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Output format
Description
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 output
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:
Bonferroni single-step
- Choose sorting of numerical values:
decreasing
- Sequence identifier for relative numbering:
none
- Type of logo:
significant positions
Heatmap highlighting significant sites
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| 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. |
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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.
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Sequence logo of significant sites
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Figure 3. Sequence logo quantifying strength of residue association [4]. 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. [1]
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References
[1] Lund,O., Nielsen,M., Lundegaard,C., Kesmir,C. and Brunak,S. (2005) Immunological Bioinformatics. The MIT Press, Cambridge, Massachusetts London, England.
[2] 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.
[3] Shafer,R.W. (2006) Rationale and Uses of a Public HIV Drug-Resistance Database. J Infect Dis., 194, S51-S58.
[4] 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.
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