Nucleic Acids Research Advance Access originally published online on May 21, 2009
Nucleic Acids Research 2009 37(Web Server issue):W95-W100; doi:10.1093/nar/gkp377
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Nucleic Acids Research, 2009, Vol. 37, No. suppl_2 W95-W100
© 2009 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Articles |
primers4clades: a web server that uses phylogenetic trees to design lineage-specific PCR primers for metagenomic and diversity studies
1Estación Experimental de Aula Dei, Consejo Superior de Investigaciones Científicas, Zaragoza, 2Fundación ARAID, Paseo María Agustín 36, Zaragoza, Spain and 3Centro de Ciencias Genómicas, Universidad Nacional Autónoma de México, Cuernavaca, Morelos, Mexico
*To whom correspondence should be addressed. Tel: +52 777 3175867; Fax: +52 777 3175581; Email: vinuesa{at}ccg.unam.mx
Received January 31, 2009. Revised April 16, 2009. Accepted April 27, 2009.
Primers4clades is an easy-to-use web server that implements a fully automatic PCR primer design pipeline for cross-species amplification of novel sequences from metagenomic DNA, or from uncharacterized organisms, belonging to user-specified phylogenetic clades or taxa. The server takes a set of non-aligned protein coding genes, with or without introns, aligns them and computes a neighbor-joining tree, which is displayed on screen for easy selection of species or sequence clusters to design lineage-specific PCR primers. Primers4clades implements an extended CODEHOP primer design strategy based on both DNA and protein multiple sequence alignments. It evaluates several thermodynamic properties of the oligonucleotide pairs, and computes the phylogenetic information content of the predicted amplicon sets from Shimodaira–Hasegawa-like branch support values of maximum likelihood phylogenies. A non-redundant set of primer formulations is returned, ranked according to their thermodynamic properties. An amplicon distribution map provides a convenient overview of the coverage of the target locus. Altogether these features greatly help the user in making an informed choice between alternative primer pair formulations. Primers4clades is available at two mirror sites: http://maya.ccg.unam.mx/primers4clades/and http://floresta.eead.csic.es/primers4clades/. Three demo data sets and a comprehensive documentation/tutorial page are provided for easy testing of the server's capabilities and interface.