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Smriti 05-28-2015 12:00 PM

Is Nextera useful for Pac bio in future?
Hi There,

I am working with Nextera workflow. I was wondering if Nextera library prep can be somehow extended to Pac bio platform. My understanding is Nextera insert size is much smaller than what Pac bio can sequence. Is there a way Nextera transposon can fragment a bigger size insert, add adapters and then it gets sequenced on Pac bio.

This way, we can use advantage of both the technologies, low imput starting material and sequencing it real-time.

Any suggestions?

SNPsaurus 05-28-2015 09:27 PM

I suspect that if you were to reduce the amount of transposase compared to DNA then you would see an increase in the bias of the fragmentation, as the preferred sites would use up the low amount of reagent.

Nextera is nice because it allows you to skip end-repair and ligation of adapters. But if you want to make really long DNA fragments, it is probably preferable to just end repair and ligate and not add a fragmentation step.

nucacidhunter 05-28-2015 10:14 PM

It can be done in two ways. One is to do tagmentation with more input DNA or reduced enzyme to obtain large tagmented DNA for amplification as suggested by SNPsaurus. Other is to tagment DNA as before but fill in the gap and end repair without amplification. One also can introduce size selection either way. Nextera mate pair tagmentation system also can be used but it is less cost effective.

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