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Old 01-11-2010, 02:08 PM   #1
MGH Man
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Smile Number of PCR How many PCR cycles to enrich adapter-modified DNA fragments

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?
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MGH Man

(Sorry if this issue has been posted before )
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Old 01-14-2010, 06:39 AM   #2
GW_OK
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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.
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Old 01-14-2010, 09:31 AM   #3
jpsmith
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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.
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Old 05-14-2010, 06:55 AM   #4
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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
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Old 05-18-2010, 06:04 AM   #5
greigite
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Quote:
Originally Posted by JUdw View Post
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.
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Old 07-26-2010, 05:15 AM   #6
JUdw
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Quote:
Originally Posted by greigite View Post
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.
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