private static BiasResults GetProbePoolBiasScore(VariantComparisonCase Case, CalledAllele Consensus,
float ProbePoolBiasThreshold, VariantCallingParameters variantCallingParameters, int AltCountA, int AltCountB, int DepthA, int DepthB, Genotype Genotype, bool AltChangeToRef)
{
double ProbePoolPScore = 0; //no bias;
double ProbePoolGATKBiasScore = -100; //no bias;
int NoiseLevel = Consensus.NoiseLevelApplied;
BiasResults PB = new BiasResults();
if ((AltChangeToRef) || (Case == VariantComparisonCase.AgreedOnReference))
{
PB.GATKBiasScore = ProbePoolGATKBiasScore;
PB.BiasScore = ProbePoolPScore;
return(PB);
}
if ((Case == VariantComparisonCase.OneReferenceOneAlternate) ||
(Case == VariantComparisonCase.CanNotCombine))
{
Consensus.Filters.Add(FilterType.PoolBias);
PB.GATKBiasScore = 0;
PB.BiasScore = 1;
return(PB);
}
if (Case == VariantComparisonCase.AgreedOnAlternate)
{
int[] supportByPool = new int[]
{
AltCountA, AltCountB, 0
};
int[] covByPool = new int[]
{
DepthA, DepthB, 0
};
BiasResults ProbePoolBiasResults =
StrandBiasCalculator.CalculateStrandBiasResults(
covByPool, supportByPool,
NoiseLevel, variantCallingParameters.MinimumFrequency, ProbePoolBiasThreshold, StrandBiasModel.Extended);
ProbePoolGATKBiasScore = Math.Min(0, ProbePoolBiasResults.GATKBiasScore); //just cap it at upperbound 0, dont go higher.
ProbePoolGATKBiasScore = Math.Max(-100, ProbePoolGATKBiasScore); //just cap it at lowerbound -100, dont go higher.
ProbePoolPScore = Math.Min(1, ProbePoolBiasResults.BiasScore);
if (!ProbePoolBiasResults.BiasAcceptable)
{
Consensus.Filters.Add(FilterType.PoolBias);
}
}
PB.GATKBiasScore = ProbePoolGATKBiasScore;
PB.BiasScore = ProbePoolPScore;
return(PB);
}
/// <summary>
/// Combine two variants.
/// Variant B can be null.
/// We never try to combine two different variant alleles.
/// </summary>
/// <param name="VariantsA"></param>
/// <param name="VariantsB"></param>
/// <param name="ComparisonCase"></param>
/// <param name="Consensus"></param>
public AggregateAllele CombineVariants(CalledAllele VariantA, CalledAllele VariantB,
VariantComparisonCase ComparisonCase)
{
SampleAggregationParameters SampleAggregationOptions = _options.SampleAggregationParameters;
var Consensus = new AggregateAllele(new List <CalledAllele> {
VariantA, VariantB
});
int DepthA = 0;
int DepthB = 0;
//(A) set the reference data.
//this should be the same for both.
if (VariantA != null)
{
DoDefensiveGenotyping(VariantA);
Consensus.Chromosome = VariantA.Chromosome;
Consensus.ReferencePosition = VariantA.ReferencePosition;
Consensus.ReferenceAllele = VariantA.ReferenceAllele;
DepthA = VariantA.TotalCoverage;
}
if (VariantB != null)
{
DoDefensiveGenotyping(VariantB);
Consensus.Chromosome = VariantB.Chromosome;
Consensus.ReferencePosition = VariantB.ReferencePosition;
Consensus.ReferenceAllele = VariantB.ReferenceAllele;
DepthB = VariantB.TotalCoverage;
}
//normally the reference data is the same for both, no matter what the case.
//but if we have one deletion and one not, we might have different ref alleles.
//So we need to get this right.
