library prep by transposition

[upenn_ngs]

http://www.epibio.com/nextera/nextera.asp?gclid=CMWY7-mtx58CFRE55QodmjUO3A

what are thoughts on this new method? no need for a covaris, and low input. interested in using before capture protocol, i am concerned the transposome complex will not yield fragments representative of entire genome.

[ECO]

Moving this to the library prep forum as it's applicable to 454 (and probably solid) as well.

[Tom Haltern]

size distribution using transposition

I heard about the new technology as well. I was just wondering what the size distribution looks like afterwards. As far as I know there is no need to do it. Thanks.

[upenn_ngs]

here is the distribution with bioanalyzer for a full reaction with 10 pcr cycles:



and for 1/5 scaled down reaction with 15 pcr cycles:

[upenn_ngs]

here is the distribution with bioanalyzer for a full reaction with 10 pcr cycles:

[IMG][/IMG]

and for 1/5 scaled down reaction with 15 pcr cycles:

http://www.postimage.org/image.php?v=PqE_Lh9[IMG][/IMG]

[krobison]

You should check with your capture reagent vendor -- one of the problems seen with Illumina libraries is "daisy-chaining" of fragments via the adapters which brings in off-target material. Nextera uses custom adapters, so any blocking oligos supplied in the kit won't be correct.

[flxlex]

@upenn_ngs: Were this size distributions for Illumina libraries?

[mclp]

what about ends fragments ?

Hi all,
This fragmentation seems very interesting by its low input DNA and its relative simplicity!
I am wondering about the non representativity of ends fragments with this fragmentation except with a previous step like circularization or adding primers.
Is that makes sense ?

[upenn_ngs]

yes, this is illumina library prep. although there is a great time saving, a fragmentation and ligation protocol with NEB enzyme is still much more cost effective.