Pooling for emPCR

[JPC]

Does anyone struggle with the SOLiD™ Library TaqMan® qPCR Module for quantification of SureSelect Exomes?

We are finding that our coverage results correlate closely with our bioanalyser traces but not at all with the qPCR.

JPC

[JPC]

I found out that other 5500 users have had similar problems with the qPCR quantification kit so we've been pooling samples based on the quantity shown on the bioanalyser. Our balance between samples has improved but I wonder if it could be better (I hope it can).

In our latest run we have 10 human exomes with the following coverage
49, 50, 51, 56, 57, 66, 68, 80, 86, 105.
With better balance we could get 12 or 13, 50X exomes which would help bring our costs down significantly.

Does anyone get a more uniform result from their pools? and if so, how do you do it?

JPC

[JPC]

Just to update anyone who reads this, a colleague in a different centre who has a lot of SOLiD experience has said that this sort of spread is pretty normal. they found using the Qubit for quantifying a batch of libraries gave the best results in terms of balance.

I hope I don't get in trouble for replying on my own threads, if anyone else wants to chip in it'll stop this looking like a personal blog!