Product Class: Kit

NEBNext® Immune Sequencing Kit (Mouse)


Catalog #E6330

Product Introduction

With the NEBNext® Immune Sequencing Kit (Mouse), sequence the full-length immune gene repertoires of B cells and T cells. Profile somatic mutations across all relevant contexts (e.g., V, D, and J segments and isotypes IgM, IgD, IgG, IgA, and IgE) with improved sequence accuracy. Characterize BCR light, BCR heavy, TCRα, TCRβ, TCRγ and TCRδ chains. This kit includes UMIs for source-molecule identification.

  • Unlock the immune system’s complexity with a deeper analysis of receptor sequences
  • Enrich for and sequence both B cell receptors (BCR) and T cell receptors (TCR)
  • Generate full-length immune gene repertoires of B and T cells
  • Accurately quantify transcripts with unique molecular identifiers (UMIs)
  • Analyze data using a bioinformatic workflow based on the open-source pRESTO toolkit (tutorial)

Note: We have a separate kit available for immune repertoire sequencing of Human samples.

Product Information

Description

The NEBNext Immune Sequencing Kit (Mouse) enables exhaustive profiling of somatic mutations in the full-length immune gene repertoires of B cells and T cells, via the expression of complete antibody chains. This includes modular primer sets, providing information for complete V, D, and J segments and full isotype information analysis (IgM, IgD, IgG, IgA and IgE), and TCRα, TCRβ, TCRγ and TCRδ chain characterization. A unique, UMI-based mRNA barcoding process allows PCR copies derived from an individual molecule to be converted to a consensus sequence. This improves sequence accuracy and eliminates PCR bias. Based on the open-source pRESTO toolkit, a workflow is available via the Galaxy platform to enable robust bioinformatic analysis locally or in a cloud environment. A workflow tutorial ensures that users who are not familiar with Galaxy can successfully use the analysis workflow,

Immune repertoire sequencing is frequently used to analyze immune responses, both current and distant. Areas of particular interest include characterization of autoimmune diseases, oncology, discovery of neutralizing antibodies against infectious disease, tumor-infiltrating lymphocytes and use as a tool to study residual disease. Recent improvements in read lengths and throughputs of next-generation sequencing (NGS) platforms have resulted in a rise in the popularity of immune repertoire sequencing. 

Defining features of the NEBNext Immune Sequencing method include:

  • Generation of full-length variable sequences (including isotype information), allowing downstream antibody synthesis and functional characterization not possible with approaches sequencing only the CDR3 region
  • Eliminated use of variable region primers, reducing primer pool complexity and realizing unbiased and simultaneous recovery of B cell and T cell receptor transcripts. 
  • Minimized PCR bias and improved sequencing accuracy by allowing a consensus to be generated from duplicate sequencing reads originating from the same transcript; UMIs enable accurate quantitation of each clone present in the sample. 
  • Optimized high target-capture efficiency for immune repertoire sequencing and analysis from sub-microgram quantities of total RNA.


Figure 1: Simplified representation of the structure of an antibody or TCR




Figure 2: Workflow





Figure 3: Utilization of Unique Molecular Identifier (UMI) provides superior accuracy of clonotype detection 



Mouse TCR clonotype frequency comparison between analysis with or without UMI annotation. The top 100 highly expressed clones were ranked by clonotype frequency detected with UMI. Clonotype frequency correlation on overlapped clonotypes between technical replicates using UMI or without UMI for analysis. Each bar or circle represents a unique clone from total read analysis, or filtered using UMI. The use of UMI allows for absolute quantitation of starting RNA molecules present in the original sample. When ranking the clones from most abundant clone to least abundant, the data based on total reads without UMI annotation results in PCR or sequencing amplification bias that can mislead interpretation of clonotype frequency in the samples. The use of UMI corrects the bias by generating absolute quantitation of RNA present in the starting sample. Use of UMI also produces better correlation between replicates, especially for low input. Each library was down sampled to 500,000 reads. 




Figure 4: The NEBNext Immune Sequencing Kit (Mouse) is able to generate both mouse BCR and mouse TCR libraries in one tube 



Mouse BCR+TCR libraries were constructed from 10, 100 and 1,000 ng mouse spleen total RNA (Takara Bio #636605) with replicates for each input. Libraries were downsampled to 1,000,000 reads for all the libraries. pRESTO tools were used for quality filtering of reads, sequence assemble, and generation of consensus sequence based on UMIs. V, D and J assignment was done using MiGMAP. (A) Number of clonotypes detected for each mouse spleen  total RNA input. (B) B cell gene heavy chain (IGH) and light chain (IGK and IGL) and T cell gen alpha chain (TRA), beta chain (TRB), delta chain (TRD) and gamma chain (TRG) percentages in each library.




 
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
  • E6330S     Multi-temperature    
     

    NEBNext® Immune Sequencing Kit RT and PCR Reagents

    		 E6325-1 -20 1 x 24 reactions 24 reactions
      NEBNext® IS 4 X RT Buffer E6321AVIAL -20 1 x 0.12 ml 4 X
      NEBNext® Cell Lysis Buffer   E6322AVIAL -20 1 x 0.012 ml Not Applicable
      dNTP Solution Mix E6323AVIAL -20 1 x 0.108 ml 10 mM
      NEBNext® IS RT Primer E6324AVIAL -20 1 x 0.024 ml 10 µM
      NEBNext® IS TS Oligos E6326AVIAL -20 1 x 0.024 ml 30 µM
      NEBNext First Strand Synthesis Enzyme Mix E7761AVIAL -20 1 x 0.048 ml Not Applicable
      Q5® Hot Start High-Fidelity DNA Polymerase E6327AVIAL -20 1 x 0.06 ml 0.002 units/ml
      Q5® Reaction Buffer E6328AVIAL -20 1 x 0.6 ml 5 X
      NEBNext® IS PCR2 Universal Primer E6329AVIAL -20 1 x 0.072 ml 10 µM
      0.1X TE E6331AVIAL -20 1 x 2.28 ml Not Applicable
      Nuclease-free Water E6332AVIAL 4 1 x 1.41 ml Not Applicable
      NEBNext® i701 Primer E6333AVIAL -20 1 x 0.008 ml 10 µM
      NEBNext® i702 Primer E6334AVIAL -20 1 x 0.008 ml 10 µM
      NEBNext® i703 Primer E6335AVIAL -20 1 x 0.008 ml 10 µM
      NEBNext® i704 Primer E6336AVIAL -20 1 x 0.008 ml 10 µM
      NEBNext® i705 Primer E6337AVIAL -20 1 x 0.008 ml 10 µM
      NEBNext® i706 Primer E6338AVIAL -20 1 x 0.008 ml 10 µM
      NEBNext® i501 Primer E6339AVIAL -20 1 x 0.036 ml 10 µM
      NEBNext® i502 Primer E6340AVIAL -20 1 x 0.036 ml 10 µM
      NEBNext® i503 Primer E6341AVIAL -20 1 x 0.036 ml 10 µM
      NEBNext® i504 Primer E6342AVIAL -20 1 x 0.036 ml 10 µM
      NEBNext® Immune Sequencing Kit cDNA Purification Reagents E6325-2 4 1 x 24 reactions 24 reactions
      Streptavidin Magnetic Beads E6355AVIAL 4 1 x 0.36 ml Not Applicable
      NEBNext®  IS 2X Bind/Wash Buffer E6356AVIAL 4 1 x 11.28 ml 2 X
      0.1% Tween E6357AVIAL 4 1 x 2.96 ml 0.1 %
      NEBNext® Sample Purification Beads E6358AVIAL 25 1 x 2.76 ml Not Applicable
      NEBNext® Immune Sequencing Primers (Mouse)  E2629S -20 1 x 24 reactions 24 reactions
      NEBNext® IS BCR Primers (Mouse) E2624AVIAL -20 1 x 0.048 ml 1 µM
      NEBNext® IS TCR Primers (Mouse) E2625AVIAL -20 1 x 0.048 ml 1 µM
  • E6330L     Multi-temperature    
     

