New England Biolabs, E3410L, NEBNext® Library Quant Kit for Ultima Genomics®

The NEBNext Related Categories NGS Library Quantitation,, Next Generation Sequencing Library Preparation FAQ Q: What is the NEBNext® Library Quant Kit for Ultima Genomics® (NEB #E3410)? A: The NEBNext Library Quant Kit for Ultima Genomics is a specialized qPCR-based assay for accurately quantitating PCR-free next-generation sequencing (NGS) libraries prepared using the UG 100 SolarisTM Free workflow. Q: 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? A: 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: A single extension time can be used for all libraries, regardless of size. 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). 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. The NEBNext Library Quant Kit for Ultima Genomics comes with a convenient, concentrated Library Dilution Buffer. Q: How much should I dilute my library to run in the NEBNext® Library Quant Kit for Ultima Genomics® (NEB #E3410)? A: Libraries should be diluted to a concentration within the dynamic range of the standards provided in the NEBNext Library Quant Kit for Ultima Genomics (0.003pM-30pM). A typical starting dilution is between 1:4000 and 1:10,000 but adjustments may be necessary based on the initial concentration of your library. Q: What is the workflow time for the NEBNext® Library Quant Kit? A: The NEBNext Library Quant Kit workflow takes approximately 1h 50min. Of this, hands-on time is ~50min, and 1h will be machine time taken up by the qPCR run. Q: Do you provide any analysis tools to help with the quantitation when using the NEBNext® Library Quant Kit for Ultima Genomics® (NEB #E3410)? A: Yes, NEB provides software tools and templates to assist with analyzing and quantitating libraries using the NEBNext Library Quant Kit for Ultima Genomics, which can simplify data processing and result interpretation. The analysis tool can be found under the NGS Library Quant section of NEBioCalculator. Q: Does the library quant require a size correction when using the NEBNext® Library Quant Kit for Ultima Genomics® (NEB #E3410)? A: Yes. The intercalating dye used in the NEBNext Library Quant Kit for Ultima Genomics binds DNA in proportion to the number of base pairs per double-stranded DNA product, so it is necessary to correct for product length by normalizing to the size of the DNA standard, 200 bp. All quants are simply multiplied by a correction factor, 200/library size. This is done automatically using our online tool for NGS Library Quant. Q: How many libraries can I quantitate at a time when using the NEBNext® Library Quant Kit for Ultima Genomics® (NEB #E3410)? A: We recommend running 2 dilutions of each library and running each dilution in triplicate during the development/optimization stage. Therefore, 12 libraries can be quantitate in a 96-well plate using 5 standards and 1 no-template control (NTC). Once a workflow is well established, a single dilution could be run, allowing quantitation of 24 libraries per 96-well plate using 5 standards and 1 NTC. Q: What other materials/information do I need to run an NEBNext® Library Quant Kit? A: It’s necessary to know the average library size, so analysis with an Agilent Bioanalyzer, TapeStation or similar instrument is helpful. A concentrated library dilution buffer is provided and this should be diluted using nuclease-free water. A qPCR instrument with appropriate plates and seals are required, and standard laboratory equipment such as pipettes, tips, and conical tubes are also necessary. Q: Do I need to add ROX when using the NEBNext® Library Quant Kit for Ultima Genomics® (NEB #E3410)? A: No. Luna® Universal qPCR Master Mix contains a universal passive reference dye that enables compatibility with various real-time instruments, including those that use no passive reference normalization and those that use a low or high amount of passive reference dye (ROX). Q: Can I run the NEBNext® Library Quant Kit for Ultima Genomics® (NEB #E3410) on my qPCR machine? A: The NEBNext Library Quant Kit for Ultima Genomics has been tested and found compatible with the following qPCR instruments, using either standard or fast temperature ramping protocols: Bio-Rad CFX96TM Touch Applied Biosystems® 7500 Fast Life Technologies QuantStudioTM 6 Amplification efficiency estimates may vary across qPCR instruments due to differences in hardware and analysis software. Q: Is the NEBNext® Library Quant Kit for Ultima Genomics® (NEB #E3410) compatible with Solaris Free and Flex workflows on the Ultima Genomics platform? A: The NEBNext Library Quant Kit for Ultima Genomics is compatible with libraries prepared using the UG SolarisTM Free workflow. For libraries prepared using the UG Solaris Flex workflow, it's recommended to use alternative quantitation methods.