HTseq Segmentation fault - Page 2

DrS · 08-20-2013, 07:41 AM

Aha - on your website it only has up to p2, and the p2 was a Windows fix, where I'm on Linux. I'll download and let you know, but it may take a little while (working on other things now as well)

dpryan · 08-20-2013, 07:45 AM

Quote:

Originally Posted by DrS

Aha - on your website

I'm not the author, I just use htseq-count pretty frequently (I've never met the author!), so I'd like to get bugs fixed before they affect me too

DrS · 08-20-2013, 07:51 AM

I just downloaded and installed the one from:
https://pypi.python.org/pypi/HTSeq

The file is:
HTSeq-0.5.4p3.tar.gz

The version still reports as:
Written by Simon Anders (sanders@fs.tum.de), European Molecular Biology
Laboratory (EMBL). (c) 2010. Released under the terms of the GNU General
Public License v3. Part of the 'HTSeq' framework, version 0.5.4p1.

.... And I still get the crash.

100000 GFF lines processed.
200000 GFF lines processed.
296737 GFF lines processed.
Warning: Malformed SAM line: MRNM != '*' although flag bit &0x0008 set
Warning: Malformed SAM line: RNAME != '*' although flag bit &0x0004 set
Segmentation fault

I'm thinking I'm going to have to try another aligner and see if that works

dpryan · 08-20-2013, 07:55 AM

It's actually an easy fix to get the most recent version running. If you open the htseq-count file in a text editor, you'll see that it mentions the exact version (you can have multiple versions installed). Assuming everything with the newest version is setup correctly, you can just change "p1" to "p3" and things should work. BTW, which aligner are you using? I've had good experience with tophat2 and, more recently, STAR.

DrS · 08-20-2013, 07:58 AM

I was using Bowtie2 - paired-end genomic data from a 250x250 miSeq run.
I've asked others to chime in if they're also only having problems with paired-end data.

I'll try STAR next, but have meetings most of today so it might take a day or two to get to it.

And sorry about the mistaken identity

dpryan · 08-20-2013, 08:01 AM

Just keep in mind that you'll need rather more RAM for STAR, though it can finish a sample in the time it takes to grab a coffee.

DrS · 08-20-2013, 08:17 AM

RAM is not an issue in my group.

DrS · 09-05-2013, 08:23 AM

Just as an update - I ended up using STAR and had no problems with it. Still no idea why I was having problems with the other data.

newbietonextgen · 09-05-2013, 09:46 AM

I use GMAP to align my RNA-seq and then I use HT-Seq to get the counts. When i run the HTseq count script, it always complains of the sam file not being sorted.

I have used from picard tools, samtools and sort command to sort my SAM file but still it complains.

Picard tools (SortSAM)
SAmtools (convert from SAM to BAM, sort BAM file, Index BAM file, reconvert to BAM file).

Any reason? The HT-Seq count tools works well with tophat alignments

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09-05-2013, 09:46 AM	#29
newbietonextgen Member Location: USA Join Date: Nov 2010 Posts: 56	GMAP and HTseq problems I use GMAP to align my RNA-seq and then I use HT-Seq to get the counts. When i run the HTseq count script, it always complains of the sam file not being sorted. I have used from picard tools, samtools and sort command to sort my SAM file but still it complains. Picard tools (SortSAM) SAmtools (convert from SAM to BAM, sort BAM file, Index BAM file, reconvert to BAM file). Any reason? The HT-Seq count tools works well with tophat alignments

08-20-2013, 07:41 AM	#21
DrS Member Location: North Carolina Join Date: Mar 2013 Posts: 17	Aha - on your website it only has up to p2, and the p2 was a Windows fix, where I'm on Linux. I'll download and let you know, but it may take a little while (working on other things now as well)

08-20-2013, 07:51 AM	#23
DrS Member Location: North Carolina Join Date: Mar 2013 Posts: 17	I just downloaded and installed the one from: https://pypi.python.org/pypi/HTSeq The file is: HTSeq-0.5.4p3.tar.gz The version still reports as: Written by Simon Anders (sanders@fs.tum.de), European Molecular Biology Laboratory (EMBL). (c) 2010. Released under the terms of the GNU General Public License v3. Part of the 'HTSeq' framework, version 0.5.4p1. .... And I still get the crash. 100000 GFF lines processed. 200000 GFF lines processed. 296737 GFF lines processed. Warning: Malformed SAM line: MRNM != '' although flag bit &0x0008 set Warning: Malformed SAM line: RNAME != '' although flag bit &0x0004 set Segmentation fault I'm thinking I'm going to have to try another aligner and see if that works

08-20-2013, 07:55 AM	#24
dpryan Devon Ryan Location: Freiburg, Germany Join Date: Jul 2011 Posts: 3,480	It's actually an easy fix to get the most recent version running. If you open the htseq-count file in a text editor, you'll see that it mentions the exact version (you can have multiple versions installed). Assuming everything with the newest version is setup correctly, you can just change "p1" to "p3" and things should work. BTW, which aligner are you using? I've had good experience with tophat2 and, more recently, STAR.

08-20-2013, 07:58 AM	#25
DrS Member Location: North Carolina Join Date: Mar 2013 Posts: 17	I was using Bowtie2 - paired-end genomic data from a 250x250 miSeq run. I've asked others to chime in if they're also only having problems with paired-end data. I'll try STAR next, but have meetings most of today so it might take a day or two to get to it. And sorry about the mistaken identity

08-20-2013, 08:01 AM	#26
dpryan Devon Ryan Location: Freiburg, Germany Join Date: Jul 2011 Posts: 3,480	Just keep in mind that you'll need rather more RAM for STAR, though it can finish a sample in the time it takes to grab a coffee.

08-20-2013, 08:17 AM	#27
DrS Member Location: North Carolina Join Date: Mar 2013 Posts: 17	RAM is not an issue in my group.

09-05-2013, 08:23 AM	#28
DrS Member Location: North Carolina Join Date: Mar 2013 Posts: 17	Just as an update - I ended up using STAR and had no problems with it. Still no idea why I was having problems with the other data.