converting Ion torrent library for sequencing on HiSeq

cwachtel · 09-03-2011, 11:54 PM

Hi everyone?

Does anyone have experience preparing a library with the Ion torrent kits and then converting them to run on a Hiseq. For example, I would like to use my Ion torrent as a fast cheap way to asses the quality of my library but then run it on a HiSeq.

Thanks!

frozenlyse · 09-05-2011, 05:31 PM

Sounds like someone needs a MiSeq instead of an Ion Torrent.

krobison · 09-06-2011, 04:10 AM

You are doing things backwards from how this is usually envisioned; make your library on for the HiSeq & then convert a sample to IonTorrent.

I think IT has a kit for this, but if not you would just need to design an IT amplicon fusion primer with the constant Illumina adapter sequences as your targeting sequences.

pmiguel · 09-06-2011, 04:31 AM

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...

--
Phillip

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09-03-2011, 11:54 PM	#1
cwachtel Junior Member Location: Israel Join Date: Aug 2011 Posts: 2	converting Ion torrent library for sequencing on HiSeq Hi everyone? Does anyone have experience preparing a library with the Ion torrent kits and then converting them to run on a Hiseq. For example, I would like to use my Ion torrent as a fast cheap way to asses the quality of my library but then run it on a HiSeq. Thanks!

09-05-2011, 05:31 PM	#2
frozenlyse Senior Member Location: Australia Join Date: Sep 2008 Posts: 105	Sounds like someone needs a MiSeq instead of an Ion Torrent.

09-06-2011, 04:10 AM	#3
krobison Senior Member Location: Boston area Join Date: Nov 2007 Posts: 682	You are doing things backwards from how this is usually envisioned; make your library on for the HiSeq & then convert a sample to IonTorrent. I think IT has a kit for this, but if not you would just need to design an IT amplicon fusion primer with the constant Illumina adapter sequences as your targeting sequences.

09-06-2011, 04:31 AM	#4
pmiguel Senior Member Location: Purdue University, West Lafayette, Indiana Join Date: Aug 2008 Posts: 1,698	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... -- Phillip