Exome multiplexing

[erlandsen]

Hi,

have anyone tried to multiplex samples and targeted for higher coverage in samples vs. controls. Eg. as in the truseq exome enrichment one is expected to pool 500 ng of each library, but instead use 25 % control and 75% sample?

Another question: is it possible to go down in concentration say to 250 ng into the exome enrichment protocol, or is 500 ng an absolute lower limit?

Regards,

see

[kwaraska]

I know with Sure Select you can pool in any proportion you want, but the lower limit really is the least you shoudl work with. Keep in mind that they obtain their values from nanodrop which is roughly twice what you will see on a bioanlyzer. So you can use 250ng from a bioanlyzer concentration but 500ng from a nanodrop.

At least, that has been my experience.

[erlandsen]

Hi kwaraska, and thanks for comment.

I only use Qbit for cuantifying the libraries on this step. But I have checked NanoDrop, Qbit and qRT-PCR quantification on the Truseq protocol, and my experience is that NanoDrop "overestimate" ca 20 % compared to Qbit and Qbit again "overestimate" ca 10-15% compared to qRT-PCR. I trust qRT-PCR to be most reliable. I never use Bioanalyzer for quantification.

[kwaraska]

Agreed that is the most reliable.

But when they tell you that you need 500ng, they are assuming data from a nondrop. I have used 250ng from a bioanlyzer and done well.

If you feel the difference is 20% then you can use 400ng instead of 500ng.

It is assumed that you will not use qPCR at this step because it will not be reliable for what you are trying to generate. You simply need sufficient DNA library to capture.

[mveerapen]

I am using the SureSelect 50 Mb capture - would I be able to multiplex my samples. I was thinking of including a 1:1 (in terms of QuBit concentration) in the capture. Would I be able to computationally tease out which variants belong to which sample?

On another note, if I have a mixed sample, where the DNA was extracted from a fixed tissue that has a heterogenous tissue (where I assume 0.25:0.75 ratio of tissue 1:tissue 2), would I be able to tease out variants belonging to tissue 1 and 2 separately?

If I had posted this question in a wrong thread, please correct me? I am new in SEQanswers.