Help & Tutorials

Tutorials

The Human Splicing Finder (HSF) system combines 12 different algorithms to identify and predict mutations’ effect on splicing motifs including the acceptor and donor splice sites, the branch point and auxiliary sequences known to either enhance or repress splicing: Exonic Splicing Enhancers (ESE) and Exonic Splicing Silencers (ESS).

These algorithms are based on either PWM matrices, Maximum Entropy principle or Motif Comparison method. For each of them we defined a consensus value threshold and a score variation threshold, based on litterature datatests:

Type of signal Algorithm type Prediction algorithm CV threshold Variation threshold Comment
Donor or acceptor splice site Position Weight Matrices HSF 65 +/-10% Consensus values go from 0 to 100 for HSF, -20 to +20 for MaxEnt. The threshold is defined at 65 for HSF, 3 for MaxEnt. This means that every signal with a score above the threshold is considered to be a splice site (donor or acceptor).
When a mutation occurs, if the WT score is above the threshold and the score variation (between WT and Mutant) is under -10% for HSF (-30% for MaxEnt) we consider that the mutation breaks the splice site. In the other case, if the WT score is under the threshold and the score variation is above +10% for HSF (+30% for MaxEnt) we consider that the mutation creates a new splice site.
Maximum Entropy MaxEntScan 3 +/-30%
Branch point site Position Weight Matrices HSF 67 +/-10% Consensus values go from 0 to 100 and the threshold is defined at 67. This means that every signal with a score above 67 is considered to be a potential branch point.
When a mutation occurs, if the WT score is above 67 and the score variation (between WT and Mutant) is under -10% we consider that the mutation breaks the branch point.
Exonic Splicing Enhancers (ESE) Position Weight Matrices HSF 9G859.24 Yes/No Consensus values go from 0 to 100 and the threshold is defined differently for each algorithm. Every signal with a score above the defined threshold is considered to be a potential ESE.
When a mutation occurs, if the WT score is above the threshold and the Mutant score is under it we consider that the mutation breaks the ESE.
Tra2-β75.96
ESE Finder SF2/ASF72.98
SF2/ASF(IgM)70.51
SC3575.05
SRp4078.08
SRp5573.86
Motif Comparison method RESCUE ESE hexamers Present/Absent If the tested motif exists in the database, it is considered to be a potential ESE.
When a mutation occurs, if the WT motif is present in the database and the Mutant one is absent we consider that the mutation breaks the ESE.
Exonic Splicing Silencers (ESS) Position Weight Matrices HSF hnRNP-A1 65.476 Consensus values go from 0 to 100 and the threshold is defined differently for each algorithm. Every signal with a score above the defined threshold is considered to be a potential ESS.
When a mutation occurs, if the WT score is under the threshold and the Mutant score is above it we consider that the mutation creates a new ESS.
Sironi motifs 60
Motif Comparison method ESS decamers from Wang et al. Present/Absent If the tested motif exists in the database, it is considered to be a potential ESS.
When a mutation occurs, if the WT motif is absent in the database and the Mutant one is present we consider that the mutation creates a new ESS.
Both ESEs and ESSs PESE & PESS Octamers If the tested motif exists in the database, it is considered to be a potential ESE or ESS.
When a mutation occurs, if the WT motif is present in the database and the Mutant one is absent we consider that the mutation breaks the ESE. Else if the WT motif is absent in the database and the Mutant one is present we consider that the mutation creates a new ESS.
ESR Sequences
EIEs & IIEs Hexamers

The "Interpreted data" section relies on an expert system for data interpretation. This algorithm uses a decision tree based on mutation position: intronic vs. exonic, localization or not in wild type splice sites or branch point. Based on these criteria, context-irrelevant signals are automatically ruled out to focus on relevant ones to evaluate the potential impact of mutations.

Now in this example, the mutation occurs in the exon (DMD c.4250T>A). The system predicts only 3 signals out of all to be possibly relevant, a new acceptor site created on position 7 of the exon, a new ESS created between position 13 and 21 of the exon, and an ESE site broken on position 13.

Here's what the columns stand for:

  • Predicted signal:
    Indicates the type of the signal to be altered by the mutation. It could be a boken WT splice site (donor or acceptor), a new cryptic site created (donor or acceptor), a broken WT branch point, an auxiliary splicing signal broken or cretaed (ESE or ESS)
  • Prediction algorithm:
    This section displays the algorithm(s) that predicted the alteration, at least 2 different algorithms (out of 6) need to overlap to consider an ESE or ESS signal to be relevant. The little number preceding the algorithm name indicates the graphic representation of the signal in the next column.
  • cDNA Position:
    Displays a graphic representation of the region in which the mutation occurs. If 2 or more algorithms have predictions for the same signal, each of them would appear with a line in different color (position and motif length can differ from an algorithm to another). On the top part of the graph, the sequence is represented with the WT nt in green (if broken site) or the mutant nt in red (if new site created). Finally the positions on the bottom are exonic, if the mutation occurs in the intron, + or - sign would appear.
  • Interpretation:
    A hint about the predicted signal and its possible effect on splicing.

Webservice

Our team is actively working on a new webservice, which will allow implementation of the HSF system in NGS pipelines. It will take in charge VCF files and render results in JSON format.

We will let you know as soon as it will be available.

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