125-P: NEXT GENERATION HLA SEQUENCING ON THE Illumina MiSeq

Aim Develop a streamlined next generation sequencing assay that enables allele level HLA typing of 16 samples at HLA-A, B, C, DRB1, DQB1 and DQA1 loci in a single Illumina MiSeq run. Methods 16 samples are amplified at 6 loci (HLA-A, B, C, DRB1, DQB1 and DQA1) by long range PCR using custom designed primers that delineate full length HLA genes (5’UTR to 3’UTR) with the exception of DRB1 for which the amplicon spans intron 1 to intron 4. Individual libraries of each amplicon are prepared by enzymatic fragmentation, end repair, adenylation and ligation of indexed adaptors. The 96 indexed libraries are pooled prior to paired-end 2 x 250 bp sequencing on an Illumina MiSeq. 10,000 paired sequence reads per library are analyzed with NGSengine (GenDx) and HLA Target (Omixon). The full workflow from amplification to HLA genotype is completed in 4 days. Sample prep is performed in a 96 well plate, enabling simple manual or automated sample preparation. Results We have successfully sequenced up to 96 libraries (16 samples x 6 loci) in a single MiSeq run. Sequence output ranged from 2.4-7.3 Gb of sequence (4.7-14 million sequence reads) per run. The quality of sequence obtained was 83.4% of bases ⩾Q30. Average depth of coverage was 461 from the alignment of 10,000 reads per locus. The accuracy of genotype results was 96.7%. Results were comparable between manual and automated library preps. Conclusions We have developed an optimized assay for the Illumina MiSeq that is a simple, robust, and scalable. The method delivers allele level HLA typing without ambiguity. Full genomic sequence is obtained for all loci with the exception of DRB1. The method can be performed manually yet is amenable to automation. Here we describe the means to HLA type 16 samples at 6 loci on the Illumina MiSeq. Given that