No further enrichment after the 2nd Truseq exome capture

[christophpale]

Hi,
We have just started to make our own TruSeq libraries and do the Truseq multiplex exome capture. We found that the second enrichment did not have any effect on the input (i.e. the amount we put in was the amount we got out after the second enrichment reaction).
Can some one please comment on their experience with TruSeq exome capture.
1. If you put in 500ng (x 6 samples = 300ng) of the pooled TruSeq library for Truseq multiplex exome capture, what is the yield after the first capture?
2. What is the yield after the second capture?
3. How much of the capture material do you need for sequencing on a hiseq?
thanks
Christoph

[sehrrot]

Hi Chris

I haven't measured the yield after the first capture. But the 2nd capture's yield before amplification was around 13ng/ul in 10 ul and then after amplification the yield was 5 ng/ul in 30 ul (higher concentration on pre-amplification may be due to some un-cleaned beads in samples?). This is a singlet capture.

[Maqbool]

Hi Sehrrot,

We get the same amount of captured library as you have written, 4-5 ng/ul in 30 ul.

What is your experience for cluster density adjustment? I need to load 3-4 times more than a regular genomic library. I would also like to know your experience with data analysis regarding the PCR duplicates and the fold coverage depth.

[ageneheo]

Sehrrot,

It is hard to understand the yield of exome capture library before PCR and after PCR.

Did you find any answer about that?