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Nucleic Acids Research Advance Access originally published online on September 16, 2009
Nucleic Acids Research 2009 37(19):6291-6304; doi:10.1093/nar/gkp659
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Nucleic Acids Research, 2009, Vol. 37, No. 19 6291-6304
© The Author(s) 2009. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Computational Biology

Multiplex primer prediction software for divergent targets

Shea N. Gardner1,*, Amy L. Hiddessen2,*, Peter L. Williams1, Christine Hara2, Mark C. Wagner1 and Bill W. Colston, Jr3

1Computations/Global Security, 2Physical & Life Sciences, Lawrence Livermore National Laboratory and 3QuantaLife, Inc., Livermore, CA, USA

*To whom correspondence should be addressed. Tel: +1 925 422 4317; Fax: +1 925 423 6437; Email: gardner26{at}llnl.gov

Correspondence may also be addressed to Amy L. Hiddessen. Tel: +1 925 422 4787; Fax: +1 925 423 8920; Email: hiddessen1{at}llnl.gov

Received December 18, 2008. Revised July 22, 2009. Accepted July 24, 2009.

We describe a Multiplex Primer Prediction (MPP) algorithm to build multiplex compatible primer sets to amplify all members of large, diverse and unalignable sets of target sequences. The MPP algorithm is scalable to larger target sets than other available software, and it does not require a multiple sequence alignment. We applied it to questions in viral detection, and demonstrated that there are no universally conserved priming sequences among viruses and that it could require an unfeasibly large number of primers (~3700 18-mers or ~2000 10-mers) to generate amplicons from all sequenced viruses. We then designed primer sets separately for each viral family, and for several diverse species such as foot-and-mouth disease virus (FMDV), hemagglutinin (HA) and neuraminidase (NA) segments of influenza A virus, Norwalk virus, and HIV-1. We empirically demonstrated the application of the software with a multiplex set of 16 short (10 nt) primers designed to amplify the Poxviridae family to produce a specific amplicon from vaccinia virus.


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