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,
Next Generation Sequencing Library Preparation,

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]

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