Exome capture validation

[arvi8689]

Hey I am using the agi exome capture kit. My capture is done and now I would like to confirm if my capture worked. What is the best validation method to do so?

I thought of using a qPCR with primers against 3 exons and 3 introns and compare the dna samples before and after capture. The ideal result should be that the exons are enriched in samples after capture as against introns. But the issue is since I dont have a control I dont know how to calculate a delta delta ct. I am missing a loading control.
Any suggestions?

Thanks
Arvi

[Heisman]

Is your goal to validate that the capture worked prior to submitting it for sequencing? If you're going to submit it regardless it will be easy to see how well it worked when you analyze the data. If you want to check before sequencing that is much harder. Did your sample pass the various QC steps throughout the process?

[TonyBrooks]

The Nimblegen EZ Exome has a simple qPCR QC step which you could adapt
Pick a few genes that are on your exome capture and design SYBR qPCRs (or use the sequences in the Nimblegen protocol)
Run 5ng (by Qubit) of pre-capture library (in triplicate) against 5ng (by Qubit) of post-capture (in triplicate). You shouldn't need to know an exact value for E as enrichment should be in the >100 fold region so any differences should be obvious. We just use e=1.9

~ Enrichment = 1.9^(ct_of_pre -ct_of_post)