SeqTools: visual tools for manual analysis of sequence alignments

Abstract

Background: Manual annotation is essential to create high-quality reference alignments and annotation. Annotators need to be able to view sequence alignments in detail. The SeqTools package provides three tools for viewing different types of sequence alignment: Blixem is a many-to-one browser of pairwise alignments, displaying multiple match sequences aligned against a single reference sequence; Dotter provides a graphical dot-plot view of a single pairwise alignment; and Belvu is a multiple sequence alignment viewer, editor, and phylogenetic tool. These tools were originally part of the AceDB genome database system but have been completely rewritten to make them generally available as a standalone package of greatly improved function.

Findings: Blixem is used by annotators to give a detailed view of the evidence for particular gene models. Blixem displays the gene model positions and the match sequences aligned against the genomic reference sequence. Annotators use this for many reasons, including to check the quality of an alignment, to find missing/misaligned sequence and to identify splice sites and polyA sites and signals. Dotter is used to give a dot-plot representation of a particular pairwise alignment. This is used to identify sequence that is not represented (or is misrepresented) and to quickly compare annotated gene models with transcriptional and protein evidence that putatively supports them. Belvu is used to analyse conservation patterns in multiple sequence alignments and to perform a combination of manual and automatic processing of the alignment. High-quality reference alignments are essential if they are to be used as a starting point for further automatic alignment generation.

Conclusions: While there are many different alignment tools available, the SeqTools package provides unique functionality that annotators have found to be essential for analysing sequence alignments as part of the manual annotation process.

Fig. 1

Blixem in protein mode. Blixem showing SwissProt alignments for human chromosome 4. The three-frame translation of the reference sequence is shown in yellow. The match sequence residues are highlighted in cyan for an exact match, blue for a conserved match and grey for a mismatch. Insertions are indicated by a vertical purple bar and deletions by a dot

Fig. 2

Dotter in nucleotide mode. Top left the dot-plot. Top-right the grey-ramp tool, used for dynamically adjusting the stringency cut-offs. Bottom the alignment tool, showing the sequence data at the current cross-hair position; both strands of the horizontal sequence are displayed, and residues are coloured according to how well they match

Fig. 3

Belvu colour-by-conservation mode. Multiple sequence alignment in Belvu with residues coloured by average similarity by BLOSUM62. Cyan indicates conservation of 3.0 or greater, blue 1.5, and grey 0.5. Colours and thresholds are user-configurable

Fig. 4

Belvu in colour-by-residue mode. Multiple sequence alignment in Belvu with residues coloured by residue type. Colours are user-configureable and thresholds can be specified to only colour residues with a given percent identity or higher

Fig. 5

Belvu conservation profile. The maximum conservation for each column is plotted against the column number. The red line shows the average conservation, and a sliding-window of three has been applied for smoothing

Fig. 6

Belvu phylogenetic tree. Neighbour-joining tree using Scoredist correction