PhiSiGns: an online tool to identify signature genes in phages and design PCR primers for examining phage diversity

Abstract

Background: Phages (viruses that infect bacteria) have gained significant attention because of their abundance, diversity and important ecological roles. However, the lack of a universal gene shared by all phages presents a challenge for phage identification and characterization, especially in environmental samples where it is difficult to culture phage-host systems. Homologous conserved genes (or "signature genes") present in groups of closely-related phages can be used to explore phage diversity and define evolutionary relationships amongst these phages. Bioinformatic approaches are needed to identify candidate signature genes and design PCR primers to amplify those genes from environmental samples; however, there is currently no existing computational tool that biologists can use for this purpose.

Results: Here we present PhiSiGns, a web-based and standalone application that performs a pairwise comparison of each gene present in user-selected phage genomes, identifies signature genes, generates alignments of these genes, and designs potential PCR primer pairs. PhiSiGns is available at (http://www.phantome.org/phisigns/; http://phisigns.sourceforge.net/) with a link to the source code. Here we describe the specifications of PhiSiGns and demonstrate its application with a case study.

Conclusions: PhiSiGns provides phage biologists with a user-friendly tool to identify signature genes and design PCR primers to amplify related genes from uncultured phages in environmental samples. This bioinformatics tool will facilitate the development of novel signature genes for use as molecular markers in studies of phage diversity, phylogeny, and evolution.

Figure 1

An outline of the PhiSiGns workflow. PhiSiGns consists of two critical, interlinked processes: 1) the identification of signature genes conserved amongst a group of phages, and 2) the design of PCR primers for the amplification of these signature genes. Red stars indicate results that can be downloaded to a local machine

Figure 2

Web interface for the identification of signature genes. The interface shows 1) the options to limit the display of available phage genomes, 2) a list of phage genomes meeting the selection criteria, 3) a list of the user-selected phage genomes from step (2) for signature gene identification and primer design, 4) the BLAST E-value cut-off for similarity searches, and 5) the BLAST alignment coverage cut-off for similarity searches. Users can click on the question marks for additional information on a given parameter

Figure 3

Web interface for designing primers on a selected signature gene. The interface shows 1) an input box for minimum and maximum values for the primer parameters, 2) a list of genes within the selected signature gene group (users have the option to select/deselect genes from the table for alignment and primer design), 3) an option to download the sequence FASTA file, 4) an option to view the program-generated CLUSTALW alignment, and 5) an option to upload a user-generated alignment for the selected signature gene, to be used for primer design

Figure 4

Phylogenetic tree of T7-like primase/helicase sequences amplified from sewage samples with degenerate primers designed with PhiSiGns. The eight core T7-like phages and three cyanophage P60-like phages are shown in red and green, respectively. The sewage sequences amplified in this study are shown in blue. The SEWAGE clade represents the compressed view of 49 closely related sequences recovered from sewage in this study. Internal nodes with bootstrap support ≥ 70% are shown with the corresponding bootstrap value indicated. The scale bar represents the number of nucleotide substitutions per site.