Quick Reference and Tutorial

Here we provide an overview of the how to use and interpret the results of Skippy. On the variant submission page, we have provided three examples of published variants that cause exon skipping which can be submitted to gain an understanding as to how Skippy should be used. In addition, one of these variants (a variant in the ATR gene on chromosome 3) also creates a 5' ectopic splice site in addition to causing exon skipping.

  1. Submit the coordinates of the variants in correct format. You may choose to score for features of exon-skipping and/or for the creation of ectopic splice site creation. If you choose to score for the creation of ectopic splice sites, you must click the appropriate box in the submission form.
  2. Results are returned in tabular format. No single feature is diagnostic for a skipping or ectopic variant, however the strength of the evidence will be presented.
  3. When possible, a comparison set is provided. These are drawn from the collection of human SNPs in HapMap and are matched for the size of the exons containing the variants in question or in other cases by variants with similar distances from the splice junction.
  4. Throughout the report, variants that look more like a skipping variant than a common polymorphism are color-coded red or blue, respectively.
  5. The bottom half of the report uses color coding as a measure of the difference between the candidate variant and the common human SNPs. Colors represent 1, 2, or 3 standard deviations from the mean.

Results by Category

Variant and Exon Summaries

Features should be confirmed for accuracy. For example, the correct variant position, gene name, nucleotide change, and length of exon are all critical to the results.

Changes in Exonic Splicing Regulatory Elements

The number of losses or gains in predicted ESEs or ESSs is reported. These are based on the neighborhood inference dataset. Typically, SAVs are more likely to affect two or more predicted elements. The directionality of the changes is important for prediction of a SAV. Losses of ESEs and gains of ESSs are most relevant for weakening an exon's identity. Changes in ESRs include transformation from a functional site to a neutral site, a neutral site to a functional site, or a direct switch from one functional type to the other. Predicted sites often represent overlapping hexameric ESR sites, each of which could represent a functionally distinct regulatory site for splicing.

A Log Odds Ratio is calculated to indicate the potential of the combination of ESR changes to represent a SAV. These are color coded red or blue to indicate whether variants are more or less likely to affect exon skipping, respectively. The more positive the LOR score, the more likely a variant is to be splice-affecting.

Graphical View of ESR Changes in Exonic Context

This image illustrates the types of changes within the context of the exon. ESR changes that occur close to the splice junction (within 150 bp) have been published more often than further distances.

Other Variant-based Features

Distance of the Variant from a Splice Junction: As noted in the help window, scores closer to zero indicate a variant close to the splice junctions and scores closer to 1 indicate variants located close to the center of the exon. The three example variants are found throughout their exons, which are 150 bp or less in length. For comparison, average scores for human SNPs from HapMap are provided. These are matched for the same length of the candidate set.

Regulatory Constraint (RC) Score

This score represents the regulatory potential of a site as determined through negative evolutionary selection. Within coding exons regulatory conservation near a variant is assessed by comparison to the expected level of conservation for the codon position throughout the genomic alignments of human, mouse, rat and dog. The presence of conservation that is greater than expected around the variant suggests regulatory conservation. This can be done by comparing the score to scores for regions surrounding HapMap SNPs.

Exonic Environment

Splice Junction Strengths: MaxEnt score <5 at 5' and 3' splice sites are known to predispose exons to the effects of skipping variants

Exonic ESE/ESS Density: Side-by-side comparison of the ESR density for candidate skipping variants compared to common human SNPs.

Change in Splice Site (SS) Score Surrounding Variant (for ectopic site detection): If an ectopic splice site is created by a variant, a delta score is reported for the 3' or 5' splice junction. This score represents the maximum difference between the wild type and ectopic splice junctions, and should be larger than 1 to implicate an ectopic splice site. If the delta SS score is larger than 1, the MaxEnt score of the novel splice site is reported. It can be compared to the MaxEnt score generated for the natural splice site, which is reported in the Splice Junction Strength column on the left.

Intronic Environment

Side-by-side comparison of intronic ESRs to common human SNPs. Both upstream and downstream regions flanking the exon are reported.

Summary