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Hi everyone,
I've been having issues with the quantification of my library pool, and I'm hoping you'll have some helpful suggestions on what is causing the problem.
My pool is 3.5 ng/uL based on HS Qubit. I ran a HS tapestation, and found an average fragment size of 364 bp with no adapter-dimer peak present. So my pool should be 14.6 nM. However, qPCR has consistently yielded results that are ~30 nM - double what I expected.
Since there's no adapter-dimer peak on the tapestation trace, any ideas what could be causing the problem?
The WGS libraries were KAPA Hyper preps with amplification, followed by a target enrichment with MyBaits. There was an additional amplification after the capture assay.
There is a double peak on my qPCR melt curve for this sample, but no evidence of adapter-dimer on the tapestation, so I'm not sure how much this relates to my quantification discrepancy.
Any input would be greatly appreciated! Thanks!
I've been having issues with the quantification of my library pool, and I'm hoping you'll have some helpful suggestions on what is causing the problem.
My pool is 3.5 ng/uL based on HS Qubit. I ran a HS tapestation, and found an average fragment size of 364 bp with no adapter-dimer peak present. So my pool should be 14.6 nM. However, qPCR has consistently yielded results that are ~30 nM - double what I expected.
Since there's no adapter-dimer peak on the tapestation trace, any ideas what could be causing the problem?
The WGS libraries were KAPA Hyper preps with amplification, followed by a target enrichment with MyBaits. There was an additional amplification after the capture assay.
There is a double peak on my qPCR melt curve for this sample, but no evidence of adapter-dimer on the tapestation, so I'm not sure how much this relates to my quantification discrepancy.
Any input would be greatly appreciated! Thanks!