FAQ: What are the advantages of the qPCR-based NEBNext^® Library Quant Kit for Ultima Genomics^® (NEB #E3410) compared to other methods of quantitating NGS libraries?

qPCR is considered the most accurate and effective method of library quantitation. Quantitation consistency and reproducibility are considerably higher with qPCR than with electrophoresis or spectrophotometry, which measures 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, several additional improvements have been incorporated into the NEBNext Library Quant Kit for Ultima Genomics to increase convenience:

1. A single extension time can be used for all libraries, regardless of size.
2. Luna^® Universal qPCR Master Mix is formulated to require only the addition of primers (no additional pipetting step is required to add water to each reaction).
3. Luna Universal qPCR Master Mix contains a universal passive reference dye, eliminating the need for ROX addition even in real-time instruments that require ROX normalization.
4. The NEBNext Library Quant Kit for Ultima Genomics comes with a convenient, concentrated Library Dilution Buffer.