SEQanswers

Go Back   SEQanswers > Bioinformatics > Bioinformatics



Similar Threads
Thread Thread Starter Forum Replies Last Post
Paired-end Solexa data mapping wit Bowtie rebrendi Bioinformatics 21 03-30-2012 12:31 PM
Discrepancy in paired-end Illumina data kopardev Bioinformatics 1 01-03-2012 11:23 PM
Bowtie Illumina paired end reads alignment empyrean Bioinformatics 3 09-20-2011 09:51 AM
illumina GA2, 75bp paired-end data yasutake Bioinformatics 0 02-02-2009 06:39 PM
Paired-end Illumina data mchaisso Bioinformatics 7 07-17-2008 11:52 AM

Reply
 
Thread Tools
Old 01-03-2012, 11:59 AM   #1
kopardev
Member
 
Location: VA, USA

Join Date: Oct 2011
Posts: 18
Post Aligning paired end Illumina data with Bowtie

I have 2 fastq files read1.fastq and read2.fastq.
If I align read1.fastq to the reference genome, I get a set of readids that align. Lets say a1, a2, a3, a4, a5, a6, a7, a8, a9 for simplicity.
If I align read2.fastq to the reference genome, I get another set of readids: a1, a2, a4, a5, a6, a8, a9, a10.
But when I align both together as paired end data using -1 and -2 Bowtie command line options, I get only a1, a9 (I expected a1, a2, a4, a5, a6, a8, a9, i.e. common to read1 and read2) along with a bunch of memory warnings that look like this:

Warning: Exhausted best-first chunk memory for read HWI-XXXXX:103046AACXX:5:1101:4313:3969 1:N:0:CAGATC/1 (patid 7142); skipping read

Can someone explain this?
Thanks,
Vishal
kopardev is offline   Reply With Quote
Old 01-03-2012, 12:55 PM   #2
swbarnes2
Senior Member
 
Location: San Diego

Join Date: May 2008
Posts: 912
Default

I don't know bowtie well, but what do the single end .sam entries look like for A2-8? Are they repetative? Are they way far apart?
swbarnes2 is offline   Reply With Quote
Old 01-03-2012, 08:40 PM   #3
xinwu
Member
 
Location: Beijing

Join Date: Jul 2010
Posts: 33
Default

It seems that bowtie does not guarantee to find the best match, after several tries, it may give up.
xinwu is offline   Reply With Quote
Old 01-03-2012, 11:27 PM   #4
simonandrews
Simon Andrews
 
Location: Babraham Inst, Cambridge, UK

Join Date: May 2009
Posts: 871
Default

A couple of the default bowtie parameters for paired end data can cause you to lose hits you might want to keep.

The 'exhausted memory' error can be fixed by adding --chunkmbs 512 to the bowtie options.

Bowtie also has a strict limit (250bp) on the distance between paired sequences which is quite low. We tend to use --maxins 1000 to allow hits which are a bit further apart.
simonandrews is offline   Reply With Quote
Old 01-26-2012, 02:44 AM   #5
nilmot13
Member
 
Location: Leeds, UK

Join Date: Jan 2011
Posts: 19
Default

I'm having similar problem. Currently using Bowtie for Illumina on Galaxy

Single-end mapping with each dataset yield high number (similar number) of reads and coverage. However when I used paired-end mapping number of stacks were dramatically reduced (after applying filter). Regions of genome where you expect to have stacks aren't there?

Tried increasing the insert size, it did yield more coverage.
Is it because it needs to be in a proper pair?

Also does seed length affect the outcome? E.g. sequences trimmed to 40bp and 25bp, for read1 and read2, respectively. When -l = 28, would read2 be considered as valid sequences?
nilmot13 is offline   Reply With Quote
Old 03-29-2012, 08:46 AM   #6
schipma
Junior Member
 
Location: Chicago

Join Date: Aug 2008
Posts: 1
Default

Yes, proper pairing does indeed matter. When both paired-ends are aligned together, the results will include only the alignments in which the paired-end are within a certain distance apart, and in the proper orientation. With SOLiD reads, properly paired-end reads must be directed toward each other, and on opposite strands. I'm not surprised that increasing the insertion size improves the results slightly. But when the reads of a pair are mapped to different chromosomes, then increasing the insertion size won't help.

If your results are in BAM format, try running "flagstat" from samtools on it. This will give you pairing stats. In my experience, when the percentage of properly paired reads is very low (e.g. 5% - 10%), that indicates that the sequences may not be good.

Adjusting the seed length probably won't improve the paired-end results. If you increase the seed length from 25 to 28, then read2 will probably have fewer alignments -- but they will be more confident alignments. If you shorten the seed length, then the reads will align in more places, and the number of paired-reads may increase slightly. But I believe that this approach simply creates false positives.
schipma is offline   Reply With Quote
Reply

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 05:37 AM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2019, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO