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BMC Bioinformatics. 2017 Jan 5;18(1):14. doi: 10.1186/s12859-016-1453-3.

MPD: multiplex primer design for next-generation targeted sequencing.

Author information

1
Division of Neurology, Atlanta VA Medical Center, Decatur, 30033, GA, USA. thomas.wingo@emory.edu.
2
Department of Neurology, Emory University School of Medicine, Atlanta, 30322, GA, USA. thomas.wingo@emory.edu.
3
Department of Human Genetics, Emory University School of Medicine, 615 Michael Street NE, Atlanta, GA, 30322, USA. thomas.wingo@emory.edu.
4
Department of Human Genetics, Emory University School of Medicine, 615 Michael Street NE, Atlanta, GA, 30322, USA.

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.

KEYWORDS:

DNA-sequencing; Next-generation sequencing; Primer design; Targeted resequencing

PMID:
28056760
PMCID:
PMC5217220
DOI:
10.1186/s12859-016-1453-3
[Indexed for MEDLINE]
Free PMC Article

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