FAQ: What are the advantages of the qPCR-based NEBNext Library Quant Kit compared to other methods of quantitating NGS libraries?

qPCR is considered the most accurate and effective method of library quantitation. Quantitation consistency and reproducibility is considerably higher with qPCR than electrophoresis or spectrophotometry, which measure total nucleic acid concentration. Amplification-based methods quantitate only those molecules that contain both adaptor sequences, providing a more accurate estimate of the library concentration that can be sequenced. Because qPCR experiments can be set up in 96-well plates, it’s also more adaptable to high-throughput workflows. Compared to other qPCR methods, a number of additional improvements have been incorporated into the kit to increase convenience:
  1. The NEBNext kit uses only 4 standards, allowing more plate space and reagents for libraries 
  2. The NEBNext kit comes with a convenient, concentrated Library Dilution Buffer
  3. The NEBNext Library Quant Master Mix is formulated to require only the addition of primers (no additional pipetting step is required to add water to each reaction)
  4. A single extension time can be used for all libraries, regardless of size.