Droplet-based high-throughput T cell phenotyping and clonotyping method with target gene-specific barcode beads

Identification: Pessa, Heli


Droplet-based high-throughput T cell phenotyping and clonotyping method with target gene-specific barcode beads

Heli Pessa, Dawit Yohannes, Lisa Jahn, Benedek Poor and Päivi Saavalainen

Research Programs Unit, Immunobiology, University of Helsinki, Helsinki, Finland

The highly variable and somatically recombined T cell receptor (TCR) repertoire gives the immune system the ability to recognize a large variety of pathogens as well as self antigens, and the identification of the TCRs is important for the design of treatments for conditions such as infections, cancer and autoimmune diseases. Most T cells express a TCR consisting of two chains, the TCRa and TCRb (or TCRg and TCRd in the subset of gamma-delta T cells), and bulk mRNA sequencing does not reveal the combinations expressed by individual cells. The pairing information can be obtained from single-cell sequencing, but the low sequencing depth typical of single-cell experiments can provide information only on the most abundant mRNAs. Furthermore, high-throughput 3’ barcoding methods such as Drop-seq combined with shortread Illumina sequencing do not reach the most variable complementarity determining region 3 (CDR3) located closer to the 5’ end of the TCR transcripts. Thus, improved enrichment methods are required to probe the full clonality and phenotype of T cells. To do that, we partly modified barcoded Drop-seq beads with TCR gene-specific extension sequences that bring the cell and molecule barcodes closer to the CDR3 domain of TCRs. We used Drop-seq capture on human peripheral blood mononuclear cells (PBMCs) both with the extension beads and standard Drop-seq beads, and sequenced the libraries in 150bp+150bp Illumina NextSeq500 paired end run. The preliminary data demonstrated a significant enrichment of TCR specific reads in the data from the extension beads, and we are currently optimizing the method with larger panels of marker genes as well as developing specific analysis tools for this targeted gene method. Our protocol shows promise not only for large scale T and B cell repertoire studies but more broadly for panels of any target genes of interest independently from poly-A tailing.


Credits: None available.

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