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Advances in Bioinformatics
Volume 2014 (2014), Article ID 101894, 8 pages
http://dx.doi.org/10.1155/2014/101894
Research Article

Multiplex Degenerate Primer Design for Targeted Whole Genome Amplification of Many Viral Genomes

1Computations, Lawrence Livermore National Laboratory (LLNL), Livermore, CA 94550, USA
2Physical and Life Sciences/Global Security, Lawrence Livermore National Laboratory (LLNL), Livermore, CA 94550, USA

Received 30 May 2014; Accepted 14 July 2014; Published 3 August 2014

Academic Editor: Paul Harrison

Copyright © 2014 Shea N. Gardner et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background. Targeted enrichment improves coverage of highly mutable viruses at low concentration in complex samples. Degenerate primers that anneal to conserved regions can facilitate amplification of divergent, low concentration variants, even when the strain present is unknown. Results. A tool for designing multiplex sets of degenerate sequencing primers to tile overlapping amplicons across multiple whole genomes is described. The new script, run_tiled_primers, is part of the PriMux software. Primers were designed for each segment of South American hemorrhagic fever viruses, tick-borne encephalitis, Henipaviruses, Arenaviruses, Filoviruses, Crimean-Congo hemorrhagic fever virus, Rift Valley fever virus, and Japanese encephalitis virus. Each group is highly diverse with as little as 5% genome consensus. Primer sets were computationally checked for nontarget cross reactions against the NCBI nucleotide sequence database. Primers for murine hepatitis virus were demonstrated in the lab to specifically amplify selected genes from a laboratory cultured strain that had undergone extensive passage in vitro and in vivo. Conclusions. This software should help researchers design multiplex sets of primers for targeted whole genome enrichment prior to sequencing to obtain better coverage of low titer, divergent viruses. Applications include viral discovery from a complex background and improved sensitivity and coverage of rapidly evolving strains or variants in a gene family.