public static void Process(CalledAllele allele,
float minFrequency, int?lowDepthFilter, int?filterVariantQscore, bool filterSingleStrandVariants, float?variantFreqFilter, float?lowGqFilter, int?indelRepeatFilter,
RMxNFilterSettings rMxNFilterSettings, float?noCallFilter, float?ampliconBiasFilter, ChrReference chrReference, bool isStitchedSource = false)
{
allele.SetFractionNoCalls();
ApplyFilters(allele, lowDepthFilter, filterVariantQscore, filterSingleStrandVariants, variantFreqFilter, lowGqFilter, indelRepeatFilter, rMxNFilterSettings, noCallFilter, ampliconBiasFilter, isStitchedSource, chrReference);
}
private static void ApplyFilters(CalledAllele allele, int?minCoverageFilter, int?variantQscoreThreshold, bool filterSingleStrandVariants, float?variantFreqFilter, float?lowGenotypeqFilter, int?indelRepeatFilter,
RMxNFilterSettings rMxNFilterSettings, float?noCallFilter, bool hasStitchedSource, ChrReference chrReference)
{
//Reset filters
allele.Filters.Clear();
if (minCoverageFilter.HasValue && allele.TotalCoverage < minCoverageFilter)
{
allele.AddFilter(FilterType.LowDepth);
}
if (variantQscoreThreshold.HasValue && allele.VariantQscore < variantQscoreThreshold && (allele.TotalCoverage != 0))
{
//note we wont flag it for Qscore, if its got zero depth, because in that case, the Q score calc was not made anyway.
allele.AddFilter(FilterType.LowVariantQscore);
}
if (allele.Type != AlleleCategory.Reference)
{
//No call filter
if (noCallFilter.HasValue && allele.FractionNoCalls > noCallFilter)
{
allele.AddFilter(FilterType.NoCall);
}
if (!allele.StrandBiasResults.BiasAcceptable ||
(filterSingleStrandVariants && !allele.StrandBiasResults.VarPresentOnBothStrands))
{
allele.AddFilter(FilterType.StrandBias);
}
if (indelRepeatFilter.HasValue && indelRepeatFilter > 0)
{
var indelRepeatLength = ComputeIndelRepeatLength(allele, chrReference.Sequence);
if (indelRepeatFilter <= indelRepeatLength)
{
allele.AddFilter(FilterType.IndelRepeatLength);
}
}
if (RMxNCalculator.ShouldFilter(allele, rMxNFilterSettings, chrReference.Sequence))
{
allele.AddFilter(FilterType.RMxN);
}
if (variantFreqFilter.HasValue && allele.Frequency < variantFreqFilter)
{
allele.AddFilter(FilterType.LowVariantFrequency);
}
if (hasStitchedSource) //can only happen for insertions and MNVs
{
if (allele.AlternateAllele.Contains("N"))
{
allele.AddFilter(FilterType.StrandBias);
}
}
}
}
private void ExecuteRMxNTest(string variantBases, string referenceSequence, int expectedRepeatLength, AlleleCategory category, int maxRepeatUnitLength)
{
var alleleCoordinate = referenceSequence.IndexOf('*');
var cleanReferenceSequence = referenceSequence.Replace("*", "");
var refAllele = "";
var altAllele = "";
if (category == AlleleCategory.Insertion)
{
refAllele = cleanReferenceSequence.Substring(alleleCoordinate - 1, 1);
altAllele = refAllele + variantBases;
}
else if (category == AlleleCategory.Deletion)
{
altAllele = cleanReferenceSequence.Substring(alleleCoordinate - 1, 1);
refAllele = altAllele + variantBases;
}
else
{
refAllele = cleanReferenceSequence.Substring(alleleCoordinate - 1, variantBases.Length);
altAllele = variantBases;
}
var allele = new CalledAllele(category)
{
ReferenceAllele = refAllele,
AlternateAllele = altAllele,
ReferencePosition = alleleCoordinate
};
RMxNFilterSettings rmxnFilterSettings = new RMxNFilterSettings();
rmxnFilterSettings.RMxNFilterFrequencyLimit = 1.1f;
rmxnFilterSettings.RMxNFilterMaxLengthRepeat = maxRepeatUnitLength;
rmxnFilterSettings.RMxNFilterMinRepetitions = expectedRepeatLength;
// If expected repeats == N, flag
AlleleProcessor.Process(allele, 0.01f, 0, 0,
