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I would like to sequence PCR amplicons with Ilumina. The PCR amplicon are between 200bp to 450bp. These are PCR amplicons from one gene already pooled for one patient.I will index each patient. I have heard that the PCR amplicons might be to small to use with the sample prep kit from Ilumina? Also for sequencing there might be a to big difference between the amplicons to sequence them together. Anyone with experience in ampliconsequencing that can give me some good advice?
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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...