132-P: DEVELOPMENT OF AN HLA TYPING METHOD FOR THE ION TORRENT PGM NEXT GENERATION SEQUENCING PLATFORM

Abstract

The Ion Torrent PGM™ next generation sequencing platform allows for the simultaneous sequencing of up to 96 barcoded samples in a single two to four hour run, providing a method to process a large number of gene sequences in significantly less time than traditional sequencing. We are developing an HLA typing method based on the technology that enables read lengths of 400 bases on the PGM™ platform. Two different library preparation workflows have been evaluated. The first approach is based on long-range PCR (LR-PCR) followed by fragmentation and ligation of emulsion PCR adaptor sequences. This method allows coverage of an entire HLA gene with a single long-range PCR reaction for each sample. The basis for the second workflow is a tiled, multiplex, short-range PCR design. The amplicons in this design range in length from 350 - 400 base pairs and cover alternating sections of a gene in two primer pools. This approach offers a workflow that does not include the fragmentation and size selection steps required in the LR-PCR method, saving at least four hours of processing time while producing results equivalent to the LR-PCR workflow. Here we describe the development of a tiling assay that enables full coverage of the HLA-C gene. Approximately 100 primer sets were screened in designing the amplicons. Thirty samples have been sequenced using each method to compare coverage of region, read depth, accuracy, cis/trans ambiguity resolution, and ease of workflow. The current design is comprised of two PCR tubes, each with thirty-five primers, used to generate eighteen tiling amplicons. Each primer pool is designed to amplify every HLA-C gene with known genomic sequence. While the LR-PCR method has simplified primer designs, the short-range PCR approach enables a simplified workflow with equivalent genomic sequence results. The assay designs to date show the feasibility of the PGM™ platform for accurate, high throughput HLA gene sequencing. Bialozynski: Life Technologies: Employee. Dinauer: Life Technologies: Employee. Gifford: Life Technologies: Employee. Zhao: Life Technologies: Employee. Shi: Life Technologies: Employee. Radick: Life Technologies: Employee. Conradson: Life Technologies: Employee.