The output consists of a heatmap and a sequence logo, both visualizing the significant sites identified by SigniSite-2.0.
The user may specify whether the heatmap shall comprise all alignment positions or only significant positions. Per position, each residue is assigned a Z score. These Z scores are displayed in the heatmap and color-coded according to the significance level associated with it.
Example data set
100 HIV-1 protease variants and associated quantification of resistance towards inhibitor Atazanavir measured using Virco Antivirogram® assay
Compiled from the Stanford University HIV Drug Database [2,3]
The example data set, was submitted to SigniSite-2.0 with the following settings
- Significance threshold:
α = 0.05
- Method for correction for multiple testing:
- Choose sorting of numerical values:
- Sequence identifier for relative numbering:
- Type of logo:
Heatmap highlighting significant sites
|Figure 1. Heatmap color scale. A z-score lower than zlower (in this case z < -1.96) is colored blue. A z-score higher than zupper (in this case z > 1.96) is colored red. For z-scores larger than zlower, but smaller than zupper colors inbetween are used. For z-scores equal to zero, a grey color is used. The zlower and zupper bounds are determined by the level of significance, e.g. α = 0.05 corresponds to the bounds used in this example, i.e. zlower = -1.96 and zupper = 1.96, α = 0.01 corresponds to zlower = -2.58 and zupper = 2.58 etc.
Figure 2. Heatmap visualisation of strength of residue association.. Black cells denote abscence of amino acid residue. A grey cell denotes a residue with a z-score of 0 (i.e. p-value = 1). If there is only one grey cell at a position, the position is completely conserved harbouring only this residue. If more than one grey cell are present, the p-value for this residue has become p=1 after correction for multiple comparisons. Each column corresponds to one of the 20 proteinogenic amino acids and each row to a position in the submitted multiple sequence alignment. If any of the positions are numbered negatively, this means that a reference sequence was chosen for numbering, and the particular position lies within a gapped region. -1 is the first gap, -2 the second and so on.
Sequence logo of significant sites
Figure 3. Sequence logo quantifying strength of residue association . Amino acid residues on the positive y-axis are associated with strong phenotype values and residues on the negative y-axis, with weak phenotype values.
The amino acids are colored according to their chemical properties as follows: Acidic [DE]: red, Basic [HKR]: blue, Hydrophobic [ACFILMPVW]: black and Neutral [GNQSTY]: green. 
 Lund,O., Nielsen,M., Lundegaard,C., Kesmir,C. and Brunak,S. (2005) Immunological Bioinformatics. The MIT Press, Cambridge, Massachusetts London, England.
 Rhee,S.Y., Gonzales,M.J., Kantor,R., Betts,B.J., Ravela,J., and Shafer,R.W. (2003) Human immunodeficiency virus reverse transcriptase and protease sequence database. Nucleic Acids Res., 30(1), 298-303.
 Shafer,R.W. (2006) Rationale and Uses of a Public HIV Drug-Resistance Database. J Infect Dis., 194, S51-S58.
 Thomsen,M.C.F. and Nielsen,M. (2012) Seq2Logo: a method for construction and visualization of amino acid binding motifs and sequence profiles including sequence weighting, pseudo counts and two-sided representation of amino acid enrichment and depletion. Nucleic Acids Res., 40(Web Server Issue), W281-W287.