2.5. Challenges of AIR Profiling

Studies show that healthy individuals have at least 100-200 million different TCR and BCR clonotypes circulating in peripheral blood, and about 1-10% of these clonotypes are shared between individuals [15-17]. As a result, comprehensive characterization of the complete immune repertoire would require at least 10 billion lymphocyte cells (e.g., from leukapheresis blood samples) which is not practical for most applications. Therefore, comprehensive AIR profiling is more challenging than conventional RNA-seq due to the enormous complexity of different clonotype sequences (100-200 million) compared to the number of protein-coding genes (around 20,000).

AIR profiling data shows a long-tailed clonotype distribution, with most reads corresponding to low-abundant clonotypes (present at single-cell levels), resulting in reduced efficiency of detecting medium-abundant clonotypes only a few molecules may represent in the RNA sample. Therefore, with a limited amount of total RNA, we can reliably detect only the most abundant (high- to medium-abundant) clonotypes while losing a significant fraction of low-abundant clonotypes, which are present at a single mRNA molecule level (Fig. 13). For biological samples with a very small number of lymphocytes (e.g., specific tumor samples, some immune cell fractions, FFPE, etc.) or single-cell AIR assays, comprehensive quantitation and detection of both high and medium-abundant clonotypes represent a severe technical challenge and in most cases is not feasible.

Additionally, the cost of NGS sequencing can be a challenge. When working with a large number of samples, if the goal of the experiment is to profile the most frequent clonotypes, you can run more samples in the flow cell with lower reads per sample, as shown in the table below:

For detailed instructions for sequencing, please refer to the Product User Manual