Consensus.ReferenceAllele = CombineReferenceAlleles(VariantA, VariantB, ComparisonCase);
// (B) set the Alternate data
Consensus.AlternateAllele = CombineVariantAlleles(VariantA, VariantB, ComparisonCase);
// (C) set filters, etc.
Consensus.Filters = CombineFilters(VariantA, VariantB);
// (E) set GT data, includes calculating the probe-pool bias and quality scores
RecalculateScoring(VariantA, VariantB, ComparisonCase, Consensus, SampleAggregationOptions, _options.VariantCallingParams);
return(Consensus);
}
private static void CheckSimpleCombinations(
CalledAllele VarA, CalledAllele VarB, ConsensusBuilder cb,
Genotype ExpectedGT, string ExpectedAlt,
VariantComparisonCase ComparisonCase)
{
var AandB = cb.CombineVariants(VarA, VarB, ComparisonCase);
var BandA = cb.CombineVariants(VarB, VarA, ComparisonCase);
var AandNull = cb.CombineVariants(VarA, null, VariantComparisonCase.CanNotCombine);
var NullAndB = cb.CombineVariants(null, VarB, VariantComparisonCase.CanNotCombine);
int ExpectedAlleleCount = VarA.AlleleSupport + VarB.AlleleSupport;
if (ComparisonCase == VariantComparisonCase.OneReferenceOneAlternate)
{
//for this test, we presume we are calling alternate.
ExpectedAlleleCount = (VarA.Type == AlleleCategory.Snv) ? VarA.AlleleSupport : VarB.AlleleSupport;
}
//check alt-and-nocalls
Assert.Equal(ExpectedGT, AandB.Genotype);
Assert.Equal(VarA.ReferenceAllele, AandB.ReferenceAllele);
Assert.Equal(ExpectedAlt, AandB.AlternateAllele);
Assert.Equal(VarA.TotalCoverage + VarB.TotalCoverage, AandB.TotalCoverage);
Assert.Equal(ExpectedAlleleCount, AandB.AlleleSupport);
Assert.Equal(ExpectedGT, BandA.Genotype);
Assert.Equal(VarA.ReferenceAllele, BandA.ReferenceAllele);
Assert.Equal(ExpectedAlt, BandA.AlternateAllele);
Assert.Equal(VarA.TotalCoverage + VarB.TotalCoverage, BandA.TotalCoverage);
Assert.Equal(ExpectedAlleleCount, BandA.AlleleSupport);
Assert.Equal(ConsensusBuilder.DoDefensiveGenotyping(VarA), AandNull.Genotype);
Assert.Equal(VarA.ReferenceAllele, AandNull.ReferenceAllele);
Assert.Equal(VarA.AlternateAllele, AandNull.AlternateAllele);
Assert.Equal(VarA.TotalCoverage, AandNull.TotalCoverage);
Assert.Equal(VarA.AlleleSupport, AandNull.AlleleSupport);
Assert.Equal(ConsensusBuilder.DoDefensiveGenotyping(VarB), NullAndB.Genotype);
Assert.Equal(VarB.ReferenceAllele, NullAndB.ReferenceAllele);
Assert.Equal(VarB.AlternateAllele, NullAndB.AlternateAllele);
Assert.Equal(VarB.TotalCoverage, NullAndB.TotalCoverage);
Assert.Equal(VarB.AlleleSupport, NullAndB.AlleleSupport);
}
private void WriteVarsToVennFiles(VariantComparisonCase ComparisonCase,
CalledAllele VariantA, CalledAllele VariantB)
{
AggregateAllele AggregateA = AggregateAllele.SafeCopy(VariantA, new List <CalledAllele> {
VariantA, VariantB
});
