

hi Anelda,

I made the genome.fa and gtf file in the same order, but still I got the " Segmentation fault" error in the step of " Learning bias parameters" if I use -b option. Without the "-b" option, I don't get the error. So, I think the bug is in the "-b" option. Hopefully cufflinks team can get attention to the problem.
sterding is offline   Reply With Quote
Old 01-12-2015, 12:12 PM   #12
biocomputer
Member
 
Location: Canada

Join Date: Dec 2013
Posts: 62
Default

Quote:
Originally Posted by sterding View Post
hi Anelda,

I made the genome.fa and gtf file in the same order, but still I got the " Segmentation fault" error in the step of " Learning bias parameters" if I use -b option. Without the "-b" option, I don't get the error. So, I think the bug is in the "-b" option. Hopefully cufflinks team can get attention to the problem.
I'm using Cufflinks 2.2.1 and having the same problem with cufflinks and cuffdiff. -b causes a segfault, it works fine without it. I ensured the genome.fa and gtf file have their chromosomes in the same order and contain the same chromosomes and there is lots of free memory available.
biocomputer is offline   Reply With Quote
Old 01-21-2015, 06:25 AM   #13
offspring
Member
 
Location: Lund, Sweden

Join Date: Mar 2013
Posts: 32
Default

Please file an issue report at https://github.com/cole-trapnell-lab/cufflinks containing a description of the problem and how to reproduce it, otherwise the cufflinks team won't even be aware of the problem.
offspring is offline   Reply With Quote
Old 01-21-2015, 10:45 AM   #14
biocomputer
Member
 
Location: Canada

Join Date: Dec 2013
Posts: 62
Default

I was able to solve my problem. Despite the "-b genome.fa" seemingly being the cause of the problem it's actually the .gtf file. See here how to modify the .gtf file:

https://groups.google.com/d/msg/tuxe...c/p47AwnCXxvwJ

https://groups.google.com/d/msg/tuxe...U/LJhbCHBsITAJ

Last edited by biocomputer; 01-21-2015 at 11:28 AM.
biocomputer is offline   Reply With Quote
Reply

Tags
cufflinks 2.0.2, error, rna-seq, segmentation fault, tophat 2.0.6

Thread Tools

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off




All times are GMT -8. The time now is 02:32 AM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2020, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO