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NEBNext® Immune Sequencing Kit (Mouse)

Catalog # Concentration Size List Price Quantity Your Price
E6330L 96 reactions $9,031.00
$8,127.90
E6330S 24 reactions $2,635.00
$2,371.50
Catalog # Size List Price Your Price
E6330L 96 reactions $9,031.00
$8,127.90
E6330S 24 reactions $2,635.00
$2,371.50
Catalog #
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*On-line ordering is for Canadian customers only. Web pricing is applicable only to orders placed online at www.neb.ca

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.

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    Using large scale B cell receptor repertoire sequencing to further understand human autoimmune disease

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.




 
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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.

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

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