GUIDANCE: a web server for assessing alignment confidence scores

Abstract

Evaluating the accuracy of multiple sequence alignment (MSA) is critical for virtually every comparative sequence analysis that uses an MSA as input. Here we present the GUIDANCE web-server, a user-friendly, open access tool for the identification of unreliable alignment regions. The web-server accepts as input a set of unaligned sequences. The server aligns the sequences and provides a simple graphic visualization of the confidence score of each column, residue and sequence of an alignment, using a color-coding scheme. The method is generic and the user is allowed to choose the alignment algorithm (ClustalW, MAFFT and PRANK are supported) as well as any type of molecular sequences (nucleotide, protein or codon sequences). The server implements two different algorithms for evaluating confidence scores: (i) the heads-or-tails (HoT) method, which measures alignment uncertainty due to co-optimal solutions; (ii) the GUIDANCE method, which measures the robustness of the alignment to guide-tree uncertainty. The server projects the confidence scores onto the MSA and points to columns and sequences that are unreliably aligned. These can be automatically removed in preparation for downstream analyses. GUIDANCE is freely available for use at http://guidance.tau.ac.il.

Figure 1.

An example of the GUIDANCE output. (a) Residue confidence scores are projected onto the MAFFT alignment of Vpu protein sequences from human and simian immunodeficiency viruses (HIV and SIV). Confidently aligned residues are colored in shades of magenta and pink, while uncertain residues are colored in shades of blue. Column scores are plotted below the alignment. (b) Dramatically improved alignment confidence after filtering low-scoring sequences and re-running GUIDANCE. Note the color-coding next to the sequence names before and after re-alignment.

Figure 2.

Average run-time performance as a function of the number of sequences. Simulated protein sequences roughly 300 amino acids long were aligned using MAFFT and analyzed by GUIDANCE (blue diamonds) or HoT (red squares). In addition, running time for GUIDANCE on PRANK alignments is plotted with green triangles. Each data point represents ten replicates.