Has anyone used Nextera library prep for illumina? I'm looking to test but any info on biases or other data would be welcome.
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We tried it. If you don't have a Covaris it might be worthwhile. It is very easy and fast but it doesn't give a tight size distribution. -
Thanks.
We do have a covaris but it's the price. We have to charge a wet lab hourly rate. If nextera can cut 2-4 hours off library making it might be useful.
I suppose we could use an ez-gel to tighten the size range.Comment
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I would use the HMW buffer, never the LMW buffer for Illumina prep. The peak at 200 represents 135 bp of the Nextera adapters and 65 bp of insert. I have had to use the HMW buffer and do size selection. Be careful if you do Size Selection post PCR, you do not have overages to re-amplify and the standard PE PCR Primers will not work.Comment
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Starting with the recommended amount of DNA for Nextera (50 ng), you should get 300-500 ng of "tagmented" DNA after 9 cycles of PCR. We have some customers who don't bother with size selection, and get good sequencing results--they find that a tight size distribution is not necessary for Illumina sequencing in their application. However, it's your choice.
Also, in response to the original point, Nextera will definitely save 2-4 hours compared to Covaris + adaptor ligation.Comment
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