So with the release of the 2012-05-12 "Customer Sequence Letter" on the Illumina web site, do we know everything we need to know to construct Nextera-style dual index amplicons?
Would be advantageous to do so, because then we could design experiments with 96 index amplicons requiring only 20 oligos to be synthesized. By extending the index sequences to 16 x 24 we could do 384 index amplicons by purchasing only 40 oligos. (Not counting the locus-specific primers.)
That is, fuse the read 1 and read 2 "Nextera transposase sequences" with your locus specific primers forward and reverse primers, respectively. Then order a batch of the Nextera Index Kit-PCR primers. Do primary PCR on using the locus specific oligos, then a second PCR reaction with the flowcell/index/etc. primers.
Am I missing anything? Do I need to methylate the "A" of the DpnI site of the flow cell part? Anything else?
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Phillip
Would be advantageous to do so, because then we could design experiments with 96 index amplicons requiring only 20 oligos to be synthesized. By extending the index sequences to 16 x 24 we could do 384 index amplicons by purchasing only 40 oligos. (Not counting the locus-specific primers.)
That is, fuse the read 1 and read 2 "Nextera transposase sequences" with your locus specific primers forward and reverse primers, respectively. Then order a batch of the Nextera Index Kit-PCR primers. Do primary PCR on using the locus specific oligos, then a second PCR reaction with the flowcell/index/etc. primers.
Am I missing anything? Do I need to methylate the "A" of the DpnI site of the flow cell part? Anything else?
--
Phillip
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