APARENT Saturation Mutagenesis

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Predict the change in cut distribution and isoform abundance due to every possible SNV of the input pA sequence.

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In-Silico Saturation Mutagenesis

Explanation of values, fields and graphics presented in the tool.

  • Reference Cleavage %
    The wildtype 3' cleavage distribution is shown as a black curve. The values (Cleavage %) are non-normalized (they do not sum to 100%). Rather, Cleavage % is scaled with respect to the strength of the PAS relative to the average affinity of a distal, well-separated PAS.
  • Variant Cleavage %
    The variant 3' cleavage distribution is shown as either a red or green curve (depending on the effect of the SNV).
  • Isoform Window
    The window frame shown on top of the cleavage distribution defines what cleavage positions define the isoform of interest. All isoform metrics (e.g. the variant isoform fold change) is calculated by aggregating the cleavage within this window.
  • Heatmap color
    The APA isoform fold change of each SNV is depicted (in log scale) as blue/red color intensities in the heatmap.
  • Variant details
    The hg19 coordinate, strand, reference and variant alleles of the selected SNV is shown below the heatmap. Additionally, if the variant is recorded in ClinVar, its identifier and clinical significance are shown.
  • Fold Change
    The APA isoform fold change of the selected SNV is shown in log scale (same value as the corresponding heatmap cell).
  • Wildtype score
    The aggregate APA isoform log odds ('strength') of the wildtype sequence, with respect to the average affinity of a well-separated, distal PAS. The histogram to the right shows the distribution of native human pA sites and their isoform scores.

Instructions on how to use the tool.

  • Choose gene and pA site
    Select the gene of interest in the dropdown list above the cleavage curve. Choosing a gene populates the dropdown to the right with the annotated pA sites of that gene. We currently provide integration with APADB (V2) and PolyADB (V3).
  • (Optional) Specify custom pA sequence
    Instead of choosing from a list of native human pA sites, you can paste your own custom pA sequence in the text area below the heatmap. Note: The sequence must be 205 nucleotides.
  • Define isoform window
    Drag the tabs of the isoform window over the region of the cleavage distribution according to what pA positions you wish to include in the isoform of interest.
  • Zoom and pan the sequence
    Zoom and pan into specific regions of the sequence by pressing the zoom buttons above the cleavage curve, or simply by scrolling and dragging with the mouse over the heatmap.
  • Inspect variants
    By hovering with the mouse over the heatmap, the cleavage alteration and APA isoform fold change of the corresponding variant are shown. ClinVar variants are annotated with a rectangular box surrounding the heatmap cell.
  • Select a variant
    Lock on a specific SNV by clicking the corresponding heatmap cell. Click anywhere else in the heatmap to clear the selection.
  • (Optional) Mutate the wild type sequence
    Clicking on an already selected heatmap cell causes the wildtype sequence to incorporate the SNV and re-loads the updated mutation map.