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Old 06-20-2012, 08:28 PM   #1
sasignor
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Default methods for targeted resequencing

My work is a little outside the general audience for targeted resequencing technology (disease work it seems). As such its difficult to understand the differences between all the methods, and which method would be best for our application.

We would like to sequence two regions of 50 kb from ~20 to 30 populations of non model Drosophila. Money is an issue, but we do have a wet lab capable of of any sort of sample prep. However, investing in the technique isn't really on the table because we will likely never use it again as a lab.

Any suggestions?
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Old 06-20-2012, 08:59 PM   #2
ECO
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Your target size is pretty small for most of the commercial array/pulldown techniques...and with that small sample number you'll probably pay $300-500/sample unless you really work hard on the negotiation.

The most straightforward homebrewy option is probably long/mid-range PCR, followed by concatenation/shearing and shotgun library prep...You'd probably spend the most time on PCR design and optimization to cover your whole region robustly...but if you had overlapping ~3-5kb PCR fragments it's not that many amplicons to cover 100kb redundantly. (~48 per sample)

I have no experience with it yet...but Agilent just bought Halo Genomics, whose enrichment technology is purportedly quite fast and excels at small designs...might be worth checking out but may still be pricey: http://www.halogenomics.com/haloplex/how-it-works The advantage of this is you have some recourse if it doesn't perform.

Depending on how homebrewy you want to get, it is very straightforward to brew your own pulldown set with commercial oligo syntheses...however I think given your low sample number this will end up cost prohibitive (as individually synthesized probes are expensive for one offs...but provide nearly infinite amounts for multiple captures if you had higher sample numbers).

Good luck!
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