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Old 02-28-2013, 06:43 AM   #1
ChrE
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Default NimbleGen SeqCap EZ Choice Library-preparation for the PGM

Hello all.

From a previous project, we have a 'NimbleGen SeqCap EZ Choice Library'-design that we would now like to use for a new project on our Ion Torrent PGM.
I am very new to next-gen sequencing, but it looks like I should be able to prepare a library (fragmented to appropriate PGM-size, PGM-adapter ligated, nick translated, size selected, amplified, and purified) according to the PGM protocols and then do the capture following NimbleGens protocol, followed by template preparation and sequencing according to the PGM protocols. The main problem seems to be the differences in the oligos used, i.e. during the NimbleGen capture 'hybridization enhancing'-oligos are added to the mixture. Can anyone please explain to me in detail what these are, how they are designed and how I might create a set corresponding to the PGM-adapters used?

Also, according to the NimbleGen protocol a ligation-mediated PCR is performed after the capture. Can't I just do a PGM-protocol amplification here and achieve the same or am I missing a point here?

Any advice would be greatly appreciated. Thanks in advance.

Best regards
Christian
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Old 02-28-2013, 07:13 AM   #2
ECO
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"Hyb enhancing" oligos are blocking oligos that anneal to the adapter sequences of your library, to prevent one properly captured library fragment (on-target) from pulling down another via annealing in the adapter regions. This dramatically increases your on-target rate.

In my experience with SureSelect, you should get >>70% on target if your design is sound, leaving out the blocking Oligos will net ~40% on-target.

There is a Nimblegen document (easily googleable, but if you any find it, I'll post it), that describes the design of the hyb enhancing oligos for Illumina. One can likely use the same principles to design blocking oligos for the IT adapters.

Alternatively, call Nimblegen. I would be surprised if they didnt have a canned PGM recommendation.

Good luck!
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Old 03-05-2013, 06:51 AM   #3
ChrE
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Thanks for the quick answer and forgive my late reply.

I didnt manage to find the mentioned paper but contacted NimbleGen directly. They confirmed that it should be possible to exchange their HE-oligos with PGM adapter-complementary oligos and achieve the same, so I will give it a try very soon :-)
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Old 03-12-2013, 10:22 PM   #4
Mahmoud
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please update us with you results, cuz I'm planning to do the same.
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Old 03-26-2013, 11:56 PM   #5
sokram
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Default SeqCap EZ on the Ion Torrent

Dear all,

I just found this on the internet, didn't have the time to read it all through but it seems that the hyb enhancing oligos sequences are in there.

Cheers,
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File Type: pdf SeqCapEZ for Torrent.pdf (718.7 KB, 132 views)
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Old 03-29-2013, 12:54 AM   #6
ChrE
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Great, this is spot on!
Thanks a lot!

Do you know who created this?

Best regards
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Old 05-29-2013, 06:03 PM   #7
cheerioet
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Quote:
Originally Posted by sokram View Post
Dear all,

I just found this on the internet, didn't have the time to read it all through but it seems that the hyb enhancing oligos sequences are in there.

Cheers,
Thanks for this information.
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