Cost-Effective and Highly Scalable Typing of HLA Classes I and II Genes of up to 96 Individuals Using Nanopore Sequencing.

HLA typing of large donor registries and biobanks as well as acute single patient/donor samples remains expensive, slow and logistically challenging, despite recent developments in the field. We have tested and validated a cost-effective, accurate and highly scalable method for typing specific genes in the HLA region. This enables HLA typing from 1 to 96 individuals simultaneously, using a targeted PCR and Native Barcoding kit from Oxford Nanopore Technologies. A primer set for seven HLA genes (HLA-A, -B, -C, -DRB1, -DQA1, -DQB1 and -DPB1) was developed to work in a multiplex PCR reaction. The resulting amplicons provide a possible four-field resolution of the HLA Class I genes and G-group resolution of the HLA Class II genes. The entire process, from DNA to HLA typing result, takes a total of 5.5-10.5 h depending on the number of samples processed simultaneously. Data analysis was conducted using NGSEngine-Turbo from GenDx (Utrecht, The Netherlands), with analysis time ranging from 1 to 5 min per sample. Samples from 96 Danish registered stem cell donors were typed using this method. One allele out of 1128 analysed alleles was inaccurately called homozygous, leading to an accuracy of 99.91%. The rapid turnaround, low cost and high accuracy make this new method highly relevant for HLA typing of large biobanks and donor registries, as well as for acute single samples. HLA typing can be obtained within 1 day, with a cost per sample of approximately €7 when 96 samples are sequenced simultaneously.