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Does anyone have any information about Nextera multiplex library prep versus the Illumina method?
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You should be able to find the information you need in the Nextera Illumina-compatible protocol (click the "protocol" link on the product page). Appendix A lists the sequences of our barcode primers; you can also design your own if you wish.
If you have any other questions or issues, feel free to contact me via PM. -
Multiplex prep
We used the Nextera multiplex primers to prepare several bacterial libraries, then pooled the libraries for one lane of Illumina sequencing. This was quite straightforward following the manufacturer's instructions. The libraries sequenced well and we could identify a perfect match to each 6 bp index sequence in 97% of the Illumina index reads. Be aware that if one is performing paired read sequencing, the second of the paired reads (actually Illumina read 3) will have low quality in the last 10-20bp because the index read (read 2) uses some of read 3's reagents. The read1 quality averaged Q20 out to 120 bp while Q20 was around base 110. We found the Nextera kit to be very, very simple and much faster compared to the standard Illumina prep, although we have not examined any potential read bias yet.Last edited by Boonie; 12-30-2010, 11:04 AM.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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