Tophat mapping

[guzhi100]

Hi,

After mapping RNA-Seq paired end reads with Tophat, I can see that most of reads fall into the right regions. However, I still can see lots of reads mapped to non-coding region (the locations where the reads are mapped to don't contain exons).

I am wondering if these "non-coding reads" will be included when cufflinks calculates transcript/gene expression.
Dying to know your opinion.

And another question is: how to know the number of reads mapped to a certain exon?

Thanks

[ETHANol]

Hi,

After mapping RNA-Seq paired end reads with Tophat, I can see that most of reads fall into the right regions. However, I still can see lots of reads mapped to non-coding region (the locations where the reads are mapped to don't contain exons).

I am wondering if these "non-coding reads" will be included when cufflinks calculates transcript/gene expression.
Dying to know your opinion.

And another question is: how to know the number of reads mapped to a certain exon?

Thanks

HTseq-count can assign your mapped reads to whatever you like as long as you have a GTF file. Check DEXseq, there's info about exon specific mapping there.

[Camg]

What organism are you mapping to?
Can you be more specific about the "non-coding regions", such as: are the reads mapping adjacent to coding regions (ie within a few hundred bp's)? are they mapping between coding regions that are close together? or are they mapping to regions that are basically in the middle or nowhere with nothing annotated nearby (at least a few kb away)? and, what kind of depth of coverage do you see in these non-coding regions?
Depending on the "compactness" of the genome you might see what looks like reads mapping to non-coding regions simply because of the proximity to coding regions. Or you might just have novel transcribed regions.

You can tell cufflinks whether you want it to assemble novel transcripts and give you counts for those (default), or whether you want it to only count expression for the genes that you give it in a GTF file (use the -G or --GTF option).

I've used HTseq and it works well.