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Math is not my strong point! I've pooled 12 samples, previously quantified by KAPA quant, based on their size-adjusted concentration. My library peaks average 700 bp. The KAPA quant of the pooled, size-adjusted library is less than I expected (not super low, just a bit lower than I was aiming for). Was the problem just pipetting or should I have pooled based on the non-adjusted concentration? I'm not sure that I haven't adjusted for size twice, and I don't want to overload the MiSeq.
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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...