SEQanswers

Go Back   SEQanswers > Bioinformatics > Bioinformatics



Similar Threads
Thread Thread Starter Forum Replies Last Post
mRNA fragmentation for 100bp paired-end reads amdic2 Illumina/Solexa 7 09-01-2013 05:54 AM
Simulating paired-end reads and bowtie alignments droog_22 Bioinformatics 0 02-09-2012 08:53 AM
RNA-Seq: Single Read and Paired End mRNA-Seq Illumina Libraries from 10 Nanograms Tot Newsbot! Literature Watch 0 11-09-2011 03:10 AM
Bowtie Illumina paired end reads alignment empyrean Bioinformatics 3 09-20-2011 10:51 AM
Bowtie output from paired end reads godzilla07 Bioinformatics 0 01-06-2011 12:36 PM

Reply
 
Thread Tools
Old 11-15-2010, 12:19 PM   #1
liux
Member
 
Location: Midwest

Join Date: Mar 2009
Posts: 30
Default use bowtie for paired-end mRNA-seq reads?

I am trying to map 10 million pairs of reads to the genome + splice junction templates. However Bowtie will not align most of the reads.

here is the command I used and the screen output (partial):
Code:
time bowtie -p 2 -v 2 -k 11 -m 10 -t --strata --best hg19.index -1 sample_10M_1.fastq -2 sample_10M_2.fastq sample.bowtie_aln.txt

# reads processed: 9999641
# reads with at least one reported alignment: 317813 (3.18%)
# reads that failed to align: 9679936 (96.80%)
# reads with alignments suppressed due to -m: 1892 (0.02%)
if I align them as single reads, majority reads are aligned:
Code:
bowtie -p 2 -v 2 -k 11 -m 10 -t --strata --best hg19.index -q sample_10M_1.fastq,sample$}_10M_2.fastq samplebowtie_aln_single.txt

# reads processed: 19999282
# reads with at least one reported alignment: 16697210 (83.49%)
# reads that failed to align: 3220837 (16.10%)
# reads with alignments suppressed due to -m: 81235 (0.41%)
I figured that it may be caused by the introns between the pair. what options I can use to make it work?
liux is offline   Reply With Quote
Old 11-15-2010, 02:36 PM   #2
Jon_Keats
Senior Member
 
Location: Phoenix, AZ

Join Date: Mar 2010
Posts: 279
Default

Why do you not want to run TopHat? Which is designed for this purpose largely using Bowtie on the front end. Alternatively, if you choose to do this for a specific reason you can try BWA instead of Bowtie, I've used it to do the same but you get lots or errors during the pairing step because of the introns. Alternatively, your strategy sounds like the one used in Alexa-Seq which works well.
Jon_Keats is offline   Reply With Quote
Old 11-15-2010, 03:02 PM   #3
liux
Member
 
Location: Midwest

Join Date: Mar 2009
Posts: 30
Default

The intention is to compare how well different mRNAseq tools perform with a common dataset. ERANGE is one in the list, and ERANGE will use bowtie alignment. I could just use bowtie to align those as single reads and then use ERANGE.
liux is offline   Reply With Quote
Old 11-16-2010, 10:18 AM   #4
liux
Member
 
Location: Midwest

Join Date: Mar 2009
Posts: 30
Default

found a solution in the manual. since erange assumes two neighoring exons from one gene should be with in 20kb, the maximum 'insert' between a pair can be set by -X option, which is 20300 for this case. Now 72.9% reads are mapped.
liux 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 05:42 PM.


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