Agilent SureSelect: problems with target enrichment

[Asereth]

Hi all,

we are using Agilent SureSelect Target Enrichment System (protocol v1.5) and plan to work with ABI SOLiD.

We have problems as early in the protocol as target enrichment up to pre-hybridization amplification. After this amplification we analyzed our PCR product but there was no DNA amount detectable with NanoDrop. The starting DNA amounts were 9µg (whole genome amplification (WgA) done before target enrichment; preferred) or 6µg (without WgA; just in comparison to WgA sample).

Has anyone similar problems or suggestions what the problem could be?
Is there a possibility to check a successful amplification beside the PCR product itself? Something like a positive control?
Has anybody done an additional amplification step to increase the PCR product amount?

Hoping for help, thanks in advance,
Theresa

[NextGenSeq]

The protocol worked first time in our hands with 2 different DNA samples. Real time PCR is more sensitive than NanoDrop. If you have access to an Realtime PCR instrument you could quantify by it. They recommend limiting PCR cycles to reduce amplification biases but you could try increasing the cycle number.

[selupe]

I had no issues the first time running protocol 1.5.1 a few months ago. I've done 2 more samples recently, but this time following the addendum to the SureSelect protocol (which was provided by my SOLiD FAS). This addendum adds a column purification step before the nick translation reaction. The annealing step at 54oC should be for 45 sec, not 15 sec as in the original protocol or the addendum. By doing that, I got 3X more amplification than before.

[Asereth]

Hi,

thanks for your replies.

I will try to quantify my samples by Realtime PCR. That’s a good idea. Did you use SYBRGreen and primers annealing to the adaptor sequences?

I already did the additional purification step last time but there was no change in my results. Perhaps there is a difference but I couldn’t detect it so far because of using NanoDrop instead of Realtime PCR.
During next performance I´ll probably change some amplification parameters.

Has anybody mentioned other problems or changed something in the protocol?
Is anybody working with the latest SureSelect protocol (v1.7) from Agilent?

Thank you

[NextGenSeq]

Note most people are switching to Roche's kit since it gives more even and higher coverage.

See

http://www.abrf.org/ResearchGroups/D...2March2010.pdf

[atgc]

Theresa,
We are just starting to work with Agilents Sure Select for sequencing with Illumina. Id like to know if your issue was resolved using RT PCR.

[hbn]

I have a related question: I would like to do target enrichment using the SureSelect system for a non-model species.
My question is; what software to use for this? Agilent advises me to use eArrayXD (local software) but I can not find this software anywhere...
Does someone have experience with this? I would appreciate any advice...