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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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Love this - good data definitely starts from good input, and poor input can only give relatively poor data. I particularly like the mention of Nanodrop/absorbance based methods for quantification. It's such a toss up if you'll get an accurate reading or what amounts to a randomly generated number, and a lot of library/sequencing related issues can be traced back to poor quant.07-01-2026, 11:43 AM
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I’m not a sequencing expert. I’m a purification scientist who uses NGS to evaluate workflows my group develops. With this perspective, we think about the sample first and the NGS workflow second. The sequencer is an exceptionally honest reporter, but it can only report on what you give it, so whether you get clean, interpretable data from an NGS workflow is largely determined before you begin.
Here are nine questions we think about, in roughly the order they matter, before...
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