Library prep kits and MYbaits compatibility

[kzarn]

Hi all,

I'm wondering if anyone has any experience using Illumina's TruSeq NANO DNA kit with MYcroarray's MYbaits. The NANO kit has been recommended to us by our rep, but Illumina's website indicates that the NANO kit does not support enrichment.

If anyone has experience or input regarding the use of the TruSeq NANO kit (or any other available kit) with MYcroarray's MYbaits, we would love to hear about it!

Thanks for reading!

[nucacidhunter]

The NANO kit has been recommended to us by our rep, but Illumina's website indicates that the NANO kit does not support enrichment.

I think that is for Illumina enrichment kit which has been discontinued. Would you be able to put a link to that Illumina web page? Under proper conditions and set up, one can use any library for enrichment.

[kzarn]

http://www.illumina.com/products/truseq-nano-dna-sample-prep-kit.ilmn

[nucacidhunter]

Thanks for the link. To my knowledge Illumina now only offers Nextera based enrichment kits and the kit that they recommend for enrichment (TruSeq DNA) in that table was discontinued while ago. If you use proper blocking oligos for Illumina adapters, Nano kit should be fine.

[kzarn]

A colleague has recommended that we use the TruSeq PCR-free kit - do you have any reason to believe that this kit might be better or worse than the Nano kit? (Assuming we order the appropriate blocking oligos of course.)

Thanks for entertaining my questions!

[kzarn]

I asked this question to our rep as well - here's what he had to say:

"...we recommend the Nano because it should work best for enrichment due to the fact that you want a high yield of your ligated library before subjecting that to the enrichment probes for pulldown.

Nano actually gives you a better yield than the PCR free option. Seems counterintuitive as the Nano starts with less input material, but it ends up with higher yield.

One critical metric in enrichment (or pulldown) methods is that you need a lot of material, and Nano gets you closest to the mark here.

Nano also appears to reproduce the former TruSeq kits performance better than TruSeq PCR free."

Additional input will be appreciated, of course!

[nucacidhunter]

In principal I agree with your rep statements. PCR free library average insert size is 550 bp. This might have a negative impact on your capture efficiency if your targets are small exonic regions, because if target sequences are in the middle of the captured fragments they will not be sequenced. I think the rcommended input into hybridization reactionis 500 ng and you are less likely to obtain that amount of yield from PCR free libraries. These can be overcome by modifying library size selection (and correspondingly shearing to maximise yield) and pooling couple of libraries to have sufficient input for capture reaction. Nano kits are robust and you may not gain much from all the trouble of modifying PCR free kit. I would recommend PCR free kit if you are missing capture from some targets because of PCR bias.