A High-Plex PCR Approach for Massively Parallel Sequencing

Current methods for targeted massively parallel sequencing (MPS) have several drawbacks, including limited design flexibility, expense, and protocol complexity, which restrict their application to settings involving modest target size and requiring low cost and high throughput. To address this, we have developed Hi-Plex, a PCR-MPS strategy intended for high-throughput screening of multiple genomic target regions that integrates simple, automated primer design software to control product size. Featuring permissive thermocycling conditions and clamp bias reduction, our protocol is simple, cost- and time-effective, uses readily available reagents, does not require expensive instrumentation, and requires minimal optimization. In a 60-plex assay targeting the breast cancer predisposition genes PALB2 and XRCC2, we applied Hi-Plex to 100 ng LCL-derived DNA, and 100 ng and 25 ng FFPE tumor-derived DNA. Altogether, at least 86.94% of the human genome-mapped reads were on target, and 100% of targeted amplicons were represented within 25-fold of the mean. Using 25 ng FFPE-derived DNA, 95.14% of mapped reads were on-target and relative representation ranged from 10.1-fold lower to 5.8-fold higher than the mean. These results were obtained using only the initial automatically-designed primers present in equal concentration. Hi-Plex represents a powerful new approach for screening panels of genomic target regions.