AggregateAllele AggregateB = AggregateAllele.SafeCopy(VariantB, new List <CalledAllele> {
VariantB, VariantA
});
if (ComparisonCase == VariantComparisonCase.AgreedOnAlternate)
{
_vennDiagramWriters["AandB"].Write(new List <AggregateAllele>()
{
AggregateA
});
_vennDiagramWriters["BandA"].Write(new List <AggregateAllele>()
{
AggregateB
});
}
if ((ComparisonCase == VariantComparisonCase.OneReferenceOneAlternate) ||
(ComparisonCase == VariantComparisonCase.CanNotCombine))
{
if ((VariantA != null) && (VariantA.Type != Pisces.Domain.Types.AlleleCategory.Reference))
{
_vennDiagramWriters["AnotB"].Write(new List <CalledAllele>()
{
VariantA
});
}
if ((VariantB != null) && (VariantB.Type != Pisces.Domain.Types.AlleleCategory.Reference))
{
_vennDiagramWriters["BnotA"].Write(new List <CalledAllele>()
{
VariantB
});
}
}
}
public void VennVcf_CombineTwoPoolVariants_Qscore_DiffentNL_Test()
{
//chr3 41266161 . A G 30 PASS DP=3067 GT:GQ:AD:VF:NL:SB 0/1:30:3005,54:0.0176:35:-100.0000
CalledAllele VarA = new CalledAllele()
{
Chromosome = "chr3",
ReferencePosition = 41266161,
TotalCoverage = 3067,
Genotype = Pisces.Domain.Types.Genotype.HeterozygousAltRef,
VariantQscore = 30,
GenotypeQscore = 30,
AlleleSupport = 54,
ReferenceSupport = 3005,
NoiseLevelApplied = 35,
StrandBiasResults = new Pisces.Domain.Models.BiasResults()
{
GATKBiasScore = -100.0000
},
ReferenceAllele = "A",
AlternateAllele = "G",
Type = Pisces.Domain.Types.AlleleCategory.Snv
};
///chr3 41266161 . A . 75 PASS DP=3795 GT:GQ:AD:VF:NL:SB 0/0:75:3780:0.0040:2:-100.0000
CalledAllele VarB = new CalledAllele()
{
Chromosome = "chr3",
ReferencePosition = 41266161,
TotalCoverage = 3795,
Genotype = Pisces.Domain.Types.Genotype.HomozygousRef,
VariantQscore = 75,
GenotypeQscore = 75,
AlleleSupport = 3780,
ReferenceSupport = 3780,
NoiseLevelApplied = 2,
StrandBiasResults = new Pisces.Domain.Models.BiasResults()
{
GATKBiasScore = -100.0000
},
ReferenceAllele = "A",
AlternateAllele = ".",
Type = Pisces.Domain.Types.AlleleCategory.Reference
};
//old answer
//chr3 41266161 . A . 100.00 PASS DP=6862;cosmic=COSM1423020,COSM1423021;EVS=0|69.0|6503;phastCons GT:GQ:AD:VF:NL:SB:PB:GQX 0/0:100:6785:0.0079:35:-100:-100.0000:100
SampleAggregationParameters SampleAggregationOptions = new SampleAggregationParameters();
SampleAggregationOptions.ProbePoolBiasThreshold = 0.5f;
SampleAggregationOptions.HowToCombineQScore = SampleAggregationParameters.CombineQScoreMethod.CombinePoolsAndReCalculate;
_basicOptions.BamFilterParams.MinimumBaseCallQuality = 20;
_basicOptions.VariantCallingParams.MinimumFrequency = 0.01f;
_basicOptions.VariantCallingParams.MinimumFrequencyFilter = 0.03f;
_basicOptions.SampleAggregationParameters = SampleAggregationOptions;
string consensusOut = Path.Combine(_TestDataPath, "ConsensusOut.vcf");
VariantComparisonCase ComparisonCase = VennProcessor.GetComparisonCase(VarA, VarB);
ConsensusBuilder consensusBuilder = new ConsensusBuilder(consensusOut, _basicOptions);
AggregateAllele consensus = consensusBuilder.CombineVariants(VarA, VarB, ComparisonCase);
Console.WriteLine(consensus.ToString());
double expectedNoiseLevel = MathOperations.PtoQ(
(MathOperations.QtoP(35) + MathOperations.QtoP(2)) / (2.0)); // 5
//GT:GQ:AD:VF:NL:SB:PB:GQX 0/0:100:6785:0.0079:35:-100:-100.0000:100
Assert.NotNull(consensus);
Assert.Equal(consensus.VariantQscore, 100);
Assert.Equal(consensus.ReferenceAllele, "A");
Assert.Equal(consensus.AlternateAllele, ".");
Assert.Equal(consensus.Genotype, Pisces.Domain.Types.Genotype.HomozygousRef);
Assert.Equal(consensus.TotalCoverage, 6862);
Assert.Equal(consensus.ReferenceSupport, 6785);
Assert.Equal(consensus.AlleleSupport, 6785);
Assert.Equal(consensus.GenotypeQscore, 100);
Assert.Equal(consensus.Frequency, 0.98877877f);
Assert.Equal(consensus.NoiseLevelApplied, ((int)expectedNoiseLevel));
Assert.Equal(consensus.NoiseLevelApplied, 5);
Assert.Equal(consensus.StrandBiasResults.GATKBiasScore, -100);
Assert.Equal(consensus.PoolBiasResults.GATKBiasScore, -100.0000);
//now check, we take the min NL score if we are taking the min Q score.
// (in this case of combined alt+ref -> ref, the q score will still need to be recalculated.
//just with the MIN NL.
SampleAggregationOptions.HowToCombineQScore = SampleAggregationParameters.CombineQScoreMethod.TakeMin;
ComparisonCase = VennProcessor.GetComparisonCase(VarA, VarB);
consensus = consensusBuilder.CombineVariants(VarA, VarB, ComparisonCase);
Assert.Equal(consensus.NoiseLevelApplied, 2);
Assert.Equal(consensus.VariantQscore, 100);
//ok, now sanity check we dont barf if either input is null:
ComparisonCase = VennProcessor.GetComparisonCase(VarA, null);
consensus = consensusBuilder.CombineVariants(VarA, null, ComparisonCase);
Assert.Equal(consensus.NoiseLevelApplied, 35);
Assert.Equal(consensus.VariantQscore, 100);
ComparisonCase = VennProcessor.GetComparisonCase(null, VarB);
consensus = consensusBuilder.CombineVariants(null, VarB, ComparisonCase);
Assert.Equal(consensus.NoiseLevelApplied, 2);
Assert.Equal(consensus.VariantQscore, 100);
//ok, lets check this again, for the PoolQScores option.
//sanity check we dont barf if either input is null:
SampleAggregationOptions.HowToCombineQScore = SampleAggregationParameters.CombineQScoreMethod.CombinePoolsAndReCalculate;
ComparisonCase = VennProcessor.GetComparisonCase(VarA, null);
consensus = consensusBuilder.CombineVariants(VarA, null, ComparisonCase);
Assert.Equal(consensus.NoiseLevelApplied, 35);
Assert.Equal(consensus.VariantQscore, 100);//low freq variant -> nocall. note, qscore would be 41 if NL = 20.
ComparisonCase = VennProcessor.GetComparisonCase(null, VarB);
consensus = consensusBuilder.CombineVariants(null, VarB, ComparisonCase);
Assert.Equal(consensus.NoiseLevelApplied, 2);
Assert.Equal(consensus.VariantQscore, 100); //sold ref
}
public void VennVcf_CombineTwoPoolVariants_Qscore_Test()
{
//Var A, kdot-43_S3.genome.vcf
//chr3 41266161 . A G 30 PASS DP=3067 GT:GQ:AD:VF:NL:SB 0/1:30:3005,54:0.0176:35:-100.0000
CalledAllele VarA = new CalledAllele();
VarA.Chromosome = "chr3";
VarA.ReferencePosition = 41266161;
VarA.TotalCoverage = 3067;
VarA.Genotype = Pisces.Domain.Types.Genotype.HeterozygousAltRef;
VarA.GenotypeQscore = 30;
VarA.AlleleSupport = 54;
VarA.ReferenceSupport = 3005;
// "VF", "0.0176"
VarA.NoiseLevelApplied = 35;
VarA.StrandBiasResults = new Pisces.Domain.Models.BiasResults()
{
GATKBiasScore = -100
};
VarA.ReferenceAllele = "A";
VarA.AlternateAllele = "G";
VarA.Filters = new List <Pisces.Domain.Types.FilterType>();
VarA.VariantQscore = 30;
VarA.Type = Pisces.Domain.Types.AlleleCategory.Snv;
//Var B, kdot-43_S4.genome.vcf
//chr3 41266161 . A . 75 PASS DP=3795 GT:GQ:AD:VF:NL:SB 0/0:75:3780:0.0040:35:-100.0000
CalledAllele VarB = new CalledAllele();
VarB.Chromosome = "chr3";
VarB.ReferencePosition = 41266161;
VarB.TotalCoverage = 3795;
VarB.Genotype = Pisces.Domain.Types.Genotype.HomozygousRef;
VarB.GenotypeQscore = 75;
VarB.AlleleSupport = 3780;
VarB.ReferenceSupport = 3780;
// "VF", "0.0040"
VarB.NoiseLevelApplied = 35;
VarB.StrandBiasResults = new Pisces.Domain.Models.BiasResults()
{
GATKBiasScore = -100
};
VarB.ReferenceAllele = "A";
VarB.AlternateAllele = ".";
VarB.Filters = new List <Pisces.Domain.Types.FilterType>();
VarB.VariantQscore = 75;
VarB.Type = Pisces.Domain.Types.AlleleCategory.Reference;
//old answer
//chr3 41266161 . A . 100.00 PASS DP=6862;cosmic=COSM1423020,COSM1423021;EVS=0|69.0|6503;phastCons GT:GQ:AD:VF:NL:SB:PB:GQX 0/0:100:6785:0.0079:35:-100:-100.0000:100
VennVcfOptions parameters = new VennVcfOptions();
parameters.VariantCallingParams.MinimumFrequencyFilter = 0.03f;
parameters.VariantCallingParams.MinimumFrequency = 0.01f;
parameters.BamFilterParams.MinimumBaseCallQuality = 20;
parameters.SampleAggregationParameters.ProbePoolBiasThreshold = 0.5f;
parameters.SampleAggregationParameters.HowToCombineQScore = SampleAggregationParameters.CombineQScoreMethod.TakeMin;
VariantComparisonCase ComparisonCase = VennProcessor.GetComparisonCase(VarA, VarB);
ConsensusBuilder consensusBuilder = new ConsensusBuilder("", parameters);
AggregateAllele consensus = consensusBuilder.CombineVariants(VarA, VarB, ComparisonCase);
//GT:GQ:AD:VF:NL:SB:PB:GQX 0/0:100:6785:0.0079:35:-100:-100.0000:100
Assert.NotNull(consensus);
Assert.Equal(consensus.VariantQscore, 100);
Assert.Equal(consensus.GenotypeQscore, 100);
Assert.Equal(consensus.ReferenceAllele, "A");
Assert.Equal(consensus.AlternateAllele, ".");
Assert.Equal(consensus.Genotype, Pisces.Domain.Types.Genotype.HomozygousRef);
Assert.Equal(consensus.GenotypeQscore, 100);
Assert.Equal(consensus.AlleleSupport, 6785);
Assert.Equal(consensus.ReferenceSupport, 6785);
Assert.Equal(consensus.TotalCoverage, 6862);
Assert.Equal(consensus.Frequency, 0.98877, 4);
Assert.Equal(consensus.NoiseLevelApplied, 35);
Assert.Equal(consensus.StrandBiasResults.GATKBiasScore, -100);
Assert.Equal(consensus.PoolBiasResults.GATKBiasScore, -100);
}
public void VennVcf_CombineTwoPoolVariants_RulesAthroughD_Tests()
{
var outDir = TestPaths.LocalScratchDirectory;
var VcfPathRoot = _TestDataPath;
string OutputPath = Path.Combine(outDir, "outEandF.vcf");
if (File.Exists(OutputPath))
{
File.Delete(OutputPath);
}
VennVcfOptions parameters = new VennVcfOptions();
parameters.VariantCallingParams.MinimumFrequencyFilter = 0.03f;
parameters.VariantCallingParams.MinimumFrequency = 0.01f;
parameters.ConsensusFileName = OutputPath;
string VcfPath_PoolA = Path.Combine(VcfPathRoot, "09H-03403-MT1-1_S7.genome.vcf");
var PoolAVariants = (AlleleReader.GetAllVariantsInFile(VcfPath_PoolA)).ToList();
string VcfPath_PoolB = Path.Combine(VcfPathRoot, "09H-03403-MT1-1_S8.genome.vcf");
var PoolBVariants = (AlleleReader.GetAllVariantsInFile(VcfPath_PoolB)).ToList();
CalledAllele VariantA = PoolAVariants[0];
CalledAllele VariantB = PoolBVariants[0];
List <CalledAllele[]> pairs = VennProcessor.SelectPairs(
new List <CalledAllele>()
{
VariantA
},
new List <CalledAllele>
{
VariantB
});
VariantComparisonCase ComparisonCase = VennProcessor.GetComparisonCase(pairs[0][0], pairs[0][1]);
ConsensusBuilder consensusBuilder = new ConsensusBuilder("", parameters);
CalledAllele Consensus = consensusBuilder.CombineVariants(
VariantA, VariantB, ComparisonCase);
//Rule "A" test
//A if combined VF<1% and less than 2.6% in each pool, call REF
//(note, we were Alt in one pool and ref in another)
Assert.Equal(VariantA.Genotype, Pisces.Domain.Types.Genotype.HomozygousRef);
Assert.Equal(VariantA.Frequency, 0.9979, 4);
Assert.Equal(VariantA.VariantQscore, 100);
Assert.Equal(VariantA.Filters, new List <Pisces.Domain.Types.FilterType> {
});
Assert.Equal(VariantB.Genotype, Pisces.Domain.Types.Genotype.HeterozygousAltRef);
Assert.Equal(VariantB.Frequency, 0.0173, 4);
Assert.Equal(VariantB.VariantQscore, 100);
Assert.Equal(VariantB.Filters, new List <Pisces.Domain.Types.FilterType> {
});
Assert.Equal(ComparisonCase, VariantComparisonCase.OneReferenceOneAlternate);
Assert.Equal(Consensus.Genotype, Pisces.Domain.Types.Genotype.HomozygousRef);
Assert.Equal(Consensus.Frequency, 0.9907, 4);
Assert.Equal(Consensus.VariantQscore, 100);
Assert.Equal(Consensus.Filters, new List <Pisces.Domain.Types.FilterType> {
}); //<-low VF tag will NOT added by post-processing b/c is ref call
//B if combined VF<1% and more than 2.6% in one pool, call NO CALL
VariantA = PoolAVariants[1];
VariantB = PoolBVariants[1];
ComparisonCase = VennProcessor.GetComparisonCase(VariantA, VariantB);
Consensus = consensusBuilder.CombineVariants(
VariantA, VariantB, ComparisonCase);
Assert.Equal(VariantA.Genotype, Pisces.Domain.Types.Genotype.HeterozygousAltRef);
Assert.Equal(VariantA.Frequency, 0.0776, 4);
Assert.Equal(VariantA.VariantQscore, 100);
Assert.Equal(VariantA.Filters, new List <Pisces.Domain.Types.FilterType> {
});
Assert.Equal(VariantB.Genotype, Pisces.Domain.Types.Genotype.HomozygousRef);
Assert.Equal(VariantB.Frequency, 0.9989, 4);
Assert.Equal(VariantB.VariantQscore, 100);
Assert.Equal(VariantB.Filters, new List <Pisces.Domain.Types.FilterType> {
});
Assert.Equal(ComparisonCase, VariantComparisonCase.OneReferenceOneAlternate);
Assert.Equal(Consensus.Genotype, Pisces.Domain.Types.Genotype.AltLikeNoCall);
Assert.Equal(Consensus.Frequency, 0.0070, 4);
Assert.Equal(Consensus.VariantQscore, 0);
Assert.Equal(Consensus.Filters, new List <Pisces.Domain.Types.FilterType>
{
Pisces.Domain.Types.FilterType.PoolBias
}); //<-low VF tag will also get added by post-processing
//Rule "Ca" test
//C-a if combined 1%<VF<2.6%
// and more than 2.6% in one pool and less than 1% in the other, call NO CALL w/PB
VariantA = PoolAVariants[2];
VariantB = PoolBVariants[2];
ComparisonCase = VennProcessor.GetComparisonCase(VariantA, VariantB);
Consensus = consensusBuilder.CombineVariants(
VariantA, VariantB, ComparisonCase);
Assert.Equal(VariantA.Genotype, Pisces.Domain.Types.Genotype.HeterozygousAltRef);
Assert.Equal(VariantA.Frequency, 0.0367, 4);
Assert.Equal(VariantA.VariantQscore, 100);
Assert.Equal(VariantA.Filters, new List <Pisces.Domain.Types.FilterType> {
});
Assert.Equal(VariantB.Genotype, Pisces.Domain.Types.Genotype.HomozygousRef);
Assert.Equal(VariantB.Frequency, 0.9976, 4);
Assert.Equal(VariantB.VariantQscore, 100);
Assert.Equal(VariantB.Filters, new List <Pisces.Domain.Types.FilterType> {
});
Assert.Equal(ComparisonCase, VariantComparisonCase.OneReferenceOneAlternate);
Assert.Equal(Consensus.Genotype, Pisces.Domain.Types.Genotype.AltLikeNoCall);
Assert.Equal(Consensus.Frequency, 0.0117, 4);
Assert.Equal(Consensus.VariantQscore, 23);
Assert.Equal(Consensus.Filters, new List <Pisces.Domain.Types.FilterType> {
Pisces.Domain.Types.FilterType.PoolBias
});
//Rule "Cb" test
//C-a if combined 1%<VF<2.6%
// and more than 2.6% in one pool and between 1% and 2.6% in the other, call NO CALL w/ no PB
VariantA = PoolAVariants[3];
VariantB = PoolBVariants[3];
ComparisonCase = VennProcessor.GetComparisonCase(VariantA, VariantB);
Consensus = consensusBuilder.CombineVariants(
VariantA, VariantB, ComparisonCase);
Assert.Equal(VariantA.Genotype, Pisces.Domain.Types.Genotype.HeterozygousAltRef);
Assert.Equal(VariantA.Frequency, 0.01725, 4);
Assert.Equal(VariantA.VariantQscore, 100);
Assert.Equal(VariantA.Filters, new List <Pisces.Domain.Types.FilterType> {
});
Assert.Equal(VariantB.Genotype, Pisces.Domain.Types.Genotype.HeterozygousAltRef);
Assert.Equal(VariantB.Frequency, 0.03667, 4);
Assert.Equal(VariantB.VariantQscore, 100);
Assert.Equal(VariantB.Filters, new List <Pisces.Domain.Types.FilterType> {
});
Assert.Equal(ComparisonCase, VariantComparisonCase.AgreedOnAlternate);
Assert.Equal(Consensus.Genotype, Pisces.Domain.Types.Genotype.AltLikeNoCall);
Assert.Equal(Consensus.Frequency, 0.02347, 4);
Assert.Equal(Consensus.VariantQscore, 100);
