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Author Notes:

Corresponding Author: Thomas Wingo Email: thomas.wingo@emory.edu Phone: 404-727-4905

TSW and DJC designed, implemented, and tested the software.

TSW and DJC designed the wet-lab experiment, analyzed the data, and wrote the manuscript.

AK designed and implemented the web interface and TSW and AK tested the web interface.

All authors read and approved the final manuscript.

We gratefully acknowledge the research volunteers at the Emory Alzheimer’s Disease Research Center and technical assistance from Zoe White and Se Min Heo.

The content is solely the responsibility of the authors and does not necessarily represent the official views of the Veterans Health Administration or National Institutes of Health.

Subjects:

Research Funding:

This work has been supported by the Veterans Health Administration (BX001820), National Institutes of Health (AG025688), and was supported in part by the Emory Integrated Genomics Core (EIGC), which is subsidized by the Emory University School of Medicine and is one of the Emory Integrated Core Facilities.

The content is solely the responsibility of the authors and does not necessarily represent the official views of the Veterans Health Administration or National Institutes of Health.

Keywords:

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

MPD: Multiplex primer design for next-generation targeted sequencing

Tools:

Journal Title:

BMC Bioinformatics

Volume:

Volume 18, Number 1

Publisher:

, Pages 14-14

Type of Work:

Article | Final Publisher PDF

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.

Copyright information:

© 2017 The Author(s).

This is an Open Access work distributed under the terms of the Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/).
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