primers4clades, a web server to design lineage-specific PCR primers for gene-targeted metagenomics
Sachman-Ruiz,B. Contreras-Moreira,B., Zozaya,E., Martínez-Garza,C. and Vinuesa,P.. primers4clades, a web server to design lineage-specific PCR primers for gene-targeted metagenomics. Chapter 51 in Frans J. de Bruijn (ed.), Handbook of Molecular Microbial Ecology vol.I: Metagenomics and Complementary Approaches. Wiley/Blackwell. (in press). : Wiley/Blackwell, 2011.
Primers4clades is an easy-to-use web server that uses phylogenetic trees to design lineage-specific PCR primers for cross-species amplification of novel sequences from metagenomic DNA, or from uncharacterized organisms. The server takes a set of protein coding genes, with or without introns, generates a codon alignment, and computes a neighbor-joining tree, which is displayed on screen for easy selection of a target sequence cluster on which to focus the design of 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. 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/. This chapter will provide an overview of the server’s capabilities by describing the design and use of rpoB primers to study the diversity of environmental mycobacteria in two Mexican tropical soils.
Primers4clades is an easy-to-use web server that uses phylogenetic trees to design lineage-specific PCR primers for cross-species amplification of novel sequences from metagenomic DNA, or from uncharacterized organisms. The server takes a set of protein coding genes, with or without introns, generates a codon alignment, and computes a neighbor-joining tree, which is displayed on screen for easy selection of a target sequence cluster on which to focus the design of 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. 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/. This chapter will provide an overview of the server’s capabilities by describing the design and use of rpoB primers to study the diversity of environmental mycobacteria in two Mexican tropical soils.