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Old 04-22-2009, 02:14 PM   #1
timread
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Default Amplicon sequencing on Illumina GA II?

Looking through the recent paper by Quail et al* I noticed this ..
Quote:
To avoid unnecessary PCR amplification steps, which would potentially exacerbate biases, we can perform extremely deep sequencing of short amplicons using locus- specific primers that possess tails that can hybridize to the oligos tethered to the flowcell surface. The tailless forward and reverse oligos are then used as primers in the sequencing steps (Supplementary Protocol 10 online).
454-like amplicon sequencing on the Illumina? Be interested to hear if anyone has tried this?

I guess you could build in the standard Illumina sequenicng primer into your amplicon if you didnt want to sequence with custom primers ...

* http://www.nature.com/nmeth/journal/...meth.1270.html
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Old 06-03-2009, 06:13 PM   #2
greigite
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I haven't tried this but am also curious to find out more. Wish they had given some more results! I tried to diagram out a bit how this would work, though, and would welcome any comments (see attachment).

I think the way it works is just to attach your specific primer to an oligo matching the tail of the flow cell oligos. You run a standard PCR, though I'd be curious how tagging on ~30 bp to your locus primers affects amplification and product distribution. You'd probably need a relatively high annealling temperature for your locus specific primer to avoid nonspecific interactions with the added sequence.

Presumably at this point you just denature your product and add it directly to a paired end flow cell. Here I'm a bit confused, because if you use the primers given in the paper, you would end up re-sequencing your amplicon specific primers. Why not just use these as sequencing primers directly? Should we interpret the primers in the supp info as the tags that are supposed to be added onto the amplicon specific primers?

The way it's described, though, I think you use the sequencing primers given in the paper rather than the standard ones. The sequencing primer 1 given in the supplementary info matches flow cell oligo C, while primer 2 matches flow cell oligo D. It looks to me like the authors have retained the A overhang on the side hybridizing to oligo C, which is confusing since this protocol would not require a ligation step. Maybe I'm missing something here.

In any case, welcome comments from folks who have tried this.
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File Type: pdf amplicon_sequencing.pdf (33.3 KB, 293 views)
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