sequencing 100kb continous region? What is the best method for amplification?

[galloucf]

Hi all,

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

[AStretton]

You could do the long range PCRs, fragment your products then use Nextera for the library prep.

[mcnelson.phd]

It seems like you're looking for a home-brewed version of Moleculo...

You could use primers that are spaced multiple Kbp apart to get a 100kbp fragment, but you would probably have to use something like phi29 to get such a large target as the longest amplicons I've ever achieved are on the range of 20Kbp, and that's still pretty hit or miss. AmpliTaq LA and a few others say that they can amplify 50Kbp fragments, but that's still half of the size you're looking at and you're probably opening yourself up to really weird chimeras forming.