Exon capture and RNA-seq

[adobrovic]

Apologies if this has been covered before but I am new to this great forum.

I want to combine exon capture which I have done on DNA with RNA seq.

If anyone can point me to any pertinent literature or make any constructive comments, these would be welcomed.

[Simon Anders]

Maybe you could first explain to us why you would want to do this? It is a rather unusual suggestion, after all.

[adobrovic]

Pathway specific RNA-Seq

I am just wanting to look at certain pathways,
This should then bring the cost persample down

[Simon Anders]

If, by "exon capture", you mean capturing all exons, you can get this easier by just pulling out polyadenylated RNA. This is what is usually done.

Capture adds all the usual biases one knows from microarrays, and hence, RNA-Seq after capture will only give you what you would have gotten easier and cheaper with expression microarrays, unless you want to study sequence changes (RNA-editing, coding SNPs, or the like).

If you are only interested in a few genes, would not qPCR (analysed e.g. with HTqPCR package) or NanoStrings be cheaper, simpler and more accurate than capture and HTS?

[adobrovic]

We are interested in the range of 200-500 genes.

Because of their restricted dynamic range, arrays are not suitable.

We have a limited amount of material, which precludes RT-qPCR.

Nanostring would be appropriate but we do not have access to this methodology.