96 or 384 Nextera-style dual indexing

[pmiguel]

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

[ECO]

Hey Philip, in my testing no methylation is necessary....the cleavage is at the uracil in the flow cell primer. I'm planning on posting my test ASAP, but all the info necessary is in the other thread (if a bit disjointed)...I'll try to do that tomorrow.

[pmiguel]

Yes, that would be great. Just to be clear, what you have created is more of a TruSeq-adapter-based methodology that is able to utilize the Nextera dual indexing "path", right?

I was liking the idea of moving amplicons to a different adapter sequence (Nextera) so they would have less chance of contaminating TruSeq experiments. On the other hand, I guess that does require a little more work if they are to be used on a HiSeq, whereas your hybrid TruSeq/Nextera libraries would not?

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Phillip

[ECO]

Yes, that would be great. Just to be clear, what you have created is more of a TruSeq-adapter-based methodology that is able to utilize the Nextera dual indexing "path", right?

Exactly. Ligatable TruSeq-esque adapter which enables a second index, sequenced with the standard 2-read/2-index protocol on the MiSeq (which currently is tied to Nextera sample prep in the official channel).

See the document which I attached to post 16 in the original thread.