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Home NEBNext® Immune Sequencing Kit (Human)

NEBNext® Immune Sequencing Kit (Human)

With the NEBNext® Immune Sequencing Kit (Human), 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 and TCRβ 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 Mouse samples.

Featured Video

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

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

Catalog # Concentration Size
E6320S Not Applicable 24 reactions
E6320L Not Applicable 96 reactions
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  • Product Information

    The NEBNext Immune Sequencing Kit (Human) 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 and TCRβ 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 



    Clonotype frequency comparison between analysis with or without UMI annotation for human TCR libraries from 10 ng and 100 ng T cell Total RNA. The top 100 highly expressed clones were ranked by clonotype frequency detected with UMI. Clonotype frequency correlation on overlapped clonotypes was plotted between technical replicates using UMI or without UMI for TCR libraries from 10 ng and 100 ng T cell Total RNA. 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 were down sampled to 500,000 reads.




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



    Human BCR+TCR libraries were constructed from 1 μg, 100 ng and 10 ng human PBMC Total RNA (Takara Bio #636592) with replicates for each input. Libraries were downsampled to 950,000 reads for all the libraries. pRESTO tools were used for quality filtering of reads, sequence assemble, and generation of consensus sequence UMIs. V, D and J assignment was done using MiGMAP. (A) Number of clonotypes detected for each human PBMC Total RNA input. (B) B cell chains and T cell chains percentages in each library.




     
    • Kit Components

      Kit Components

      The following reagents are supplied with this product:

      NEB # Component Name Component # Stored at (°C) Amount Concentration
      • E6320S     Multi-temperature    
          NEBNext® Immune Sequencing Kit  E6325-1 -20    
          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 mM
          NEBNext® IS TS Oligos E6326AVIAL -20 1 x 0.024 ml 30 mM
          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 mM
          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  E6325-2 4    
          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 (Human) 24 rxn E2628S -20    
          NEBNext® IS BCR Primers (Human) E2626AVIAL -20 1 x 0.048 ml 1 µM
          NEBNext® IS TCR Primers (Human) E2627AVIAL -20 1 x 0.048 ml 1 µM
      • E6320L     Multi-temperature    
          NEBNext® Immune Sequencing Kit  E6325-3 -20    
          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 mM
          NEBNext® IS TS Oligos E6326AAVIAL -20 1 x 0.096 ml 30 mM
          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 mM
          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  E6325-4 4    
          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 (Human) 24 rxn E2628L -20    
          NEBNext® IS BCR Primers (Human) E2626AAVIAL -20 1 x 0.192 ml 1 µM
          NEBNext® IS TCR Primers (Human) E2627AAVIAL -20 1 x 0.192 ml 1 µM
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