P146 Nanopore sequencing as a maturing platform for full-length HLA genotyping

Aim While short read sequencing technologies have proven highly successful for HLA genotyping, longer reads would be desirable to resolve phasing ambiguities. The emerging nanopore sequencing platform from Oxford Nanopore Technologies (ONT) presents a promising technology capable of sequencing unfragmented molecules in the order of tens of kilobases. We demonstrate that nanopore sequencing is an increasingly viable platform for HLA genotyping by addressing questions of throughput, mappability and genotyping accuracy. Methods We sequenced a benchmarking set of 30 samples with established full length sequence information, 10 each for HLA-A, HLA-B and HLA-C, on the MinION, a nanopore sequencing device from ONT, using 3 successive chemistries R7.3, R9 and R9.4. We mapped the resulting reads, both template-only (1D) and template-complement (2D), to the reference sequences using ‘bwa’ in its ‘ont’ mode, a set of parameters tailored for MinION sequencing reads. Consensus sequences were then derived from the SAM mapping data using a routine leveraging the pysam Python library. Results We observed progressive chemistries yielding increasing read numbers, from approximately 3000 reads to 8000 reads per sample. We also observed a successive increase in mappability from 90% to 98% for 1D reads, and from 97% to 99.8% for 2D reads. We compared the consensus sequences with the reference sequences, and observed reproducibility at 98% for 1D reads and 99.8% for 2D reads. We note here that the ONT basecaller systematically undercalls polynucleotide stretches resulting in gaps explaining the consensus sequence errors. We expect to report on this issue being ameliorated by a basecaller update scheduled for May 2017 specifically aimed at accurately calling homopolymer stretches. We evaluate if improvements in basecalling are achievable by training the neural network that underlies the ONT basecaller on curated data derived from HLA references sequences. Conclusions The sequencing yield and mappability of full-length HLA alleles using nanopore sequencing could render it a suitable platform for HLA genotyping. The platform’s major disadvantage, severe undercalling of polynucleotide stretches, is expected to improve with pending updates to the basecalling software.