1004-LBP: HLA SEQUENCE-BASED TYPING WITH NEXT GENERATION TECHNOLOGY

Abstract

Current technologies for analysis of complex polymorphic gene systems restrict the ability to routinely obtain both breadth (number of loci) and depth (allele resolution) of data. The costs of increasing the breadth of analysis are additive in that the cost for each locus is similar. The costs for high resolution analysis of highly polymorphic genes, such as HLA, are increased by the extended processes required to resolve ambiguous heterozygous typing results. In order to improve the economy of obtaining both broad and high-resolution data in both high and medium throughput settings, we have undertaken to develop next generation sequencing (NGS) reagents and software systems for analysis of complex immune genes using the Illumina MiSeq device. For the HLA complex, our original software extracts precise HLA typings from highly multiplexed raw NGS data by aligning the uniquely barcoded sequence fragments followed by analysis with an HLA allele calling script. Data for HLA-A, B, C, exons 2, 3, and 4 are obtained with phase determined between exons 2 and 3 by sequencing and with exon 4 by coincidence with established databases. Data for DRB1345, DQA1, DQB1, DPB1 are obtained from exons 2 and 3 with phase between exons determined by coincidence with established databases. DPA1 data are obtained from exon 2. We also developed an NGS KIR haplotyping system and are evaluating SNP-based chimerism analysis, both with workflows similar to the HLA approach. Beyond extensive in-house evaluation, we took our HLA system to two clinical laboratories associated with hematopoietic cell transplant programs for beta testing of our NGS approach in moderate throughput environments. We discuss these results and the proven enhanced efficacy and economy of the NGS typing approach in moderate throughput laboratories. Furthermore, we discuss future NGS applications that will become available to those laboratories to support their evolving role in the application of precision medicine.