A Simple Multiplex PCR Approach for Target Enrichment in Next-Gen Sequencing

Multiplexing PCR is a simple way to extract genomic regions of interest for various genetic tests such as variant analysis or genotyping. Somatic mutations such as SNPs are unlikely to be best detected using regular whole genome sequencing and genotyping by sequencing (GBS) in any large genome species requires reduction of genome complexity. Therefore, many current genetic test workflows start with multiplexing PCR to extract genetic marker carrying regions from whole genomes before running hybridization, sequencing, or electrophoresis tests to identify the markers.

We have developed a new multiplexing PCR approach with a significantly simplified workflow and significantly improved robustness. When applied to sequencing target enrichment application, the workflow for producing amplified targets involves only one hands-on step and one PCR run. Both sample barcodes and molecular tags are incorporated during this single PCR run. The approach is designed to require low sample input and to produce superior amplicon uniformity and sequence specificity. The approach involves a novel primer design and a proprietary reaction composition. A PCR run consists of two functionally separated reaction phases, namely target capture and library amplification, without any hands-on step in between.