Overview of methodologies for T-cell receptor repertoire analysis

Abstract

Background: The T-cell receptor (TCR), located on the surface of T cells, is responsible for the recognition of the antigen-major histocompatibility complex, leading to the initiation of an inflammatory response. Analysing the TCR repertoire may help to gain a better understanding of the immune system features and of the aetiology and progression of diseases, in particular those with unknown antigenic triggers. The extreme diversity of the TCR repertoire represents a major analytical challenge; this has led to the development of specialized methods which aim to characterize the TCR repertoire in-depth. Currently, next generation sequencing based technologies are most widely employed for the high-throughput analysis of the immune cell repertoire.

Results: Here, we report on the latest methodological advancements in the field by describing and comparing the available tools; from the choice of the starting material and library preparation method, to the sequencing technologies and data analysis. Finally, we provide a practical example and our own experience by reporting some exemplary results from a small internal benchmark study, where current approaches from the literature and the market are employed and compared.

Conclusions: Several valid methods for clonotype identification and TCR repertoire analysis exist, however, a gold standard method for the field has not yet been identified. Depending on the purpose of the scientific study, some approaches may be more suitable than others. Finally, due to possible method specific biases, scientists must be careful when comparing results obtained using different methods.

Keywords: CDR3; Clonotype; Immune repertoire; Immunogenetics; Immunogenomics; T-cell receptor (TCR); TCR profiling; TCR repertoire; Target sequencing; Vdj.

Fig. 1

Interaction between an antigen presenting cell (APC) and a T cell, and V(D)J recombination. a Interaction between the antigen–major histocompatibility complex (MHC) and the αβ T-cell receptor (TCR). b V(D)J recombination: During T cell development, the loci that encode T-cell receptor α and β-chains are rearranged. For both loci, variable (V) and joining (J) gene segments, and an additional diversity (D) gene segment for the β-chain, are recombined to form the final rearranged TCR DNA sequence. This process also involves the deletion and insertion of nucleotides at the V-D, D-J and V-J junctions (not shown). Following transcription, the sequence between the recombined V (D)J regions and the gene encoding the constant (C) region is removed by splicing. The complementarity-determining region (CDR) 3 is encoded by the V (D) J junction, whereas the CDR1 and CDR2 loops are encoded within the germline V gene

Fig. 2

Exemplary workflow of three principal methodologies for TCR library preparation. The figure depicts a simplified workflow of the library preparation procedure using multiplex PCR, targeted in-solution enrichment and 5’RACE-switch-oligo nested PCR. Multiplex PCR is suitable for both RNA and gDNA sequencing. Samples undergo cDNA synthesis and 1 or more PCR steps followed by adaptor ligation and sequencing. While the forward primers for cDNA synthesis are designed to cover all known V genes for both starting materials, the location and number of the reverse primers differs, due to introns in DNA. Target enrichment, also applicable to both gDNA and RNA, is preceded by a standard library preparation including fragmentation for gDNA or mRNA purification for RNA, followed by end-repairing, A-tailing and finally adaptor ligation. The enrichment of target sequences is then performed using RNA baits complementary to the sequence of interest. The RNA baits hybridize with molecules in the library, which are then retrieved using magnetic beads and can undergo further amplification before sequencing. Nested PCR based on the 5’RACE and switch-oligo approach (only for RNA) makes use of the incorporation of an adaptor molecule at the 5′ end of the cDNA during cDNA synthesis. The forward primer for a subsequent PCR is designed to bind to the 5′ adaptor sequence, while the reverse primer is designed to bind to the C-region of the transcript. Hence, only one primer pair is required to cover the complete spectrum of possible V genes. Subsequent nested PCRs performed in the same fashion may increase outcome specificity. Finally, adaptor ligation is performed. The procedures showed in this picture constitute only an example of the different available methods

Fig. 3

Venn diagram showing the overlap between the most abundant TCR sequences detected by each method. The threshold was defined by the method which detected fewest TCR species (UMI-corrected 5’RACE-based PCR). The diagrams show the number and relative frequency of TCR sequences detected by only one up to all four methods. Sequences that were found only by one method may still be detected by other methods, but may not appear in the most abundant species and are thus not represented here. Data are shown for one patient for both (a) α and (b) β chains

Fig. 4

Alpha chain Variable (V) gene usage among methods and replicates. The heat map shows the gene usage proportion inside each sample for the V genes listed at the bottom on the figure. Each of the three parts of the heat map is representative of one of the methods. The samples are described by the patients “P1/2”, the method “iRepertoire/BGI/5’RACE/5’RACE + UMI” and the replicate “1 or 2” or “0” in case of BGI which does not include replicates. The figure was generated using the “ggplot2” R package

Fig. 5

Dissimilarity dendrogram for alpha chain. The distance between patients, methods, and replicates, calculated using the Morisita index, is represented by the dendrograms. The samples are described by the patients “P1/2”, the method “iRepertoire/BGI/5’RACE/5’RACE + UMI” and the replicate “1 or 2”. The figure was generated using the “ggplot2” and the “ggdendro” R packages