Dual indexing construct for MiSeq - where to put the barcode for index2?

[ECO]

Oh and page 5 of this document has as nice diagram of the process, so far the chemistry has seemed the same on the MiSeq.

[ECO]

Hey guys. I'm in the process of making my own Truseq-esque double indexing adapters for MiSeq, and (as you've seen) Illumina will not be forthcoming with the sequences...so I had to figure it out for myself and will share it with you.

I just sequenced a standard single-indexed TruSeq library with the Nextera/Amplicon protocol and the below describes my observations.
It may not help with figuring out the nextera sequences but probably contains some information that will help.

I'm just about to run libraries with my own homebrew double indexing design, will post when/if it turns out well.

[koadman]

The video in that training is the best explanation I've seen yet of the dual index adapter structure and sequencing process. Interesting that the 2nd index is sequenced while the template molecule is bridged. Thanks Vinz!

[Vinz]

Thanks for the link to the paper of Kircher et al. That is indeed helpful.
The way the 2nd barcode is sequenced is nicely described in the Dual Indexing Training for all that have access. It just does not show any sequences.

[koadman]

Hi Vinz, I looked into this and the information that Illumina distributes to customers does not contain the full length adapter sequences as far as I know. You might also be interested in this thread where csquared helpfully suggested that the 2nd barcode is inside the other adapter and is primed directly from the flowcell annealing sequence. Reading Illumina's instrument operation manuals appears to confirm this, they suggest there are some chemistry-only dark cycles prior to reading the other index in their standard protocol and these presumably synthesize the bases between the flow cell annealing region and the index region.

However, if you are doing amplicon sequencing you probably have the flexibility to design your own sequencing primers in which case you might be able to follow a design like what Kircher et al have described. That would have the advantage of not wasting 9 cycles of reagents.

[Vinz]

We would like to use the second indexing read on the MiSeq for amplicon sequencing (not using the Illumina Custom Amplicon Sequencing). Has anyone successfully designed the adaptors containing index2? Where would you put the barcodes?
I assume "TrueSeq Universal Adapter" from the "Illumina-Customer-Sequence-Letter" would be the sequence to start.
Thanks
Vinz