Next-generation sequencing (NGS) has opened the door for comprehensive analysis of the adaptive immune repertoire (IR). However, several aspects of sampling can affect the experimental output. Therefore, it’s important to consider three aspects of the experimental design prior to running the assay:
Archer® Immunoverse™ TCR and BCR kits use RNA as the input material. RNA has several advantages over genomic DNA, making it the preferred material for exploring functional aspects of specific TCR and/or BCR variants. Briefly, RNA interrogation allows for:
For more detailed information on the advantages of Archer Anchored Multiplex PCR (AMP™) chemistry using RNA input for IR sequencing, read the blog.
T and B cell content varies by sample source and can thus affect the ability to generate high-quality and unbiased sequencing reads. For example, the T cell content is greater in peripheral blood mononuclear cell (PBMC) samples than in whole blood samples (see table below). The table below shows typical cell concentrations based on cell type and source, which can be a useful resource when determining the scope and depth of your assay requirements.
|Cell type||Sample source||Cell content||Frequencies|
T cell receptor (TCR) and B cell receptor (BCR) mRNA levels in T and B cells, respectively, should also be considered when designing your assay. TCR mRNA levels in T cells show little variation from cell-to-cell or between subtypes. For example, quiescent T cells, which include naïve and memory cells, express nearly identical TCR mRNA levels, while effector T cells may only fluctuate by plus or minus two-fold from clone-to-clone. On the other hand, BCR mRNA levels in plasma B cells can be 30-fold greater than in memory or naïve B cells (Turchaninova, et al., 2016; Schrum, et al., 2003; Cho, et al., 1999). Because of this discrepancy in BCR expression levels among B cell subtypes, the cell type of interest should be isolated via sorting (e.g., FACS or magnetic bead separation) prior to analysis to greatly reduce erroneous clonotype variation.
NGS is a powerful method to investigate the immune repertoire, and using RNA as the starting material has several advantages over DNA. Plus, sample-specific cell distributions and cell type-specific mRNA variations should be considered when analyzing the functional immune repertoire to minimize erroneous clonotyping.
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