SEQanswers

Go Back   SEQanswers > Bioinformatics > Bioinformatics



Similar Threads
Thread Thread Starter Forum Replies Last Post
Need to extract fastq of specific region in bam file jmartin Bioinformatics 10 06-05-2015 12:48 PM
Counting Reads in BAM file by Region CodeHippo Bioinformatics 4 03-24-2014 01:03 PM
Extract aligned reads from a BAM file above a certain threshold The Snow Bioinformatics 4 07-29-2013 03:02 AM
Too many reads mapping towards intronic region sanush SOLiD 6 04-14-2010 04:23 AM
Too many reads mapping towards intronic region sanush RNA Sequencing 1 04-13-2010 11:10 AM

Reply
 
Thread Tools
Old 10-18-2015, 04:57 AM   #1
ClemBuntu
Member
 
Location: Lyon

Join Date: Dec 2014
Posts: 37
Default How to extract only intronic reads or region from BAM file?

Hello all,

I used picard tool CollectRnaSeqMetrics on my RNA-seq fastq files and I found a lot (> 50%) of intronic region.

I wonder if these intronic regions are equally distributed among the genome or otherwise if my reads are aligned one only few genes.

How can I see it ? Is there a way to extract only intronic regions from a BAM file ?

Or maybe there is a way to extract only reads which mapped on intronic regions and see where they're aligned ?
ClemBuntu is offline   Reply With Quote
Old 10-18-2015, 05:21 AM   #2
GenoMax
Senior Member
 
Location: East Coast USA

Join Date: Feb 2008
Posts: 7,088
Default

Look at the samtools view -L option. Provide your intronic regions as a bed file and extract reads aligned in those areas into a new file.
GenoMax is offline   Reply With Quote
Old 10-19-2015, 01:30 AM   #3
WhatsOEver
Senior Member
 
Location: Germany

Join Date: Apr 2012
Posts: 215
Default

First, depending on the organism you are working with, it's not totally surprising that a lot of reads map to introns.
Second, if you have a lot of genes with different isoforms in your sample, the number of "intronic reads" may be influenced by the annotation file you are using.

There is at least one easy way I can think of to get non-exon regions from a BAM file. bedtools intersect (http://bedtools.readthedocs.org/en/l...intersect.html) allows you to select for reads that don't overlap defined regions (-v parameter). But, you will eventually get the inter-gene reads as well... So you would actually need to define gene regions first (maybe from 5'UTR start to 3'UTR end) and then use this file together with an exon defining file in bedtools intersect -v. This will give you an intron-only file which you can use for detecting unequal mapping distributions.
WhatsOEver is offline   Reply With Quote
Old 10-19-2015, 02:41 AM   #4
ClemBuntu
Member
 
Location: Lyon

Join Date: Dec 2014
Posts: 37
Default

Hello,

Thank you guys for your answers, all I have is a GTF file with the transcripts coordinates (3th column contains only "CDS" "exon" "start_codon" and "stop_codon") so bedtools intersect with -v option seems more suitable for me.

Do you think a grep "*_codon" on my transcripts file will be do the trick to have only the gene regions ?

So if I well understood I should do something like that :
bedtools intersect -abam -a myfile.bam -b onlyGeneCoordinate.gtf > bamVSgene.bed #first I want the intersection between my reads and genes

bedtools intersect -v -abam -a bamVSgene.bed -b genesAndTranscripts.gtf > intronOnlyFile.bed #filter the exonic regions

bedtools intersect -abam -a myfile.bam -b intronOnlyFile.bed > onlyIntrons.bed #finally get only the intronic reads

I'm not sure the 2nd step will work because there is not intronic regions in the "genesAndTranscripts.gtf" file.
ClemBuntu is offline   Reply With Quote
Old 02-14-2017, 02:04 AM   #5
minie
Junior Member
 
Location: INDIA

Join Date: Aug 2016
Posts: 1
Default

Hi how did you get the intron.bed file? I have gff/gtf file with transcripts/exon/gene/cds information snd trying to parse intronic region. any help ?
minie 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 07:49 AM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2021, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO