Profile Coverage Of Rnaseq Samples?

Question

[daniel.soronellas]

Hi all,

I have a quick question:

How can I visualize aligned paired-end reads from RNAseq datasets in UCSC browser?

I already mapped the reads and assembled the transcripts with Tophat/Cufflinks but I'm not sure how to proceed to visualize the mappings

After sorting the BAM files and fixing the mate pairs, I tried to compute the coverage using the following commands:

genomeCoverageBed -bg -split -ibam F.T0.rep2-accepted_hits-fS.bam -g ~/conversion_util/chrom.hg19.sizes > F.T0.rep2-accepted_hits-fS.bg 
bedGraphToBigWig F.T0.rep2-accepted_hits-fS.bg ~/conversion_util/chrom.hg19.sizes F.T0.rep2-accepted_hits-fS.bw

But I was not able to visualize properly the mappings. Here I paste a screenshot of how it looks like:

Do you know where is the mistake?

Thanks!

[Aaronquinlan]

The -split option should exclude the introns. What version of bedtools is this? The latest is 2.17.0

[daniel.soronellas]

I had been using version 2.16 and didn't worked, but once I changed to version 2.17 worked fine! Thanks!

Answer 1

[David Langenberger]

Tophat returns splitted reads (reads covering splice junctions) with Ns.

Here is an example of how the CIGAR string can look like: 78M2523N22M

I actually don't know, how genomeCoverageBed works, but it looks like, it gives coverage to Ns.

[daniel.soronellas]

Thanks for your clarification! I appreciate!

But how can I manage to visualize this paired-end mRNA using browser? There's another way to do it?

[Istvan Albert]

visualizing read pairs dependes on the tool, I don't know if the UCSC browsers allows that - but you can certainly do that in IGV by right-clicking and selecting the options accordingly