NemaFootPrinter: a web based software for the identification of conserved non-coding genome sequence regions between C. elegans and C. briggsae

Abstract

Background: NemaFootPrinter (Nematode Transcription Factor Scan Through Philogenetic Footprinting) is a web-based software for interactive identification of conserved, non-exonic DNA segments in the genomes of C. elegans and C. briggsae. It has been implemented according to the following project specifications:a) Automated identification of orthologous gene pairs. b) Interactive selection of the boundaries of the genes to be compared. c) Pairwise sequence comparison with a range of different methods. d) Identification of putative transcription factor binding sites on conserved, non-exonic DNA segments.

Results: Starting from a C. elegans or C. briggsae gene name or identifier, the software identifies the putative ortholog (if any), based on information derived from public nematode genome annotation databases. The investigator can then retrieve the genome DNA sequences of the two orthologous genes; visualize graphically the genes' intron/exon structure and the surrounding DNA regions; select, through an interactive graphical user interface, subsequences of the two gene regions. Using a bioinformatics toolbox (Blast2seq, Dotmatcher, Ssearch and connection to the rVista database) the investigator is able at the end of the procedure to identify and analyze significant sequences similarities, detecting the presence of transcription factor binding sites corresponding to the conserved segments. The software automatically masks exons.

Discussion: This software is intended as a practical and intuitive tool for the researchers interested in the identification of non-exonic conserved sequence segments between C. elegans and C. briggsae. These sequences may contain regulatory transcriptional elements since they are conserved between two related, but rapidly evolving genomes. This software also highlights the power of genome annotation databases when they are conceived as an open resource and the possibilities offered by seamless integration of different web services via the http protocol.

Availability: The program is freely available at http://bio.ifom-firc.it/NTFootPrinter.

Figure 1

Web Interface Scheme. NemaFootPrinter scheme: starting from gene name submission ('Start analysis' on the top-left of the scheme), gene name and organism are submitted to the GENEFINDER script (red-border box) that verifies if the given gene has an ortholog and displays the clone name. GENEFINDER also allows not-interactive selection of n base pairs upstream and downstream of the given genes. After gene-name retrieval the user can choose a display mode (green-border boxes): TEXT-MODE allows non-interactive selection of subsequences; the SLIDER-MODE uses an Applet Java to select interactively sub-sequences; the FRAME-MODE combines the slider and the result page on two horizontal frames. On the RESULTS page (blue-border boxes) users can find images of genes structure and boundaries generated on the fly, links to the sequences (FASTA format) and links to a series of bioinformatics tools (yellow-border boxes): BLAST 2 sequences is used for a first screening of the two sequences for similarities; Dotmatcher generates a dot plot and associate an image for direct graphical visualization of regions of similarity. By clicking on a point into the Dotmatcher image and extending the selection for n base pairs, it is possible to align small regions with the Smith and Waterman algorithm. One can then send Fasta files and Blastz alignments to the rVista server (the two subsequences and the relative Blastz alignment generated on the local server). Through this public database it is possible to identify the transcriptional regulatory elements, if any, associated with conserved subsequences.

Figure 2

Select subsequences. CBrothersSlider.java is a small application written in Java to interactively display gene structures with intron/exon structure and to select subsequences. The interface display clone identifiers (A) and gene images generated "On-the fly" (B). Shifting the sliders (C) or submitting directly chromosome coordinates (D), the user is able to select a subsequence. A small control panel (E) can be used to select only the region on the left of the gene (left checkbox) or on the right of the gene (right checkbox); if both the checkboxes are selected, the application selects only the gene sequence. After sequence manipulation, using the Submit button (F), user can post the selected subsequence coordinates to the main script that generates new FASTA files and display the Results page.

Figure 3

dotmatcher. A Dotplot image: C. elegans and C. briggsae sequences are placed on the axes. The gene structure generated on the fly help the Investigator to orient itself in the rectangular image. The top of the image shows parameters given by the investigator (windowsize and threshold). This web images are clickable by the user, the single click is extended in both directions for n base pairs. Both segments (one for C. elegans and one for C. briggsae) are sent to the Ssearch control page, in order to align segments with the Smith and Waterman algorithm.