P100 A novel sequence enrichment protocol allowing HLA-typing on 5-cells

Aim Limited genomic DNA template in a sample can make accurate HLA-typing difficult. We therefore aimed to develop a method that allows HLA-typing using next generation sequencing (NGS) up to p-group resolution with starting DNA template of only 5 cells. Methods Aliquots of 5 cells were sorted from aneuploid and euploid cell lines (Coriell Institute of Medical Research, USA). Whole genome amplification was performed using the DOPlify™ TSE (Targeted Sequence Enrichment) protocol (RHS Ltd, Australia) with the inclusion of SBTessenz® primers (GenDx, the Netherlands) to increase amplification of exon 2 to 3 of the HLA-A locus. Enrichment was determined using semi-quantitative PCR for HLA-A. The HLA-A amplicon was subsequently pooled with the original DOPlify™ TSE WGA DNA in a 1:10 and 1:20 ratio before library preparation. After indexing and multiplexing of 40 samples for sequencing on a MiSeq platform according to standard 2 × 75 bp protocol (Illumina), the NGS data was analyzed and typed by the HLA-typing software NGSengine®. Results Targeted sequence enrichment increased the breadth and depth of coverage of exon 2, intron 2 and exon 3 for the HLA-A locus, with concurrent detection of aneuploidy. When the HLA-A amplicon was pooled with the DOPlify™ TSE WGA DNA, coverage of 100% was achieved and the average read depth following NGS was greater than 200. Amplification of both alleles was confirmed with a ratio of approximately 30:70% which enabled clear detection and analysis of the heterozygous positions. The quality of the sequence data allowed resolution of all ambiguities that are present in exon 2, intron 2 and exon 3; including the sequence up front of exon 2 required to exclude several null alleles. Conclusions We developed a novel sequence enrichment protocol to amplify and type HLA-A from a 5 cell sample. The data quality meets general laboratory standards for HLA-typing and allows typing up to p-group resolution. This protocol has application in preimplantation genetic diagnosis and low template input HLA typing such as buccal swabs.