Read Counts In Intronic Region

Question

[Varun Gupta]

Hi everyone. I am working on some RNA-seq data. I am interested in finding what are the read counts within an intron for my interest of genes. I have a bam file to start with in which 3rd column of the bam file is basically represented as chromosome1 , chromosome2 and chromosome3. So suppose i have some of my genes of interest on chromosome1 , some on chromosome2 and some on chromosome3, how can I find the number of read counts within the introns of those genes.

Again all i have is a BAM file. Any command line code or anything would be helpful

Thanks in advance

Answer 1

[Rm]

coverageBed -abam input.bam -b introns.bed > introns.bed.coverage

[Rm]

look in PomBase

[Varun Gupta]

Hi RM Thank you for your reply. Since i am new to bedtools, i guess i have to make this introns.bed file i guess. So can you tell me how much columns is necessary in creating an introns.bed file. Is BED3 format ok where we have name in column1 and then start and end coordinates of intronic region. Regards

[Rm]

you can download the intron information in a bed format from ucsc http://genome.ucsc.edu/cgi-bin/hgTables?command=start#Help

[Rm]

bed format: http://uswest.ensembl.org/info/website/upload/bed.html or http://genome.ucsc.edu/FAQ/FAQformat.html#format1

[Varun Gupta]

Hey RM

Thanks for the help, but since i am working on S.pombe genome it is not their in UCSC

Answer 2

[Malachi Griffith]

Picard has a tool for this that seems to work nicely and gives you this specific metric:

CollectRnaSeqMetrics

Program to collect metrics about the alignment of RNA to various functional classes of loci in the genome: coding, intronic, UTR, intergenic, ribosomal.

Also determines strand-specificity for strand-specific libraries. Full list of: RNA-Seq metric definitions.

To narrow it down to a list of regions/genes of interest, you can filter your BAM and create new BAM with reads mapping to those regions. BEDTools could be used for this step.

[Varun Gupta]

Thanks malachig

I will look into it. BTW can you provide me with a quick code i can go through.. Regards