Using small volumes of Nextera XT kit for human genome sample prep

[cjgb]

Hi

I've seen several posts about extending the lifespan of the Nextera tagmentation kits by using a fraction (1/10ish) of the reagents. However, the only published paper talks about doing this using megabase-sized genomes. As I am doing human genome analysis, I am not sure if this will work for me. Does anybody have experience using small volume Nextera reactions with human genome tagmentation?

Thanks for your help!

[nucacidhunter]

Scaled down reaction will work but input will be too low for human genome and library diversity will be low resulting in high level of duplicate reads and uneven coverage. I think 1/2 reaction for human genome would be OK if sequencing will be less than 30x.

Edit: Just noticed that your post heading is incorrect. It should be Nextera not Nextera XT

[luc]

If you are interested in an even genome coverage of big genomes (like the human one), it would be best to use other kits and not Nextera or similar rather biased tagmentation approaches.

[fanli]

If you are interested in an even genome coverage of big genomes (like the human one), it would be best to use other kits and not Nextera or similar rather biased tagmentation approaches.

I've seen this thrown around on a few boards/blogs, but does anyone have actual data showing Nextera (+/-XT) on a mammalian genome?

[nucacidhunter]

Following paper:

http://genomebiology.biomedcentral.com/articles/10.1186/gb-2010-11-12-r119

It actually shows negligible bias compared to mechanical fragmentation.