true, 0, 0, null, rmxnFilterSettings, 0.6f, new ChrReference()
{
Sequence = cleanReferenceSequence
});
Assert.True(allele.Filters.Contains(FilterType.RMxN));
// If expected repeats > N, flag
rmxnFilterSettings.RMxNFilterMinRepetitions = expectedRepeatLength - 1;
AlleleProcessor.Process(allele, 0.01f, 0, 0,
true, 0, 0, null, rmxnFilterSettings, 0.6f, new ChrReference()
{
Sequence = cleanReferenceSequence
});
Assert.True(allele.Filters.Contains(FilterType.RMxN));
// If expected repeats < N, flag
rmxnFilterSettings.RMxNFilterMinRepetitions = expectedRepeatLength + 1;
AlleleProcessor.Process(allele, 0.01f, 0, 0,
true, 0, 0, null, rmxnFilterSettings, 0.6f, new ChrReference()
{
Sequence = cleanReferenceSequence
});
Assert.False(allele.Filters.Contains(FilterType.RMxN));
// RMxN isn't valid on reference calls
rmxnFilterSettings.RMxNFilterMinRepetitions = expectedRepeatLength + 1;
AlleleProcessor.Process(new CalledAllele()
{
}, 0.01f, 0, 0,
true, 0, 0, null, rmxnFilterSettings, 0.6f, new ChrReference()
{
Sequence = cleanReferenceSequence
});
Assert.False(allele.Filters.Contains(FilterType.RMxN));
}
private void ExecuteRMxNTest(string variantBases, string referenceSequence, int expectedRepeatLength, AlleleCategory category, double allelefrequency, int maxRepeatUnitLength)
{
var alleleCoordinate = referenceSequence.IndexOf('*');
var cleanReferenceSequence = referenceSequence.Replace("*", "");
var refAllele = "";
var altAllele = "";
if (category == AlleleCategory.Insertion)
{
refAllele = cleanReferenceSequence.Substring(alleleCoordinate - 1, 1);
altAllele = refAllele + variantBases;
}
else if (category == AlleleCategory.Deletion)
{
altAllele = cleanReferenceSequence.Substring(alleleCoordinate - 1, 1);
refAllele = altAllele + variantBases;
}
else
{
refAllele = cleanReferenceSequence.Substring(alleleCoordinate - 1, variantBases.Length);
altAllele = variantBases;
}
var allele = new CalledAllele(category)
{
Reference = refAllele,
Alternate = altAllele,
Coordinate = alleleCoordinate
};
allele.TotalCoverage = 1000;
allele.AlleleSupport = (int)(1000.0 * variantFrequncy);
RMxNFilterSettings rmxnFilterSettings = new RMxNFilterSettings();
rmxnFilterSettings.RMxNFilterFrequencyLimit = 1.1f;
rmxnFilterSettings.RMxNFilterMaxLengthRepeat = maxRepeatUnitLength;
rmxnFilterSettings.RMxNFilterMinRepetitions = expectedRepeatLength;
// If expected repeats == N, flag
Assert.True(RMxNCalculator.ShouldFilter(allele, rmxnFilterSettings, cleanReferenceSequence));
// If expected repeats > N, flag
rmxnFilterSettings.RMxNFilterMinRepetitions = expectedRepeatLength - 1;
Assert.True(RMxNCalculator.ShouldFilter(allele, rmxnFilterSettings, cleanReferenceSequence));
// If expected repeats < N, flag
rmxnFilterSettings.RMxNFilterMinRepetitions = expectedRepeatLength + 1;
Assert.False(RMxNCalculator.ShouldFilter(allele, rmxnFilterSettings, cleanReferenceSequence));
// RMxN isn't valid on reference calls
rmxnFilterSettings.RMxNFilterMinRepetitions = expectedRepeatLength + 1;
Assert.False(RMxNCalculator.ShouldFilter(allele, rmxnFilterSettings, cleanReferenceSequence));
// Even if expected repeats == N, dont flag if VF is too high
rmxnFilterSettings.RMxNFilterFrequencyLimit = 0.10f;
rmxnFilterSettings.RMxNFilterMinRepetitions = expectedRepeatLength;
Assert.False(RMxNCalculator.ShouldFilter(allele, rmxnFilterSettings, cleanReferenceSequence));
// Even if expected repeats > N, flag, dont flag if VF is too high
rmxnFilterSettings.RMxNFilterFrequencyLimit = 0.10f;
rmxnFilterSettings.RMxNFilterMinRepetitions = expectedRepeatLength - 1;
Assert.False(RMxNCalculator.ShouldFilter(allele, rmxnFilterSettings, cleanReferenceSequence));
}
