Trends in Biotechnology
ReviewAdvanced Methodologies in High-Throughput Sequencing of Immune Repertoires
Section snippets
The Rapid Rise of High-Throughput Immune Repertoire Sequencing
The rapid rise of high-throughput immune repertoire sequencing (see Glossary) has led to unprecedented quantitative insight into lymphocyte diversity (Box 1) and adaptive immunity, leading to a new era of systems immunology. Here, we refer to this paradigm for T cells and B cells as T cell receptor (TCR)-seq and immunoglobulin (Ig)-seq, respectively 1, 2. In one of the first examples using TCR-seq, Robins et al. quantitatively measured human TCR diversity [3], while subsequent studies revealed
Improving Accuracy in Immune Repertoire Data
Sequencing errors and biases are generated in several ways [21]. In some cases, they can be mitigated by good laboratory practices, such as preventing degradation or contamination of the source material. However, there are inherent problems due to the underlying dependence of molecular biology-based techniques for NGS. These problems arise because the amount of starting material (genomic DNA or total RNA) is often limited and of low purity, thus requiring reverse transcriptases and/or DNA
High-Throughput Pairing of Variable Regions
Initial studies in immune repertoire sequencing relied on bulk populations of lymphocytes. However, this approach is unable to recover the pairing of variable regions because they are expressed as unique transcripts from separate chromosomes. However, having additional functional information about variable region pairing is desirable because it can be used for a variety of applications, such as mAb discovery, vaccine profiling, and analysis of tumor-infiltrating lymphocytes. This kind of
Concluding Remarks and Future Directions
The advanced methods in immune repertoire sequencing described above demonstrate the capability to obtain accurate (error and bias-corrected) and variable region paired data. These capabilities have set a new standard for the field of immune repertoire sequencing. While the technologies established for error and bias correction were largely focused on ex vivo biological repertoires, similar approaches may be adapted to synthetic repertoires (e.g., phage, bacteria, and yeast display 68, 69).
Glossary
- 5′ Rapid amplification of cDNA ends (5′ RACE)
- a library preparation protocol designed to capture mRNA sequences when either the 5′ end is unknown or the usage of a multiplex forward primer set is not desirable. In a typical protocol, mRNA is reverse transcribed to cDNA using an antisense gene-specific primer. After cDNA synthesis, homopolymeric tails are added to the 3′ end of the transcript via terminal deoxynucleotidyl transferase (TdT). The resulting amplicon can now be amplified using
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