Introduction to Immune Repertoire Sequencing

• Posted by Samantha James June 23, 2020 Filed in Arts & Culture 344 views
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  The concept of Immune Repertoire

  Immune Repertoire (IR) refers to the sum of all functionally diverse B cells and T cells in an individual's circulatory system at any given time. To study the Immune Repertoire, it is necessary to determine the gene or RNA molecular sequence of BCR and TCR in the immune system.

  There are many factors that affect the composition of the immune group library. Such as age, disease, immune memory, immunodeficiency genetic diseases, vaccines, organ transplantation, cancer treatment, etc.

  According to the different sources of lymphocyte DNA, the research on the Immune Repertoire is divided into the following categories: healthy people generally use the method of drawing peripheral blood; special populations such as leukemia people or patients with Leukemia Cell Minimal Residual Disease (MRD), bone marrow blood is also used as the source of lymphocyte DNA in order to further study the changes in their immunological pool; newly born umbilical cord blood can be used by the researcher to study the connection and difference of IR at early birth and the mother; for animals, especially small animals, the spleen is also a common source of DNA from lymphocytes.

  The way to use saliva and urine as the source of lymphocyte DNA.

  B cell receptor (BCR)

  B cell receptor (BCR), also known as immunoglobulin (IG), is a protein molecule secreted by B cells and can bind to antigen. Mature B cells can secrete IG outside the cells to neutralize the antigen. The immunoglobulin is composed of two heavy chainsand two light chainsconnected by disulfide bonds. The heavy chain (IGH) is recombined from Variable (V), Diversity, Joining (J) and Constantgene fragments; the light chain (IGL) is only recombined from V-J-C. The heavy chain (H chain) is divided into V region, C region, transmembrane region and cytoplasmic region; while the light chain (L chain) is only V and C. The V region is composed of two domains, VH and VL, each having three CDRs, namely CDR1, CDR2, and CDR3.

  The key part that determines the recognition antigen of B lymphocytes is the most diverse heavy chain CDR3 region. The diversity arises from the end of IGHV (51 functional gene fragments) -IGHD (23 functional gene fragments) -IGH] Front-end (6 functional gene fragments) gene rearrangement and non-template-dependent nucleotide insertion or cleavage and somatic high frequency mutation when VD and DJ are connected.

  T cell receptor (TCR)

  T cell receptor (TCR) is a membrane protein molecule secreted by T cells. Unlike BCR, TCR is only secreted on the cell surface and is not secreted. The TCR of a T cell can be composed of α chain, or composed of γ chain and δ chain. α chain and γ chain are recombined from the V-J-C gene fragment, and the β chain and δ chain are recombined from the V-D-J_C gene fragment, which is also similar to the heavy and light chains of BCR. The T lymphocytes involved in the specific immune response are mainly aβ T lymphocytes, accounting for 90-95% of peripheral T lymphocytes. TCRβ chain CDR3 plays a key role in antigen-specific recognition. It is a binding site directly with the antigen peptide. Its coding gene is located on human chromosome 7 (7q34), with a total length of 620kb and composed of more than 50 variable regions (V regions), 2 sets of upstream D gene fragments/downstream constant region (C region) gene fragments, and 6 to 7 J region gene fragments. These genes are not distributed continuously in the early development of T lymphocytes, and the original T is in the thymus. In the cell stage, the D-J region fragments are connected as DJ fragments; in the mature T lymphocyte stage, the V, D, J, and C region gene fragments are connected as VDJC fragments.

  It is speculated that the random combination and rearrangement of germline VDJC gene fragments and the rearrangement of TCR a/β chain and γ/δ chain V region gene sequences produce up to 105-1018 TCI clones, which constitute an extremely diverse T lymphocyte antigen Receptor library. Different T lymphocyte clones have different sequences or lengths of TCR-CDR3 genes, and different CDR3 polypeptide sequences are translated to reflect the functional status of T lymphocytes. When a specific immune response occurs, the TCR diversity changes, and oligoclonal or even monoclonal amplification occurs. With the regeneration of the thymus, a large number of original T lymphocytes proliferate, and the diversity of the TCR immune pool is restored and reconstructed.

  Diversity mechanism of BCR/TCR

  1. Diversification of different combinations of VDJ gene fragments;

  2. Random insertion and deletion when different gene fragments are connected cause diversification of connection, insertion and deletion of nucleotides in the V(D)J junction region; deletion, which is reflected in the complementarity determining region cluster CDR3 sequence diversity;

  3. Diversification of different heavy chain and light chain combinations;

  4. And random high-frequency mutations unique to B cell receptors.

  The process of studying Immune Repertoire

  1. Extract sample DNA/RNA: Design a test plan based on the problem to be studied, extract blood or tissue samples, isolate B cells or T cells, and then extract and purify DNA/RNA.

  2. Construct a sequencing library: add different amplification primers according to different experimental conditions for PCR amplification, thereby amplifying the immune gene to be sequenced, and finally construct a sequencing library.

  3. Sequencing: Use second-generation high-throughput sequencers, such as Roche 454, llumina Hi Seq2500, Illumina Mi Seq, etc. for sequencing.

  4. Informatics analysis: carry out information mining on the measured sequence data to discover the biological significance and results. Data analysis mainly includes data cleaning, quality control, data separation of different samples, sequence alignment, sequence annotation, and various downstream analyses.

  Immune Repertoire sequencing(IR-SEQ)

  Immune Repertoire sequencing (IR-SEQ) takes T/B lymphocytes as the research goal, and determines the B cell receptor (BCR) or T cell receptor (TCR) using multiple PCR technology or 5'RACE amplification. Diversity complementary decision region (CDR3 region), combined with high-throughput sequencing technology (HTS), comprehensively assess the diversity of the immune system, and dig deeper into the relationship between the immune repertoire and disease.

  Information analysis

  1. Basic data statistics

  l Data filtering, processing the original data to remove the connector contamination sequence and low-quality reads

  l Data construction, data splicing, elimination of sequencing background and effective data construction, data statistics, data output statistics, and composition and quality evaluation of sequencing data

  2. Data comparison analysis

  Alignment analysis, re-alignment with database (IMGT) V/D/] gene fragments, find the best V/D/J alignment result

  3. Sequence structure analysis

  l Analysis of CDR sequence composition and sequence base composition

  l Analysis of base insertions and deletions of CDR sequences

  l Translation of CDR-encoding sequences into amino acids and peptide chains

  4. Construction of Immune Repertoire library

  Construction of the expression profile of the IR library, statistics on the expression of the diversity of antibody library clones

  5. Difference analysis of Immune Repertoire library

  l Analysis of diversity differences between samples (Simpson coefficient, Shannon Wiener coefficient)

  l Analysis of clonal expression differences between samples (CDR3, V-D-J)

  l Analysis of clonal expression differences between grouped samples (CDR3, V-D-J)