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We have a theory that large libraries, specifically those with fragments trailing off to >2kb, do not quantitate accurately from Bioanalzyer. We think this because qPCR concentrations do not correlate. Has anyone else had this problem? It seems if we are able to remove the "tail", then qPCR matches a bit better with Bioanalzyer.
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Libraries over 2 kb don't tend to work on Illumina sequencers, so do you really care what the concentration of those molecules is?
Also, are you doing a TaqMan qPCR or SYBR? If it's SYBR, you have to factor in the average molecule size, so you're still dependent on the Bioanalyzer anyway. -
This depend on qPCR cycle settings. Using standard protocols gives enough time for extension of around up to 900 bp fragments so larger fragments are not amplified by qPCR. Since fragments above this size do not cluster efficiently they can be ignored. For accurate quantification average fragment length from the shortest fragment to 900-950 bp on the bioanalyser can be used to calculate concentration.Originally posted by kgoglin View PostComment
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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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