HLA-PopSeq: High throughput, multiplexed six-locus Human Leukocyte Antigen typing for population-scale T cell immune profiling using rapid long-read nanopore sequencing

Abstract A major technical hurdle for T cell immune profiling is the time and cost to accurately genotype Human Leukocyte Antigen (HLA) loci from peripheral blood. Here, we developed a rapid, highly multiplexed approach HLA typing using RNA <100,000 blood mononuclear cells with Oxford Nanopore Technology (ONT) Minion sequencer. This method uses selective reverse transcription of mRNA six (A,B,C, DRB1, DQB1, DPB1), followed by PCR amplification. The individual amplified cDNA was in single sequencing pool primers unique molecular identifiers, designed permit errors enhanced data capture. Pooled amplicons were sequenced ONT MK1B R10.4 flowcells, sequence Q scores> 20. Total extracted PBMC samples 12 individuals, transcribed specific primers. pooled, then 16 hours on resulting analyzed an average depth coverage 6000x observed per sample. An 1000x observed. rapid (<24h), low-cost, portable monitoring epitope identification immunologic studies. Arizona Piper Foundation