Background: Targeted resequencing offers a cost-effective alternative to whole-genome and whole-exome sequencing when investigating regions known to be associated with a trait or disease. There are a number of approaches to targeted resequencing, including microfluidic PCR amplification, which may be enhanced by multiplex PCR. Currently, there is no open-source software that can design next-generation multiplex PCR experiments that ensures primers are unique at a genome-level and efficiently pools compatible primers.
Results: We present MPD, a software package that automates the design of multiplex PCR primers for next-generation sequencing. The core of MPD is implemented in C for speed and uses a hashed genome to ensure primer uniqueness, avoids placing primers over sites of known variation, and efficiently pools compatible primers. A JavaScript web application ( http://multiplexprimer.io ) utilizing the MPD Perl package provides a convenient platform for users to make designs. Using a realistic set of genes identified by genome-wide association studies (GWAS), we achieve 90% coverage of all exonic regions using stringent design criteria. Using the first 47 primer pools for wet-lab validation, we sequenced ~25Kb at 99.7% completeness with a mean coverage of 300X among 313 samples simultaneously and identified 224 variants. The number and nature of variants we observe are consistent with high quality sequencing.
Conclusions: MPD can successfully design multiplex PCR experiments suitable for next-generation sequencing, and simplifies retooling targeted resequencing pipelines to focus on new targets as new genetic evidence emerges.
Keywords: DNA-sequencing; Next-generation sequencing; Primer design; Targeted resequencing.