Product Class: Kit

NEBNext® Library Quant Kit for Illumina®

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.

Product Introduction

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.

Additional dilution buffer can be purchased separately.

Additional standards can be purchased separately.

Catalog # Size Concentration
E7630S 100.0 reactions
E7630L 500.0 reactions

Product Information

Description



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. 


Figure 6: The NEBNext Library Quant Kit values enable optimal cluster densities

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.


This product is related to the following categories:
NGS Library Quantitation Products,
Next Generation Sequencing Library Preparation Products,

Kit Components

Kit Components

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration
  • E7630S     -20    
      NEBNext® Library Quant Master Mix E7640AVIAL -20 1 x 1.5 ml Not Applicable
      NEBNext® Library Quant Primer Mix E7639AVIAL -20 1 x 0.1 ml Not Applicable
      NEBNext® Library Dilution Buffer E7633AVIAL -20 2 x 1 ml 10 X
      NEBNext® Library Quant DNA Standard 1 E6886AVIAL -20 1 x 0.024 ml 100 pM
      NEBNext® Library Quant DNA Standard 2 E6887AVIAL -20 1 x 0.024 ml 10 pM
      NEBNext® Library Quant DNA Standard 3 E6888AVIAL -20 1 x 0.024 ml 1 pM
      NEBNext® Library Quant DNA Standard 4 E6889AVIAL -20 1 x 0.024 ml 0.1 pM
      NEBNext® Library Quant DNA Standard 5 E6890AVIAL -20 1 x 0.024 ml 0.01 pM
      NEBNext® Library Quant DNA Standard 6 E6891AVIAL -20 1 x 0.024 ml 0.001 pM
      ROX (High) E7641AVIAL -20 1 x 0.02 ml Not Applicable
      ROX (Low) E7638AVIAL -20 1 x 0.02 ml Not Applicable
  • E7630L     -20    
      NEBNext® Library Quant Master Mix E7640AAVIAL -20 1 x 7.5 ml Not Applicable
      NEBNext® Library Quant Primer Mix E7639AAVIAL -20 1 x 0.5 ml Not Applicable
      NEBNext® Library Dilution Buffer E7633AAVIAL -20 2 x 7.5 ml 10 X
      NEBNext® Library Quant DNA Standard 1 E6886AAVIAL -20 1 x 0.12 ml 100 pM
      NEBNext® Library Quant DNA Standard 2 E6887AAVIAL -20 1 x 0.12 ml 10 pM
      NEBNext® Library Quant DNA Standard 3 E6888AAVIAL -20 1 x 0.12 ml 1 pM
      NEBNext® Library Quant DNA Standard 4 E6889AAVIAL -20 1 x 0.12 ml 0.1 pM
      NEBNext® Library Quant DNA Standard 5 E6890AAVIAL -20 1 x 0.12 ml 0.01 pM
      NEBNext® Library Quant DNA Standard 6 E6891AAVIAL -20 1 x 0.12 ml 0.001 pM
      ROX (High) E7641AAVIAL -20 1 x 0.1 ml Not Applicable
      ROX (Low) E7638AAVIAL -20 1 x 0.1 ml Not Applicable

Properties & Usage

Materials Required but not Supplied

  • Nuclease-free water
  • qPCR machine
  • qPCR plates and seals
  • PCR strip tubes or microcentrifuge tubes
  • Conical centrifuge tubes

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

Product Notes

  1. 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.

  2. For Reagents Supplied:

    If you need dilution buffer in addition to what is provided in the kit, it can be purchased separately.

Protocols, Manuals & Usage

Protocols

  1. Where can I find protocols for using the NEBNext Library Quant Kit?

Manuals

The Product Manual includes details for how to use the product, as well as details of its formulation and quality controls.

Application Notes

FAQs & Troubleshooting

FAQs

  1. How are the new 6 standards different from the old 4 standards?
  2. What are the advantages of the qPCR-based NEBNext Library Quant Kit compared to other methods of quantitating NGS libraries?
  3. What libraries can be quantitated with the Quant Kit?
  4. How much should I dilute my library to run in the Quant Kit?
  5. Can I run the NEBNext Library Quant Kit on my qPCR machine?
  6. Which ROX should I use for my qPCR machine?
  7. Are 4 standards enough for a qPCR quantitation?
  8. Does the library quant require a size correction?
  9. What other materials/information do I need to run the kit?
  10. How many libraries can I quantitate at a time?
  11. How long is a run of the Quant Kit?
  12. Do you provide any analysis tools to help with the quantitation?
  13. Can I see adaptor dimers as a measure of library quality?

Quality, Safety & Legal

Quality Assurance Statement

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.

Specifications

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]

Certificate Of Analysis

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]

Safety DataSheets

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

Legal and Disclaimers

Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email 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.

New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.

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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