19-OR: TOWARDS ALLELE LEVEL HLA GENOTYPING: A COMPARISON OF NEXT-GENERATION SEQUENCING PLATFORMS – ION TORRENT PGM AND ILLUMINA MiSeq

Aim To evaluate newly available high-throughput sequencing technologies for HLA genotyping. Methods Two samples were amplified at HLA-A, B, C, DRB1, and DQB1. The full length of HLA Class I genes were amplified with custom designed primers from 5′UTR to 3′UTR. HLA Class II were amplified from Exon 2 to Exon 4 using primers from GenDx. A separate DNA library was prepared from each long-range PCR amplicon using platform-specific library prep kits (NEB, Ipswich, MA). For the Ion Torrent run, the pool of 10 libraries was clonally amplified by emulsion PCR and was sequenced using the 200 bp kit and a 316 chip on the Ion Torrent PGM (Life Technologies). Paired-end 2 × 150 bp sequencing of the Illumina libraries was performed on the MiSeq system. Sequence alignment utilized BWA-SW software. Further analysis was conducted using SAM tools and custom written code. Sequence analysis was performed in parallel with NGSEngine (GenDx). Results Sequencing and alignment metrics were as follows:[ Table 1 ] Consensus sequence of aligned reads was 100% accurate for both platforms. Definitive HLA genotypes were obtained without any ambiguity (alternative genotypes) and were concordant with the known alleles of these samples. Conclusions Newly developed high-throughput sequencing systems hold great promise for a new method of HLA genotyping. These new technologies are capable of eliminating all ambiguity currently encountered with existing methods. As such the advancement from high resolution to allele level HLA typing is approaching. Rozemuller: GenDx: Employee. Penning: GenDx: Employee. Mulder: GenDx: Employee.