New England Biolabs, E7630L, NEBNext® Library Quant Kit for Illumina®

Accurate quantitation of next-generation sequencing (NGS) libraries is essential for maximizing data output and quality from each sequencing run. For Illumina Related Categories NGS Library Quantitation,, Next Generation Sequencing Library Preparation Specification Materials Required but not Supplied Nuclease-free water qPCR machine qPCR plates and seals PCR strip tubes or microcentrifuge tubes Conical centrifuge tubes FAQ Q: How are the new 6 standards different from the old 4 standards? A: The new 6 standards are an expansion of the old 4 standards, which increases the dynamic quantitation range by 100 fold, from 0.01pM – 10 pM to 0.001 pM – 100 pM. Please note that the new standards 2, 3, 4 and 5 are equivalent to the old standards 1, 2, 3 and 4 (with NEB #E7534, #E7535, #E7536 and #E7537, respectively). Q: What are the advantages of the qPCR-based NEBNext Library Quant kits 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 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 kits to increase convenience: The NEBNext Library Quant kits come with a convenient, concentrated Library Dilution Buffer. The qPCR master mixes are formulated to require only the addition of primers (no additional pipetting step is required to add water to each reaction). A single extension time can be used for all libraries, regardless of size. Q: What libraries can be quantitated with the NEBNext® Library Quant Kit for Illumina®? A: Any library containing the standard Illumina P5 and P7 adaptor sequences can be quantitated with the kit. Libraries can be prepared using any desired method as long as these adaptor sequences are introduced. Quantitation with the Quant Kit has been successfully performed using a number of library construction kits, including those listed below: Manufacturer Library Prep Kit New England Biolabs® NEBNext® Ultra™ II DNA NEBNext Ultra II FS NEBNext Enzymatic Methyl-seq (EM-seq™) NEBNext Immune Sequencing NEBNext ARTIC SARS-CoV-2 NEBNext Ultra II Directional RNA NEBNext Ultra II RNA NEBnext Single Cell/Low Input RNA Clontech SMARTer® Stranded Total RNA KAPA™ Hyper HyperPlus Thermo Scientific® MuSeek Illumina® Nextera Rapid Capture ScriptSeq™ v2 TruSeq DNA PCR-free TruSeq Nano® TruSeq RNA v2 TruSeq RNA Access TruSight® Rapid Capture Illumina PCR-free Takara Prep™ ILM DNA A wide variety of input genomic material is compatible with the Quant Kit. Kit performance was validated using libraries ranging in size from 140–1000 bp and 20–70% GC. Input material can be sRNA, FFPE DNA, or gDNA libraries from any source of interest including GC extremes, e.g. Plasmodium and Rhodopseudomonas. Q: How much should I dilute my library to run in the Quant Kit? A: We recommend an initial 1:1,000 dilution of the library using the provided Library Dilution Buffer. For most library preps this will result in >10 pM library, so we recommend two more 1:10 serial dilutions resulting in final 1:10,000 and 1:100,000 dilutions. For new protocols and libraries, we recommend running both of these dilutions in the Quant Kit. With more established workflows or to maximize the number of libraries quantitated, it is often possible to use only the 1:100,000 dilution for quantitation. If custom dilutions are desired, please ensure that final diluted libraries fall within the range of the standards, 100 pM – 1 fM. Q: Can I run the NEBNext Library Quant Kit on my qPCR machine? A: The Quant Kit is compatible with all qPCR instruments tested thus far, using either standard or fast temperature ramping protocols. For machines that use a reference dye for normalization, please consult the kit manual or the FAQ on ROX for appropriate ROX selection. The Quant Kit has been run successfully on the following qPCR machines: Manufacturer Instrument Applied Biosystems® StepOne™ StepOnePlus™ Prism® 7000 Prism 7900HT 7500 Fast ViiA™ 7 Bio-Rad iQ® 5 CFX96™ CFX384™ Life Technologies QuantStudio™ 7 Roche LightCycler® 96 LightCycler 480 Please contact us at info@neb.com if you would like to discuss performance on any qPCR instrument not listed above. Q: Which ROX should I use for my qPCR machine? A: We provide separate tubes of ROX normalization dye at two concentrations (Low, High) to enable use on all qPCR platforms. Please refer to the table below to determine whether ROX is required, and if so, which concentration should be used: qPCR Instrument ROX Bio-Rad® iQ™5, CFX96, CFX384, Opticon Roche Lightcycler® Qiagen Rotor-Gene™ Eppendorf Mastercycler® Cepheid® SmartCycler® Not recommended Applied Biosystems® 7500, QuantStudio™, ViiA7™ Agilent Mx™ Low ROX Applied Biosystems® 7000, 7300, 7700, 7900HT, StepOne™, StepOnePlus™ High ROX Q: Are 4 standards enough for a qPCR quantitation? A: Yes. Although we recommend using 6 standards (100 pM – 0.001pM), the standard curves generated from 4 standards (10 pM – 0.01pM) are sufficient to give a reliable measurement of library concentration. Even if one standard is an outlier from the curve due to problems in setup, the other 3 can be used with confidence. The range of standard concentrations provided with the kit will cover all libraries prepared using standard methods and kits following the kit guidelines for making library dilutions. Q: Does the library quant require a size correction? A: Yes. The intercalating dye used in the kit 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, 399 bp. All quants are simply multiplied by a correction factor, 399/library size. This is done automatically using our online calculator tool. 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: How many libraries can I quantitate at a time? A: We recommend running 2 dilutions of each library, and running each dilution in triplicate. Therefore 12 (using 6 standards) or 13 (using 4 standards) libraries can be quantitated in a 96-well plate. Once a workflow is well established, a single dilution could be run, allowing 25 (using 6 standards) or 27 (using 4 standards) libraries per 96-well plate. Q: How long is a run of the Quant Kit? A: We estimate a total end-to-end run time of 1h 45min. Of this, hands-on time is <45min, and 1h will be machine time taken up by the qPCR run. Q: Do you provide any analysis tools to help with the quantitation? A: Yes, we have a simple Library Quant qPCR calculator with our suite of NEBioTools. To use the qPCR quant tool, you need only the Cq for each standard and library, the dilution factor for each sample, and the size of the library for size correction. The tool is self-contained and data does not leave your browser. Q: Can I see adaptor dimers as a measure of library quality? A: Yes. While a melt curve is not required for the kit, and omitting it will reduce run times, data from the melt analysis can be used for library quality information. When adaptor dimers are present at >5% of the library sample, a single, sharp melt peak can be observed at 81.5 °C. In all other cases, a broad or multi-peaked melt curve will be produced, indicative of the range and diversity of the library being amplified. Q: How long can I store the Master Mix after adding the Primers? A: Once Quant Kit Primers are added to the Master Mix, the Mix+Primers solution is stable for 6 weeks at 4 °C for convenience. If you anticipate longer times between runs, store at –20°C for up to 7 months. Q: Can the NEBNext Library Dilution Buffer be purchased separately? A: Yes, in addition to the dilution buffer provided in the kit, it can be purchased separately. The part number is B6118S. It is a 7.5 ml bottle and can be purchased here.