Open Access Highly Accessed Methodology

Automated degenerate PCR primer design for high-throughput sequencing improves efficiency of viral sequencing

Kelvin Li1*, Susmita Shrivastava1, Anushka Brownley2, Dan Katzel1, Jayati Bera1, Anh Thu Nguyen3, Vishal Thovarai1, Rebecca Halpin1 and Timothy B Stockwell1

  • * Corresponding author: Kelvin Li

  • † Equal contributors

Author Affiliations

1 The J. Craig Venter Institute, 9704 Medical Center Drive, Rockville, MD, 20850, USA

2 BioTeam Inc., 7 Derosier Drive, Middleton, MA, 01949, USA

3 Department of Biology, University of Virginia, 485 McCormick Road, Charlottesville, VA, 22908, USA

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Virology Journal 2012, 9:261  doi:10.1186/1743-422X-9-261

Published: 6 November 2012



In a high-throughput environment, to PCR amplify and sequence a large set of viral isolates from populations that are potentially heterogeneous and continuously evolving, the use of degenerate PCR primers is an important strategy. Degenerate primers allow for the PCR amplification of a wider range of viral isolates with only one set of pre-mixed primers, thus increasing amplification success rates and minimizing the necessity for genome finishing activities. To successfully select a large set of degenerate PCR primers necessary to tile across an entire viral genome and maximize their success, this process is best performed computationally.


We have developed a fully automated degenerate PCR primer design system that plays a key role in the J. Craig Venter Institute’s (JCVI) high-throughput viral sequencing pipeline. A consensus viral genome, or a set of consensus segment sequences in the case of a segmented virus, is specified using IUPAC ambiguity codes in the consensus template sequence to represent the allelic diversity of the target population. PCR primer pairs are then selected computationally to produce a minimal amplicon set capable of tiling across the full length of the specified target region. As part of the tiling process, primer pairs are computationally screened to meet the criteria for successful PCR with one of two described amplification protocols. The actual sequencing success rates for designed primers for measles virus, mumps virus, human parainfluenza virus 1 and 3, human respiratory syncytial virus A and B and human metapneumovirus are described, where >90% of designed primer pairs were able to consistently successfully amplify >75% of the isolates.


Augmenting our previously developed and published JCVI Primer Design Pipeline, we achieved similarly high sequencing success rates with only minor software modifications. The recommended methodology for the construction of the consensus sequence that encapsulates the allelic variation of the targeted population and is a key step prior to designing degenerate primers is also formally described.

High-throughput computational degenerate PCR primer design; sequencing viral isolates