Mark W Watson Ph.D. B.Sc.

Next generation Sequencing (NGS) has enabled high throughput analysis of T cell receptor (TCR) repertoires, which are useful for monitoring antigen- specific T cell responses. Integrating TCR sequencing with expression levels of genes that characterise T cell phenotype at the single cell level allows comprehensive analysis of T cell function and specificity. Single cell TCR sequencing on the Illumina platform enables the identification of paired TCR α/β chains and T cell phenotype from sorted antigen-specific T cell populations. Here we highlight novel techniques available for T cell phenotyping and TCR repertoire analysis.

Hepatitis C is a global health issue with approximately 3% of the worlds’ population estimated to be infected with the hepatitis C virus (HCV) Inefficiencies in treatment has led to development of direct-acting antivirals (DAAs) that specifically target HCV proteins involved in the virus’s lifecycle1. One of the major concerns arising from the use of the DAAs is the emergence of resistance-associated variants (RAVs) that affect the efficacy of the drugs. RAVs are generally associated with a fitness cost and the use of ultra-deep pyrosequencing technology has shown that in most treatment naïve subjects low frequency circulating strains carry RAVs2. The aim of the study was to investigate i) the clinical relevance of low frequency RAVs; ii) the persistence of RAVs and iii) compensatory mutations in a subset of subjects who had failed boceprevir (SCH503034; protease inhibitor).

Several disease association studies indicate that, the interactions between the KIR and HLA loci may have a role in infectious diseases, autoimmune disorders, cancer and reproduction. Current typing methods for HLA Loci use PCR for whole gene amplification or selected exon amplification. The Killer cell immunoglobulin-like receptors (KIR) genes are polymorphic and the gene complex is polygenic with a varying number of inhibitory (iKIRs) and activating (aKIRs) receptors. Due to this complexity of the KIR locus, genotyping is not performed as routinely.

As the cost of NGS has decreased, the library preparation cost has become a larger portion of the total expenditure. This is especially true for high-throughput applications, such as single-cell analysis. Therefore, there is a need to develop methods that can not only study the transcriptomes of single cells, but can also feasibly analyze large numbers of single cells. Miniaturizing the sample preparation volume provides the opportunity for significant cost savings. Using TTP Labtech’s mosquito liquid handlers, reagent and sample quantities can be scaled down to picogram values.