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Old 09-06-2011, 04:31 AM   #4
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Location: Purdue University, West Lafayette, Indiana

Join Date: Aug 2008
Posts: 2,313

I saw a protocol for removing adapters from library molecules. Slips my mind where. You denature your library molecules, swamp with primers complementary to your adapters allowing a limited time to anneal. Use a ds nucleases to degrade away the most quickly hybridizing segments. Clean up then ligate on new adapters.

I think this will be much less efficient than a "fusion primer" method. But going from another amplicon type to HiSeq is problematic without removing the old primers. Illumina sequencers do not handle all amplicons starting with the same sequence well. But if you don't mind losing the first X bases of your sequence to your old adapter sequence, and your libraries are indexed, you could mix your fusion library with another library--in the same lane.

Finally, I believe it is possible to use "custom" primer for HiSeq sequencing. So you could go the fusion primer route, but then sequence with the IT sequencing primer. Not something I have ever done, but I am new to the Illumina arena...

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