best way to prep for miseq a 100kb continous region of DNA?

[galloucf]

Hi all,

I also posted this on the Illumina thread since I didn't know in which section it should go, but here is the situation:

We are looking to sequence a 100 kb region of the human genome for 400 samples. We are not sure what is the current recommended method for amplifying a region of this size for illumina sequencing.

We are looking into Illumina Tru seq custom amplicon kit, but would this be possible to do with a more conventional long range pcr by designing primer for large regions (such as 20KB each), preping that DNA and then sending it off?

Any input would be of help.

Thanks

[exo]

Hi,

I really doubt pcring out 20kb regions is optimal. Well, actually i doubt it will work that way at all - but thats just me. You will either have to make smaller amplicons, which will be tedious for a 100kb region.
Have you thought about cloning them into BACs? I think thats a more relevant approach, at least from my point of view.

best,
Elias

[frozenlyse]

100kb is a tricky area to target at the moment... If you have a lot of DNA for your samples you could look into doing multiplexed sequence capture ie make barcoded libraries for each sample, pool x number of libraries together then do an agilent or nimblegen sequence capture, then HiSeq

[ECO]

Order a set of Lockdown probes from IDT. ~$15/probe. Works well enough for most applications.

https://www.idtdna.com/pages/products/nextgen

*I'm a user, and have no financial interest in this product.