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 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.
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.
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.
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.
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 (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.
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.
Watch our new webinar and learn to carry out your own high-throughput Rep-Seq data analysis even without bioinformatics skills.