AIRR Community Meeting VI: Exploring New Frontiers

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Objective: B-cell receptor (BCR) repertoire profiling is increasingly used in health and pathogenic contexts with the goal of biomarker discovery. However, current sequencing technologies are limited in their ability to generate data accurately and reproducibly for all BCR isotypes. To overcome these limitations, we have developed a new kit to accurately profile all heavy (A, D, E, G, M) and light-chain (K, L) isotypes—an end-to-end solution, from library preparation to streamlined data analysis. Here we present data on an updated approach for efficient and high-throughput BCR repertoire profiling of human samples.
Methods: Libraries were prepared from human peripheral blood cells (10 ng–1 μg total RNA) or from B-cells (1 ng–100 ng total RNA) using our new human BCR repertoire profiling kit (~2.5 hours hands-on time). Prepared libraries were then analyzed on the Illumina® Miseq® benchtop sequencer using 300-bp paired-end reads.
Results: For each library, >90% of sequencing reads were on- target while the most highly represented clonotype was found to remain consistent among technical duplicates across a range of input amounts. In comparison to the previous version of our BCR-sequencing kit, the new approach enabled a ~4x increase in total clonotype count observed across various RNA inputs. Furthermore, a sensitivity assay demonstrated that B-cell RNA corresponding to a single clonotype could be detected above background levels when spiked into input total RNA at a relative concentration of 0.001%.
Conclusions: Our new human BCR repertoire profiling kit was found to accurately and reproducibly profile B-cell clones and provide information on the diversity of BCR repertoire in human samples.

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Non-human primates (NHP) such as the rhesus macaque (Macaca mulatta) have long been key translational models in biomedical research because of their genetic and physiological similarity to humans. Studies using rhesus macaques have contributed significantly to our understanding of T-cell responses to vaccines, cancer, and infectious diseases. More recently, these NHP have emerged at the forefront of COVID-19 vaccine research.

Increasingly complex information can now be gleaned from immune system processes. High-throughput TCR sequencing (TCR-seq) profiles T-cell responses in exquisite detail. A comprehensive understanding of immune responses in such a closely related organism as the rhesus macaque would be a significant advance in science.

Numerous tools exist for performing TCR-seq in human samples, but equivalent tools for rhesus samples have been lacking. Because rhesus macaques often serve as surrogates in the lead-up to human studies, there is an industry need for a complete TCR-seq solution for these NHP samples.

Due to strong species homology, our SMARTer Human TCR a/b Profiling Kit v2 (TCRv2) can generate high-quality TCR sequencing libraries using human or rhesus macaque RNA.