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Check the concentration of PhiX by qPCR (e.g. KAPA). I don't think I've yet seen a tube from Illumina come in at 10 nM. It's usually much higher, in my experience.
Also, I've noticed that if you make a 20 pM denatured PhiX stock ahead of time and use it over a period of time, the %aligned/%expected will drop with each run. Once I mistakenly used a tube of 20 pM that was left out overnight and the %aligned was massively off. I'm suspecting that much of it had renatured.
Also, I'm in the habit of aliquoting 0.2 N NaOH into 50 uL aliquots, freezing them, and only using them a couple of times. Same for the 20 pM PhiX. After the 5 min denaturing step I follow that with 1 min at 95 C. Illumina recommends an additional heating step for high GC samples, but IMO it's a good insurance policy against an aliquot of weak NaOH. Plus, it shouldn't hurt the samples/PhiX...
Also, I've noticed that if you make a 20 pM denatured PhiX stock ahead of time and use it over a period of time, the %aligned/%expected will drop with each run. Once I mistakenly used a tube of 20 pM that was left out overnight and the %aligned was massively off. I'm suspecting that much of it had renatured.
Also, I'm in the habit of aliquoting 0.2 N NaOH into 50 uL aliquots, freezing them, and only using them a couple of times. Same for the 20 pM PhiX. After the 5 min denaturing step I follow that with 1 min at 95 C. Illumina recommends an additional heating step for high GC samples, but IMO it's a good insurance policy against an aliquot of weak NaOH. Plus, it shouldn't hurt the samples/PhiX...
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