OR38 Whole gene HLA class I genotyping with the minion nanopore sequencer

Abstract

In 2014 Oxford Nanopores Technologies released the MinION, a portable miniaturized sequencer delivering multi-kb DNA reads based on nanopore sensing technology. Sequencing accuracy is improved by consecutive sequencing both strands of the DNA (2D reads). Being part of the MinION early access program we evaluated this new technology for whole gene HLA sequencing. As the sequencing yield exceeds the requirements for a targeted approach we developed a strategy for barcoding and demultiplexing of samples. Barcoded amplicons were generated by two subsequent long range PCRs covering the complete genes of HLA-A, -B and -C. Amplicons were pooled and library preparation was performed according to the Oxford Nanopore protocol (SQK-MAP006). Sequencing was performed on a MinION MKI with R7.3 flow cells. NGSengine from GenDX was used for sequence analysis and HLA genotyping. 96 randomly picked class I samples were pooled and sequenced. Over 170,000 reads were generated, approximately 65% of them as 2D reads. Two thirds of the 2D reads could be assigned to a unique barcode sequence. The HLA genotyping results were compared to allelic resolution genotypes obtained by full length sequencing the samples on both Illumina and PacBio instruments. The ONT typing results had a 95% concordance rate when considering 1st to 3rd field. Even when taking the fourth field in account concordance rate was above 90%. Given that the nanopore technology is rapidly evolving and the genotyping software has not yet been optimized for nanopore data, this study clearly underlines the great potential of the MinION for high resolution HLA genotyping.