Input considerations for TCR and BCR sequencing

By Kiri Burrow, Ph.D. on Mon Oct 2, 2017


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:

1. RNA as starting material

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:

  • Increased sensitivity
  • Greater functional relevance of IR status
  • Isotype assignment
  • IgHV somatic hypermutation status

For more detailed information on the advantages of Archer Anchored Multiplex PCR (AMP™) chemistry using RNA input for IR sequencing, read the blog.

2. T and B cell content varies by sample

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-cells Sorted >90%
PBMC 40-65% 0.5-2x106/mL
Whole blood 10-30% 0.5-2x106/mL
Lymphoid tissue 40-60%
Non-lymphoid tissue 1-15%
B-cells Sorted >90%
PBMC 1-15%
Whole blood 1-10% 0.5x106/mL

3. mRNA levels vary by cell type

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.

Summary

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.

References

Cho, B.K., Wang, C., Sugawa, S., Eisen, H.N. & Chen, J. Functional differences between memory and naive CD8 T cells. Proc. Natl. Acad. Sci. USA 96:2976-2981 (1999).

Schrum, A.G., Turka, L.A. & Palmer, E. Surface T-cell antigen receptor expression and availability for long-term antigenic signaling. Immunol. Rev. 196:7-24 (2003).

Turchaninova, M.A., Davydov, A., Britanova, O.V., Shugay, M., Bikos, V., Egorov, E.S., Kirgizova, V.I., Merzlyak, E.M., Staroverov, D.B., Bolotin, D.A., Mamedov, I.Z., Izraelson, M., Logacheva, M.D., Kladova, O., Plevova, K., Pospisilova, S. & Chudakov, D.M. High-quality full-length immunoglobin profiling with unique molecular barcoding. Nature Protocols 11:1599-1616 (2016).



About Kiri Burrow, Ph.D.

Kiri earned her Ph.D. in Behavioral Genetics and Neuroscience from the University of Colorado at Boulder. Her research involved identifying novel genetic mutations associated with addictive behaviors. Her interest in cancer genomics led her to join the ArcherDX team in May, 2017. In her free time, Kiri enjoys hiking with her dog, Frankie, and spending time with her family.

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For Research Use Only. Not for use in diagnostic procedures. For Research Use Only. Not for use in diagnostic procedures.