Repertoire sequencing

A next-generation understanding of immune response

  • The adaptive immune system is critical in safeguarding our everyday health. It helps ward off infections and even takes the fight to cancer. It is one of the most dynamic systems in the human body and is involved in a wide range of homeostatic activities. Our understanding of its function and dysfunction are crucial to the development of new drugs and therapeutics.

  • Key players in the adaptive immune response are B and T cells. These cells bear unique receptors which enable them to recognize an astonishing variety of pathogens, as well as “altered-self cells” in certain types of cancer. The collection of B and T cell receptors is referred to as an “immune repertoire” and contains a wealth of information about immune responses, past and present.

  • T cells express T cell receptors (TCRs) in the form of an alpha and a beta chain, or a gamma and a delta chain, while B cell receptors (also called immunoglobulins) are composed of a heavy and a light chain (lambda or kappa).

  • The diversity of B and T cell repertoires stems from the random recombination of variable (V), diversity (D), and joining (J) genes. Additional complexity is added by the insertion of non-templated nucleotides at the gene junctions. The resulting number of possible receptor rearrangements is immense and is a defining characteristic of the adaptive immune system.

  • Unlike traditional technologies such as Sanger, high-throughput sequencing allows for the characterization of millions of receptors in parallel, providing unprecedented opportunities to capture the dynamic landscape of immune responses.


Below you find a typical repertoire sequencing workflow. Click on one of the steps to get more information. 

Understanding the immune system
5. Deriving insights

Understanding the immune system is central to several of the most important and therapeutically relevant developments in modern medicine. Check out our application page to learn about the added value of immune repertoire analysis at all stages of biomedical research. 

Repertoire sequencing experiments
4. Bioinformatics analysis

Repertoire sequencing experiments usually generate thousands to millions of sequencing reads per sample. Powerful and specialized bioinformatics pipelines are warranted to accurately interpret large-scale repertoire data and extract biologically relevant insights. Check out our data analysis page to learn more about the analysis of repertoire sequencing data.  

Sequencing depth
3. High throughput sequencing

The sequencing depth influences the possible profiling resolution: superficial ‘screening’ saves costs and can be used to identify abundant clonotypes, whereas deep sequencing, albeit more expensive, provides more confidence on the less abundant clonotypes and is therefore more suited for repertoire-wide analyses such as diversity.

Sequencing platforms
3. High throughput sequencing

A number of sequencing platforms are available that differ in features such as read length, sequencing depth, cost, and run time. Illumina is the most commonly used platform, as it offers the highest read depth and supports paired-end read sequencing, ensuring superior read quality.

2. Library preparation

Unique molecular identifiers (UMIs) are short sequences or molecular “tags” that can be added during library preparation to mark individual molecules and help to control for PCR errors. The length and composition of the UMI affect the analysis and should be designed carefully. Shorter UMIs lead to more non-unique attachment, where the same UMI sequence gets attached to different template molecules. Longer UMIs increase the risk of primer dimer formation and have higher chances of error during amplification and sequencing. A UMI length between 8 and 12 nucleotides is currently considered optimal.

Material selection
2. Library preparation

There are two starting materials that can serve as the initial template to sequence immune repertoires: genomic DNA (gDNA) and messenger RNA (mRNA). Choosing the appropriate template is an important step in the repertoire sequencing pipeline and depends on the research goals, as well as the available sample material. 

The sample type
1. Sample collection

The sample type (e.g. blood, tissue or formalin-fixed paraffin-embedded material) influences the efficiency of the repertoire analysis and therefore the type of biological information that you recover. Word on lymphocyte isolation. 

Single-cell sequencing

BCRs and TCRs are composed of multiple chains that together form the antigen-binding site; having the information on paired alpha and beta for TCR or heavy and light for BCR is essential for the characterization of therapeutic molecules. Until recently, the majority of high-throughput sequencing approaches produced unpaired repertoires, as generating paired information requires obtaining data at the individual cell level. Recently, techniques that isolate individual cells, have demonstrated the potential for obtaining pairing information in a high-throughput manner. This nuanced understanding of the immune repertoire improves our ability to manipulate the immune system and fully exploit its unique therapeutic potential.