library prep using sonicator - indexing

[kerryp]

I'm currently using Nextera XT to prepare libraries for MiSeq. This involves 'tagmentation' which simultaneously fragments (enzymatically) and adds a tag sequence. We then amplify using Nextera XT indices.

My question is - if we were to fragment our dsDNA by sonication, how would we then amplify the fragments using Nextera XT indices? I assume the tag sequence is essential - is there any way around this?

Thanks in advance for any advice!

[Jessica_L]

Hi kerryp,

There's a lot of information about sample prep in the forums here and also on the Illumina website, so you should definitely check out those references.

That disclaimer aside, if I understand your question, you're asking about library preparation that doesn't use tagmentation? Sonicated DNA usually follows a different workflow (end repair, A-tailing, 5'-Phosphorylation, Adapter ligation using a DNA ligase) than the Nextera prep which effectively combines fragmentation and adapter ligation into a single step, eliminating the end repair steps.

I don't know off the top of my head whether you can use the Nextera adapters/indices in a general library prep. I want to say that there are some differences between Illumina's TruSeq adapters and the Nextera ones, but you would need to verify that.

Does that answer your question?

[kerryp]

Hi Jessica_L

Thanks - yes, I see the difference now between the library prep methods. We'll try the NEB kit and sonication and see how it compares with Nextera