T cells play a crucial role in the adaptive immune system by identifying and destroying infected or cancerous cells, and they are also involved in autoimmune diseases. Each T cell is equipped to recognize a specific threat, meaning every cell has its own antigen specificity. This specificity is determined by the sequence of the T-cell receptor (TCR). Analysing this sequence can reveal the strength, diversity, and clonal dynamics of an immune response.
Why TCR sequencing is complex
Sequencing TCRs is technically challenging. The TCR loci are composed of multiple gene segments located at different positions on the chromosome. During maturation in the thymus, these segments undergo random recombination to form a functional T cell. The Complementarity Determining Region 3 (CDR3) is the most diverse part of the receptor, and probabilistic modelling has shown that the chance of two unrelated CDR3 sequences being identical is extremely small. For this reason, the CDR3 sequence can serve as a unique identifier of a T-cell clone.
DNA or RNA?
RNA sequencing is a useful approach for studying TCRs because it captures functionally expressed receptor sequences. However, RNA is less stable than DNA, and many commercially available kits require high-quality RNA as input. In practice this is not always available, for example when working with FFPE tissue samples, where RNA is often highly degraded. This creates a clear need for robust RNA-based TCR sequencing approaches that tolerate degraded input material, together with reliable purification steps during library preparation. Cleanup solutions such as CleanNGS can support these workflows by enabling efficient purification of cDNA and PCR products throughout multi-step protocols.
The FUME-TCRseq approach for degraded RNA
The method presented in the paper (Baker et al., 2024), called FUME-TCRseq (FFPE-suitable unique molecular idEntifier-based TCRseq), was specifically developed to address sequencing from degraded RNA. It uses multiplex PCR to generate amplicons of approximately 170 base pairs. These shorter fragments, compared with other multiplex approaches, make it possible to analyse highly degraded RNA and improve the feasibility of TCR sequencing in FFPE material.
FUME-TCRseq workflow:
Reported results are promising for both degraded RNA and high-quality RNA. To read the whole FUME-TCRseq paper from Ann-Marie Baker et al., click here.
