The NEBNext® Library Quant Kit for Illumina® has been updated to include six standards, enabling a broader standard curve. For users who are familiar with the previous four standards, numbering has changed (e.g., what was once Standards 1-4 is now Standards 2-5). Both the four-standard and six-standard protocols are supported; see the Manual for details.
Accurate quantitation of next-generation sequencing (NGS) libraries is essential for maximizing data output and quality from each sequencing run. For Illumina® sequencing specifically, accurate quantitation of libraries is critical to achieve optimal cluster densities, a requirement for optimal sequence output. qPCR is considered to be the most accurate and effective method of library quantitation, providing considerably higher consistency and reproducibility of quantitation. Further, amplification-based methods quantitate only those molecules that contain both adaptor sequences, thereby providing a more accurate estimate of the concentration of the library molecules that can be sequenced. The NEBNext® Library Quant Kit has been optimized to provide substantial performance and workflow improvements to qPCR-based library quantitation.
Be confident in your quant values, with more accurate and reproducible results than other methods and kits.
Enjoy the flexibility to use either four or six standards (included).
Simplify your reaction setup with fewer pipetting steps and a single extension time for all libraries.
Use with all your libraries, regardless of insert size, GC content and preparation method.
Figure 1: Typical amplification and standard curves for the NEBNext Library Quant Kit
Typical results from the NEBNext Library Quant Kit on a Bio-Rad CFX96 Touch (A) and an Applied Biosystems 7500 Fast real-time qPCR instrument (B). Amplification curves are shown on the left and resulting standard curves on the right. All default settings were used on both platforms. ABI 7500 Fast assay included 1X ROX (Low Concentration) and the ROX normalization.
The NEBNext Library Quant Kit components have been optimized to deliver significant improvements to qPCR-based library quantitation for Illumina sequencing. The kit contains primers which target the P5 and P7 Illumina adaptor sequences, and a set of six high-quality, pre-diluted DNA standards to enable reliable quantitation of diluted DNA libraries between 150–1000 bp.
Features
Provides more accurate and reproducible quant values than alternative methods and kits
Compatible with libraries with a broad range of insert sizes and GC content, made by a variety of methods
Allows you to choose the optimal standard curve (four or six standards) for your experimental needs
Supplied with a convenient Library Dilution Buffer
The NEBNext Library Quant Master Mix requires only the addition of primers
Utilizes a single extension time for all libraries, regardless of insert size
Library quant values can be easily calculated using NEB’s online tool, at NEBioCalculator.neb.com
ROX is included in the kit, for use with qPCR instruments that require a reference dye for normalization
Each set of reagents is functionally validated together through qPCR-based library quantitation assays. Reagents pass the functional test by adhering to stringent criteria set for assay efficiency and quantitation cycles (Cq) for each DNA standard and no template control reaction.
Figure 2: NEBNext Library Quant Kit workflow
Figure 3: qPCR provides more consistent library quantitation results than Bioanalyzer® analysis
Concentrations of 4 libraries were determined by the NEBNext Library Quant Kit and compared to values measured using the Agilent Bioanalyzer. Compared to NEBNext’s qPCR-based method, the Bioanalyzer concentrations displayed a greater level of variation.
Figure 4: Greater reproducibility of library quantitation with the NEBNext Library Quant Kit
Three 340–400 bp libraries were quantitated by 4 different users 2–4 times using either the NEBNext or KapaTM Library Quantification Kit (Universal). A notable improvement in quantitation consistency was observed for concentrations determined by the NEBNext Kit versus those from the Kapa kit.
Figure 5: Greater lot-to-lot consistency of standards with the NEBNext Library Quant Kit
Accurate qPCR quantitation requires the use of high-quality DNA standards with known concentrations. The NEBNext Library Quant Kit contains 6 standards produced with a high level of both quantitation accuracy and consistency. This figure shows data from >70 total runs from 4 lots of both NEBNext and Kapa standards, with all Cq values plotted. Box and whiskers indicate mean and quartiles. The NEBNext Library Standards displayed much lower variation in Cq, resulting in more consistent quantitation performance.
Seven different libraries were quantitated using either the NEBNext Library Quant Kit or the Kapa Library Quantification Kit (Universal). Undiluted library concentrations ranged from 2–200 nM. Libraries were diluted to 8 pM and loaded onto a MiSeq® instrument (v2 chemistry; MCS v2.4.1.3). Libraries quantitated with the NEBNext kit resulted in a raw cluster density average of 1160 k/mm2, directly in the optimal range of 900–1300 k/mm2. In contrast, libraries loaded based on the Kapa quantitation averaged only 660 k/mm2.
Figure 7: With the NEBNext Library Quant Kit, optimal cluster density is achieved from quantitated libraries with a broad range of library size and GC content
Libraries of 310–963 bp from the indicated sources were quantitated using the NEBNext Library Quant Kit, then diluted to 8 pM and loaded onto a MiSeq (v2 chemistry; MCS v2.4.1.3). Library concentrations ranged from 7–120 nM, and resulting raw cluster density for all libraries was 965–1300 k/mm2 (ave. =1199). Optimal cluster density was achieved using concentrations determined by the NEBNext Library Quant Kit for all library sizes.
Figure 8: Accurate Library Quantitation is achieved with a broad range of library size and GC content A selection of libraries successfully quantitated with the NEBNext Library Quant Kit. Libraries are plotted by size, ranging from smaller libraries (sRNA, FFPE) at 150–230 bp to largest libraries at 980 bp. Various input sources were used, ranging from 20-70% GC content, as indicated by text color. Libraries were prepared using NEBNext, Illumina TruSeq® Nano and Kapa Hyper library prep kits (data not shown). No dependence on size or GC content was observed in library quantitation when using NEBNext.
Kit Components
The following reagents are supplied with this product:
Storage of Kit Components: All of the individual components of the NEBNext Library Quant Kit for Illumina are stable at –20°C for two years. When not in use, kit components should be stored at –20°C. All components of the kit, including the combined NEBNext Library Quant Master Mix and Primer Mix, are stable for at least 30 freeze/thaws.
Once the Primer Mix has been added to the NEBNext Library Quant Master Mix (see Protocol on page 6 of manual), the resulting mix can be stored at –20°C for seven months. For short-term storage, this mix may be stored at 4°C for up to six weeks.
Reactions can be prepared at room temperature, but unused portions of the kit reagents should be kept on ice after thawing them for use.
For Reagents Supplied:
If you need dilution buffer in addition to what is provided in the kit, it can be purchased separately.
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Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.
The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]
The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]
This product is covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB).
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For more information about commercial rights, please email us at busdev@neb.com.
This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.
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Licenses
Nucleic acid-based aptamers for use with thermophilic DNA polymerases are licensed exclusively by New England Biolabs, Inc. from SomaLogic, Inc. New England Biolabs, Inc. gives the Buyer/User a non-exclusive license to use the NEBNext® Library Quant Kit for Illumina® for Research Use Only (RUO). Commercial use of this product may require a license from New England Biolabs, Inc. For additional information or to inquire about commercial use, please contact busdev@neb.com.
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