We are pretty used to our MiSeq and have about 100 runs under the belt but after a few low cluster densities we decided to qPCR and bioanalyse some PhiX to troubleshoot. Lo and behold we get a lovely tight peak on the bionalyser but a 10pM dilution using illumina's reported PhiX stock concentration, is actually over 100pM according to the bioanalyser. Anyone else seen this? Is only 10℅ of the DNA in our PhiX stock clusterable? The qPCR results are closer to what Illumina states is the PhiX concentration in the tube, BTW, but still off slightly. Still get PhiX cluster density of ~700+ if we dilute like we're told. Any comments?
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Even though Illumina says that the phix is supplied at 10nM, I've found that the actual value does indeed seem to jump around a little bit.
I asked my FAS about that once and they confirmed that 10nM isn't a strict value but couldn't give me any more info regarding how it's made and thus how tightly the concentration is controlled.
From my experiences the supplied concentration seems to range from ~9-12, but thats mostly based on cluster densities and % aligned values which aren't very useful in determining such things.
I wouldn't trust the Bioanalyzer for quantification at all because it's very poor at that, but I wouldn't expect the concentration that Illumina supplies to be 10X greater than what they say.
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