    NEBNext® Immune Sequencing Kit RT and PCR Reagents

    		 E6325-3 -20 1 x 96 reactions 96 reactions
      NEBNext® IS 4 X RT Buffer E6321AAVIAL -20 1 x 0.48 ml 4 X
      NEBNext® Cell Lysis Buffer   E6322AAVIAL -20 1 x 0.048 ml Not Applicable
      dNTP Solution Mix E6323AAVIAL -20 1 x 0.432 ml 10 mM
      NEBNext® IS RT Primer E6324AAVIAL -20 1 x 0.096 ml 10 µM
      NEBNext® IS TS Oligos E6326AAVIAL -20 1 x 0.096 ml 30 µM
      NEBNext First Strand Synthesis Enzyme Mix E7761AAVIAL -20 1 x 0.192 ml Not Applicable
      Q5® Hot Start High-Fidelity DNA Polymerase E6327AAVIAL -20 1 x 0.24 ml 0.002 units/ml
      Q5® Reaction Buffer E6328AAVIAL -20 2 x 1.2 ml 5 X
      NEBNext® IS PCR2 Universal Primer E6329AAVIAL -20 1 x 0.288 ml 10 µM
      0.1X TE E6331AAVIAL -20 1 x 9.2 ml 0.1 X
      Nuclease-free Water E6332AAVIAL 4 1 x 5.76 ml Not Applicable
      NEBNext® i701 Primer E6333AAVIAL -20 1 x 0.032 ml 10 µM
      NEBNext® i702 Primer E6334AAVIAL -20 1 x 0.032 ml 10 µM
      NEBNext® i703 Primer E6335AAVIAL -20 1 x 0.032 ml 10 µM
      NEBNext® i704 Primer E6336AAVIAL -20 1 x 0.032 ml 10 µM
      NEBNext® i705 Primer E6337AAVIAL -20 1 x 0.032 ml 10 µM
      NEBNext® i706 Primer E6338AAVIAL -20 1 x 0.032 ml 10 µM
      NEBNext® i707 Primer E6347AAVIAL -20 1 x 0.032 ml 10 µM
      NEBNext® i708 Primer E6348AAVIAL -20 1 x 0.032 ml 10 µM
      NEBNext® i709 Primer E6349AAVIAL -20 1 x 0.032 ml 10 µM
      NEBNext® i710 Primer E6352AAVIAL -20 1 x 0.032 ml 10 µM
      NEBNext® i711 Primer E6353AAVIAL -20 1 x 0.032 ml 10 µM
      NEBNext® i712 Primer E6354AAVIAL -20 1 x 0.032 ml 10 µM
      NEBNext® i501 Primer E6339AAVIAL -20 1 x 0.144 ml 10 µM
      NEBNext® i502 Primer E6340AAVIAL -20 1 x 0.144 ml 10 µM
      NEBNext® i503 Primer E6341AAVIAL -20 1 x 0.144 ml 10 µM
      NEBNext® i504 Primer E6342AAVIAL -20 1 x 0.144 ml 10 µM
      NEBNext® i505 Primer E6343AAVIAL -20 1 x 0.144 ml 10 µM
      NEBNext® i506 Primer E6344AAVIAL -20 1 x 0.144 ml 10 µM
      NEBNext® i507 Primer E6345AAVIAL -20 1 x 0.144 ml 10 µM
      NEBNext® i508 Primer E6346AAVIAL -20 1 x 0.144 ml 10 µM
      NEBNext® Immune Sequencing Kit cDNA Purification Reagents E6325-4 4 1 x 96 reactions 96 reactions
      Streptavidin Magnetic Beads E6355AAVIAL 4 1 x 1.44 ml Not Applicable
      NEBNext®  IS 2X Bind/Wash Buffer E6356AAVIAL 4 1 x 45.2 ml 2 X
      0.1% Tween E6357AAVIAL 4 1 x 11.8 ml 0.1 %
      NEBNext® Sample Purification Beads E6358AAVIAL 25 1 x 11.1 ml Not Applicable
      NEBNext® Immune Sequencing Primers (Mouse)  E2629L -20 1 x 96 reactions 96 reactions
      NEBNext® IS BCR Primers (Mouse) E2624AAVIAL -20 1 x 0.192 ml 1 µM
      NEBNext® IS TCR Primers (Mouse) E2625AAVIAL -20 1 x 0.192 ml 1 µM

Properties & Usage

Protocols, Manuals & Usage

Protocols

  1. Where can I find protocols for using the NEBNext Immune Sequencing Kit (Mouse)? (NEB #E6330)

Manuals

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

Usage & Guidelines

FAQs & Troubleshooting

FAQs

  1. What kind of samples can be used with the NEBNext Immune Sequencing Kit (Mouse) (NEB #E6330)?
  2. Will the NEBNext Immune Sequencing Kit (Mouse) (NEB #E6330) work with non-mouse species?
  3. Can I use FFPE tissue with the NEBNext Immune Sequencing Kit (Mouse) (NEB #E6330)?
  4. What type of RNA extraction method should I use upstream of the NEBNext Immune Sequencing Kit (Mouse) (NEB #E6330)?
  5. What sequencing parameters should be used with these libraries?
  6. What sequencing depth is required for immune sequencing libraries?
  7. What barcodes should be sequenced together? 
  8. I don’t have a qPCR machine, can I still use NEBNext Immune Sequencing Kit (Mouse) (NEB #E6330)?
  9. How do I analyze the data generated from immune sequencing libraries?
  10. Do I need to perform the qPCR step every time?
  11. Do I need to gel extract my final library?
  12. What B-cell gene and T-cell gene specific primers are included in NEBNext Immune Sequencing Kit (Mouse) (NEB #E6330)? What is the format of the primers?
  13. Will NEBNext Immune Sequencing Kit (Mouse) (NEB #E6330) work with DNA?
  14. Will NEBNext Immune Sequencing Kit (Mouse) (NEB #E6330) provide heavy and light chain pairing information?

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]

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

This product is covered by one or more patents. 

This product is licensed for research and commercial use from Bio-Rad Laboratories, Inc., under U.S. Pat. Nos. 6,627,424, 7,541,170, 7,670,808, 7,666,645, and corresponding patents in other countries. No rights are granted for use of the product for Digital PCR or real-time PCR applications, with the exception of quantification in Next Generation Sequencing workflows