converting Ion torrent library for sequencing on HiSeq

[cwachtel]

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]

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

[krobison]

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]

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

[owik303]

This is super easy, we have done it multiple times with no issue besides needing to buy a longer read on Illumina. Just take your ion torrent library as is and put it through the truseq kit, having the ion adapters/barcodes on the ends isnt any different than having a DNA fragment of that size. Since the adapters are different and the barcodes are sequenced differently by illumina there is no conflict. I would not do what the post above my says unless you want to add a lot more pcr bias and unnecessary work to your life. to avoid issues with end diversity just spike in 10-15% phix into the library before sequencing, and if your still concerned we commonly use a "diversity spike" which is just a amplicon with end diversity (such as custom barcodes like 8 base barcode and the first 20 bases of the fusion primer) we spike into the sample with limited end diversity.