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  • 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?
    Best
    MGH Man

    (Sorry if this issue has been posted before )

  • #2
    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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    • #3
      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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      • #4
        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:

        Bridged amplification & clustering followed by sequencing by synthesis. (Genome Analyzer / HiSeq / MiSeq)

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        • #5
          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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          • #6
            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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