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Old 08-20-2013, 07:41 AM   #21
DrS
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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)
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Old 08-20-2013, 07:45 AM   #22
dpryan
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Quote:
Originally Posted by DrS View Post
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
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Old 08-20-2013, 07:51 AM   #23
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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
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Old 08-20-2013, 07:55 AM   #24
dpryan
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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.
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Old 08-20-2013, 07:58 AM   #25
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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
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Old 08-20-2013, 08:01 AM   #26
dpryan
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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.
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Old 08-20-2013, 08:17 AM   #27
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RAM is not an issue in my group.
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Old 09-05-2013, 08:23 AM   #28
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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.
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Old 09-05-2013, 09:46 AM   #29
newbietonextgen
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Default 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
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