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Has anyone had any luck trying to quantify Nextera XT libraries using qPCR? I have tried it twice with no success. I have been able to quantify TruSeq libraries and they have been working just right. It looks like primer sequences are correct, so I don't know what could be causing the problem.
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Yes, we got a library, already normalized and eluted as single strands using the kit protocol. The protocol called for us to just heat denature it, snap cool and immediately load into the reagent cassette for sequencing. But we did a KAPA qPCR kit (not the Illumina-specific kit, just the generic one) using normal flow cell oligo primers. The result looked fairly reasonable, so we proceeded. Basically just did what the protocol asked. (Okay, I spiked in phiX out of paranoia -- but otherwise the followed the instructions on the box.)
Worked okay.
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Phillip -
You've had luck with the bead normalization? I've never been able to get it to work. Anytime I use the beads I get a uneven distribution of up to 50%. That's why I'm relying on qPCR to quantify my library.Comment
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You get better than 0.5x variation in read numbers using qPCR? Like you have several samples in a pool and the lowest one gave you 20 million and the highest gave you 30 million? That would be close enough to perfect to satisfy me!
I did not do the bead normalization myself -- we just got a Nextera XT pool from a customer and ran it. There were >20 different index pairs in it. I generally call it a "win" if the lowest sample gives > 1/2 the number of reads as the highest sample. So by that criteria, it was good. Actually, if I remember correctly, it was better than that.
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PhillipComment
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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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