Extract junction reads from bam

1

Entering edit mode

Helen Zhou ▴ 150

@helen-zhou-2654

Last seen 9.4 years ago

United States

Dear Sir/Madam, I have a bam file with reads from a paired-end RNA-seq experiments, and I would like to extract all reads that span a particular intron/exon junction. For example where part of a read maps to exon ABC starting at chr 1 position 66909348. I do not know where the first part of the read maps; there are multiple possible exons being spliced to exon ABC. I can extract all reads mapping to and spanning for example 50nt around the junction, saying: library(GenomicRanges) loc <-RangesList('1'=IRanges(start=6690298,end=6690398))? parameter?<- ScanBamParam(which=loc, what=extract, simpleCigar=FALSE, reverseComplement=FALSE) aln <-?readGAlignmentsFromBam("test.bam", param=parameter) However, this includes all the reads that span across this region because the ends of the read map outside the area indicated in 'loc'. How can I get only the reads where (part of) the read starts mapping at the exact intron/exon location at position?66909348? Thanks you H Zhou [[alternative HTML version deleted]]

• 4.5k views

ADD COMMENT • link updated 10.3 years ago by Wei Shi ★ 3.6k • written 10.3 years ago by Helen Zhou ▴ 150

0

Entering edit mode

Michael Lawrence ★ 11k

@michael-lawrence-3846

Last seen 3.0 years ago

United States

If you've already loaded some alignments in your "aln" object, then you can extract the aligned regions (the exonic parts) with: exonic <- grglist(aln) Then, you want to see which read has an exonic region starting at position X (i.e., X is a 3' splice site), with the extra constraint that there must be at least one exonic region preceding (so we know there is a splice). trueForReadsSplicingToX <- any(which(start(exonic) == X) > 1) That may or may not be fast, but it is simple. Didn't test it. Michael On Mon, Aug 18, 2014 at 1:32 PM, Helen Zhou <zhou.helen at="" yahoo.com=""> wrote: > Dear Sir/Madam, > > I have a bam file with reads from a paired-end RNA-seq experiments, and I > would like to extract all reads that span a particular intron/exon > junction. For example where part of a read maps to exon ABC starting at chr > 1 position 66909348. I do not know where the first part of the read maps; > there are multiple possible exons being spliced to exon ABC. > > I can extract all reads mapping to and spanning for example 50nt around > the junction, saying: > library(GenomicRanges) > loc <-RangesList('1'=IRanges(start=6690298,end=6690398)) > parameter <- ScanBamParam(which=loc, what=extract, simpleCigar=FALSE, > reverseComplement=FALSE) > aln <- readGAlignmentsFromBam("test.bam", param=parameter) > > However, this includes all the reads that span across this region because > the ends of the read map outside the area indicated in 'loc'. How can I get > only the reads where (part of) the read starts mapping at the exact > intron/exon location at position 66909348? > > Thanks you > H Zhou > [[alternative HTML version deleted]] > > > _______________________________________________ > Bioconductor mailing list > Bioconductor at r-project.org > https://stat.ethz.ch/mailman/listinfo/bioconductor > Search the archives: > http://news.gmane.org/gmane.science.biology.informatics.conductor > [[alternative HTML version deleted]]

ADD COMMENT • link 10.3 years ago Michael Lawrence ★ 11k

0

Entering edit mode

Wei Shi ★ 3.6k

@wei-shi-2183

Last seen 3 months ago

Australia/Melbourne/Olivia Newton-John …

Dear Helen, If you just want to get the number of reads mapping to that junction (not the actual reads), you can try featureCounts function in Rsubread package. It has a parameter called 'countSplitAlignmentsOnly' which allows you to count exon-spanning reads only. It is extremely fast. Best wishes, Wei On Aug 19, 2014, at 6:32 AM, Helen Zhou wrote: > Dear Sir/Madam, > > I have a bam file with reads from a paired-end RNA-seq experiments, and I would like to extract all reads that span a particular intron/exon junction. For example where part of a read maps to exon ABC starting at chr 1 position 66909348. I do not know where the first part of the read maps; there are multiple possible exons being spliced to exon ABC. > > I can extract all reads mapping to and spanning for example 50nt around the junction, saying: > library(GenomicRanges) > loc <-RangesList('1'=IRanges(start=6690298,end=6690398)) > parameter <- ScanBamParam(which=loc, what=extract, simpleCigar=FALSE, reverseComplement=FALSE) > aln <- readGAlignmentsFromBam("test.bam", param=parameter) > > However, this includes all the reads that span across this region because the ends of the read map outside the area indicated in 'loc'. How can I get only the reads where (part of) the read starts mapping at the exact intron/exon location at position 66909348? > > Thanks you > H Zhou > [[alternative HTML version deleted]] > > _______________________________________________ > Bioconductor mailing list > Bioconductor at r-project.org > https://stat.ethz.ch/mailman/listinfo/bioconductor > Search the archives: http://news.gmane.org/gmane.science.biology.informatics.conductor ______________________________________________________________________ The information in this email is confidential and intend...{{dropped:6}}

ADD COMMENT • link 10.3 years ago Wei Shi ★ 3.6k

0

Entering edit mode

Dear Wei and Michael, thank you both for your helpful comments. This was just what I was looking for. H Zhou On Monday, 18 August 2014, 15:35, Wei Shi <shi at="" wehi.edu.au=""> wrote: Dear Helen, If you just want to get the number of reads mapping to that junction (not the actual reads), you can try featureCounts function in Rsubread package. It has a parameter called 'countSplitAlignmentsOnly' which allows you to count exon-spanning reads only. It is extremely fast. Best wishes, Wei On Aug 19, 2014, at 6:32 AM, Helen Zhou wrote: > Dear Sir/Madam, > > I have a bam file with reads from a paired-end RNA-seq experiments, and I would like to extract all reads that span a particular intron/exon junction. For example where part of a read maps to exon ABC starting at chr 1 position 66909348. I do not know where the first part of the read maps; there are multiple possible exons being spliced to exon ABC. > > I can extract all reads mapping to and spanning for example 50nt around the junction, saying: > library(GenomicRanges) > loc <-RangesList('1'=IRanges(start=6690298,end=6690398)) > parameter <- ScanBamParam(which=loc, what=extract, simpleCigar=FALSE, reverseComplement=FALSE) > aln <- readGAlignmentsFromBam("test.bam", param=parameter) > > However, this includes all the reads that span across this region because the ends of the read map outside the area indicated in 'loc'. How can I get only the reads where (part of) the read starts mapping at the exact intron/exon location at position 66909348? > > Thanks you > H Zhou > ??? [[alternative HTML version deleted]] > > _______________________________________________ > Bioconductor mailing list > Bioconductor at r-project.org > https://stat.ethz.ch/mailman/listinfo/bioconductor > Search the archives: http://news.gmane.org/gmane.science.biology.informatics.conductor ______________________________________________________________________ The information in this email is confidential and intend...{{dropped:9}}

ADD REPLY • link 10.3 years ago Helen Zhou ▴ 150

Login before adding your answer.