P177 NGS typing results using oxford nanopore sequencing: Can minion data be reliably used for HLA typing?

Aim Nanopore sequencing is one of the latest DNA sequencing technologies, in which single molecules are sequenced directly. It offers the potential of read lengths of tens of kilobases. However, the reads are relatively error prone. Especially the length of homopolymers and repetitive sequences cannot be determined accurately. Here we present the typing results for HLA samples using Oxford Nanopore Technologies (ONT). Methods We have developed NGSengine® (GenDx), a software package to perform HLA typing based on NGS data. NGSengine uses an integrated HLA typing approach which includes determining the HLA locus, aligning reads, phasing and typing into a single method. At DKMS Life Science Lab, the MinION sequencer from ONT has been used for full-length HLA sequencing. We have used a benchmarking set of 30 samples, 10 each for HLA-A, HLA-B and HLA-C to explore 3 successive Nanopore chemistries R7.3, R9 and R9.4. Results Using the successive ONT chemistries, we have mapped the template-complement (2D) reads to the known reference alleles using Burrows-Wheeler Aligner, resulting in a mappability increasing from 98 to 99.5%. Additionally, we have used NGSengine to map the reads to the allele database, increasing the mappability from 4 to 70%. The lower mappability with NGSengine is due to the noise present within the data and the thresholds set within NGSengine. For most of the validation samples the best match presented by NGSengine corresponds with the pretyping, but all have exon mismatches in homopolymers. Noteworthy, for the samples for which the best match did not match the pretype, a Null allele was called due to the incorrect length of a homopolymer. Conclusion With the current status of MinION data quality, the results look promising but cannot yet be applied for routinge HLA typing. 1D 2 basecalling with a slight change to the chemistry (R9.5) will be available soon. We will present an update on this chemistry, and the improvements concerning homopolymers.