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Repertoire sequencing

A next-generation understanding of immune response

  • The adaptive immune system is a miraculous phenomenon that plays a central role in human health. It is one of the most dynamic systems in the human body, involved in a wide range of homeostatic activities. As a consequence, its function and dysfunction plays a role in most, if not all, human disease states.

  • Key players in the adaptive immune response are B and T cells; highly specialized cells bearing a unique receptor to recognize an astonishing variety of pathogens, as well as “altered self-cells” as seen in cancer. This highly diverse collection of B or T cell receptors is referred to as an immune repertoire and entails a wealth of information about ongoing immune responses.

  • The majority of the T cell receptors (TCRs) are composed of an alpha chain (TRA) and a beta chain (TRB). B cell receptors (also called Immunoglobulins) are composed of a heavy chain (IgH) and a light chain (IgL).

  • V(D)J recombination is the sophisticated and elegant process of genetic recombination that gives rise to the diversity of B and T cell repertoires. In a nearly random fashion, variable (V), joining (J) and sometimes diversity (D) gene segments are rearranged, with additionally a variable number of non-templated nucleotides is inserted at the gene-junctions. The resulting number of possible rearrangements is immense and constitutes a defining feature of the adaptive immune system.

  • The immune system is precisely that: a system. Unlike traditional technologies such as Sanger, high-throughput sequencing allows for sequencing millions of receptors in parallel, providing unprecedented opportunities to truly capture the dynamic landscape of immune responses.

workflows

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.

UMIs
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

Since the 19th century, immunologists have invested great efforts in elucidating the large functional heterogeneity of immune cell receptors. The emergence of single-cell analysis has greatly increased the resolution with which we can interrogate the spatial organization, dynamics, evolution and interaction of immune cell assemblies. This nuanced understanding of cellular network improves our ability to manipulate the immune system and fully exploit its unique therapeutic potential.