I'm trying to hijack the AmpliSeq protocol and get it onto the MiSeq instead of the PGM. Has anyone figured out a good way to do this? The best way I think will be to just substitute an Illumina Adapter (blunted ended) into the AmpliSeq ligation step and then use multiplexed PCR primers in the enrichment PCR step (the 2nd round of PCR). If I go about this way, I need to know the molarity of the Adapter being added during the AmpliSeq ligation and whats the molarity of the PCR primers in the PCR step. Has anyone figured this out or have a guess?
In addition, has anyone tried using another polymerase to amplify the AmpliSeq amplicons? I was looking into Platinum® PCR SuperMix High Fidelity to use, as that is what LifeTech says to use for the Amplicon Fusion method (amplicons with the PGM sequencing adapter added to the PCR primers, so once done the amplicon PCR the resulting library is ready for sequencing without any other modifications). If I use this polymerase, I won't even need to do end repair or A-Tailing, because most of the product has an A-overhang and thus I can just ligate the Illumina adapter directly to the PCR products and then do PCR to enrich for those libraries.
Any suggestions or advice will be very helpful.
In addition, has anyone tried using another polymerase to amplify the AmpliSeq amplicons? I was looking into Platinum® PCR SuperMix High Fidelity to use, as that is what LifeTech says to use for the Amplicon Fusion method (amplicons with the PGM sequencing adapter added to the PCR primers, so once done the amplicon PCR the resulting library is ready for sequencing without any other modifications). If I use this polymerase, I won't even need to do end repair or A-Tailing, because most of the product has an A-overhang and thus I can just ligate the Illumina adapter directly to the PCR products and then do PCR to enrich for those libraries.
Any suggestions or advice will be very helpful.
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