Number of PCR How many PCR cycles to enrich adapter-modified DNA fragments

[MGH Man]

We are prepping our samples for exome sequencing (Agilent Sureselect followed by 100bp PE version 4 chemistry). The Illumina library preparation protocol includes a PCR step to enrich the adapter-modified fragments. The protocol asks for 1ul of DNA to be added and for 18 cycles of PCR. This seems a lot, especially as we are getting a lot of duplicate reads/PCR artifact in our data.

Has anyone tried a lower number of cycles, and, if so, how did it work out for you?
Best
MGH Man

(Sorry if this issue has been posted before )

[GW_OK]

The Agilent Exome seqcap protocol calls for 6 cycles of PCR to enrich for adapter modified fragments. I haven't tried it yet but I'm sure it will work.

[jpsmith]

I've done a handful of Agilent's exome sample prep and yes the 6 cycles for the initial prep is plenty. I would recommend starting with as close to 3ug as possible as when I've started with lower amounts of DNA ( around 2ug) it is more difficult to achieve the requisite 500ng to continue with the capture steps.

[JUdw]

Could you further reduce the pre-hyb PCR cycle number, e.g to 3 rounds of PCR using PE primers?

I suppose theoretically 3 rounds is the minimum, since you need the symmetric PCR product mentioned in the diagram of greigite in thread:

http://seqanswers.com/forums/showthr...?t=198&&page=2

[greigite]

Originally Posted by JUdw

Could you further reduce the pre-hyb PCR cycle number, e.g to 3 rounds of PCR using PE primers?

I suppose theoretically 3 rounds is the minimum, since you need the symmetric PCR product mentioned in the diagram of greigite in thread:

http://seqanswers.com/forums/showthr...?t=198&&page=2

I have tried going down to 4 cycles with the standard PE library prep protocol but could not see anything on bioanalyzer. However, my guess is that I did not have enough template DNA in the reaction. Really you are only limited by how sensitive your library quantification method is- qPCR is probably the most sensitive.

[JUdw]

Originally Posted by greigite

I have tried going down to 4 cycles with the standard PE library prep protocol but could not see anything on bioanalyzer. However, my guess is that I did not have enough template DNA in the reaction. Really you are only limited by how sensitive your library quantification method is- qPCR is probably the most sensitive.

I already have about 500ng of template, so i think I don't really need to do the pre-hyb PCR. For qPCR the pre-hyb PCR is however required, since these primers only anneal to the extended PCR product. Therefore i don't want to skip the pre-PCR, but i really want to use as few cycles as possible.