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Old 04-02-2014, 02:46 AM   #1
jagodoy
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Location: Sevilla, Spain

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Default Running a 454 library in Ion torrent?

Hi all,

We have a shotgun library prepared for 454 and would like to run it in ion torrent for extra data. We learned that the sequencing adaptor A is shared, but that the other adaptor is not (P1 for torrent, B for 454). The B adapter was, however, used in torrent some time ago and its sequence seems to be still present in the be bead, following P1. So the questions are:

- Should we convert by ligating just P1 adaptor?
- Any chance that we could run with no additional library conversion?
- Can an emulsion made with 454 primers and beads be loaded directly in torrent?

Any input will be most appreciated.
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Old 04-02-2014, 08:37 AM   #2
bunce
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Hi, The 454 LibL 'A' adapter is the same... but the LibA 'A' is not - you need to check this.

once you have figured this out the easiest way to adapt the library is to design a 454 "B" to ion torrent P1 fusion primer and then PCR amplify your library (together with an 'A' primer) for an appropriate number of cycles (keep it as low as possible, say 10-15 cycles). Then you can use A/P1 primers to quant the library.

We have converted a few libraries in this way.
Good luck!
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Old 04-02-2014, 03:33 PM   #3
JeremyDay
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Just to add to above (or amend) I think 10-15 cycles is prob over kill. If you have an established library already, even 6 cycles will yield 97% new ends. You can start with 5-10ng and 6 cycles will give plenty of product.
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Old 04-04-2014, 06:43 AM   #4
jagodoy
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Thanks both Bunce and JeremyDay for the input.

We were trying to avoid additional PCR cycles, but this sounds like the most promising strategy. We are indeed using 454 LilL A adapter so we'll give it a try ...

We learned that wells in torrent are smaller, so 454 beads are anticipated to work.
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Old 04-10-2014, 10:12 AM   #5
ajthomas
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I've done something similar as well. In my case, however, it was Lib-A library that I converted, so I used two different primers. I made one that used part of the Lib-A A adapter to switch it to the IT-A adapter, and did the same thing at the other end to switch the B to P1. There was still part of both of the old adapters left, but that didn't cause a problem.

You just need to be careful of your library size. The One Touch is more sensitive to library size than the 454 emPCR chemistry is, and if the fragments are too big you won't get anything.
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