What methods provide the most accurate quantitation for DNA libraries?

qPCR and fluorometry are the two methods that provide the most accurate quantitation for DNA libraries. However, it’s important to note that no single technique provides all the data you need with sufficient accuracy for normalization. While both these methods – qPCR and fluorometry - enable the most precise quantitation, neither method is able to estimate average size of DNA fragments. An additional check via electrophoresis is often necessary for estimation of fragment size. Of these two methods, only qPCR can specifically target the adaptor-ligated molecules by using primers complementary to the adaptor sequences. Quantitating only these viable sequencing templates offers the best chance at accurate normalization. Adaptor ligation efficiency between individual samples and batches can vary due to reliance on enzymatic reactions that can be affected by differences in the quality of the starting material and impurities present in the sample. In this case, fluorometry, increases the risk of over-dilution and overestimating the sequencing-competent library concentration as it involves quantitating without specificity for adaptor-ligated molecules. However, fluorometry is a faster and more cost-effective option if you use high and consistent quality starting material and the end repair/adaptor ligation step of your library prep workflow is efficient. In this case, you can expect to get results that are nearly as accurate as qPCR. Of the two methods, qPCR remains the preferred option for ultimate accuracy in quantitation.