title: MPD: multiplex primer design for next-generation targeted sequencing.
abstract: 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.
authors.affiliation: Division of Neurology, Atlanta VA Medical Center, Decatur, 30033, GA, USA. thomas.wingo@emory.edu.|Department of Neurology, Emory University School of Medicine, Atlanta, 30322, GA, USA. thomas.wingo@emory.edu.|Department of Human Genetics, Emory University School of Medicine, 615 Michael Street NE, Atlanta, GA, 30322, USA. thomas.wingo@emory.edu.
authors.forename: Thomas S
authors.initials: TS
authors.lastname: Wingo
authors.affiliation: Department of Human Genetics, Emory University School of Medicine, 615 Michael Street NE, Atlanta, GA, 30322, USA.
authors.forename: Alex
authors.initials: A
authors.lastname: Kotlar
authors.affiliation: Department of Human Genetics, Emory University School of Medicine, 615 Michael Street NE, Atlanta, GA, 30322, USA.
authors.forename: David J
authors.initials: DJ
authors.lastname: Cutler
mesh_terms: D017931:DNA Primers / Q000235:genetics*; D005091:Exons; D015894:Genome, Human; D055106:Genome-Wide Association Study; D023281:Genomics; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D020407:Internet; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide; D017422:Sequence Analysis, DNA; D012984:Software
chemical_list: D017931:DNA Primers
keywords: DNA-sequencing; Next-generation sequencing; Primer design; Targeted resequencing