The efficiency of the End Repair, dA-Tailing and Adaptor Ligation steps during library construction can be measured separately from the PCR step by doing qPCR quantitation of adaptor-ligated fragments prior to library amplification. This enables determination of the rate of conversion of input DNA to adaptor-ligated fragments, i.e., sequenceable molecules. Therefore, measuring conversion rates is another way to assess the efficiency of library construction and also provide information on the diversity of the library. Again, NEBNext Ultra II enables substantially higher rates of conversion as compared to other commercially available kits.
Minimization of PCR Cycles
In general, it is preferable to use as few PCR cycles as possible to amplify libraries. In addition to reducing workflow time, this also limits the risk of introducing bias during PCR. A consequence of increased efficiency of End Repair, dA-Tailing and Adaptor Ligation is that fewer PCR cycles are required to achieve the library yields necessary for sequencing or other intermediate downstream workflows. For applications such as exome enrichment, very high library yields (1 μg or more) are generally required as input for the enrichment step. If library preparation is inefficient, a large number of PCR cycles may be required to achieve these required yields, especially when the input amount for the original library is low. This can result in production of a library that is not representative of the original sample. View additional data on target enrichment applications.
Each reagent in the NEBNext Ultra II kit has been reformulated, resulting in a several-fold increase in yield over the original NEBNext Ultra DNA Library Prep Kit.
This video walks you through DNA Library Preparation using the NEBNext Ultra II DNA Library Prep Kit. The video also includes tips for optimization as well as safe stopping points.
These 12 quick tips for NGS library preparation are a great refresher if you're a seasoned pro, or a great introduction if you're a beginner.