Assert.Equal(Consensus.Filters, new List <Pisces.Domain.Types.FilterType> {
}); //<-low VF tag will also get added by post-processing
//Rule "D" test
//D if combined VF>=2.6% call VARIANT (PB if only present in one pool, using 1% as the cutoff)
VariantA = PoolAVariants[4];
VariantB = PoolBVariants[4];
ComparisonCase = VennProcessor.GetComparisonCase(VariantA, VariantB);
Consensus = consensusBuilder.CombineVariants(
VariantA, VariantB, ComparisonCase);
Assert.Equal(VariantA.Genotype, Pisces.Domain.Types.Genotype.HeterozygousAltRef);
Assert.Equal(VariantA.Frequency, 0.2509, 4);
Assert.Equal(VariantA.VariantQscore, 100);
Assert.Equal(VariantA.Filters, new List <Pisces.Domain.Types.FilterType> {
});
Assert.Equal(VariantB.Genotype, Pisces.Domain.Types.Genotype.HeterozygousAltRef);
Assert.Equal(VariantB.Frequency, 0.0367, 4);
Assert.Equal(VariantB.VariantQscore, 100);
Assert.Equal(VariantB.Filters, new List <Pisces.Domain.Types.FilterType> {
});
Assert.Equal(ComparisonCase, VariantComparisonCase.AgreedOnAlternate);
Assert.Equal(Consensus.Genotype, Pisces.Domain.Types.Genotype.HeterozygousAltRef);
Assert.Equal(Consensus.Frequency, 0.1716, 4);
Assert.Equal(Consensus.VariantQscore, 100);
Assert.Equal(Consensus.Filters, new List <Pisces.Domain.Types.FilterType> {
}); //<-low VF tag will also get set by post processor
}
private static Genotype GetGenotype(CalledAllele VariantA, CalledAllele VariantB, VariantComparisonCase Case,
int TotalDepth, double VarFrequency, double VarFrequencyA, double VarFrequencyB, SampleAggregationParameters SampleAggregationOptions, VariantCallingParameters variantCallingParameters)
{
var gtA = Genotype.RefLikeNoCall;
var gtB = Genotype.RefLikeNoCall;
var tempGT = Genotype.RefLikeNoCall;
if (VariantB != null)
{
gtB = VariantB.Genotype;
}
if (VariantA != null)
{
gtA = VariantA.Genotype;
}
//cases: {0/0 , 0/1, 1/1, ./.} , choose 2.
//if (A == B) GTString = A;
bool RefPresent = ((VariantA != null && VariantA.HasARefAllele) || (VariantB != null && VariantB.HasARefAllele));
bool AltPresent = ((VariantA != null && VariantA.HasAnAltAllele) || (VariantB != null && VariantB.HasAnAltAllele));
if (!AltPresent && RefPresent)
{
tempGT = Genotype.HomozygousRef;
}
else if (AltPresent && RefPresent)
{
tempGT = Genotype.HeterozygousAltRef;
}
else if (AltPresent && !RefPresent)
{
tempGT = Genotype.HomozygousAlt;
//todo, expand to cover nocalls and heterozygous calls.
}
else //(no alt and no reference detected.)
{
tempGT = Genotype.RefLikeNoCall;
}
//if its no call, thats fine. we are done
if (tempGT == Genotype.RefLikeNoCall)
{
return(tempGT);
}
//if the merged GT implies a variant call,
//it has to pass some minimal criteria, or it gets
//re-classified as a ref type or a no-call.
//So. now, check the combined result passed some minimum criteria:
//First, never call it a Variant if the combined freq
//is smaller than the reporting threshold.
//If the freq is low, we should call "0/0" or "./.". , but not "1/1" or "0/1"
//So change any "0/1"s or "1/1"s over to "./.".
//if we would have called a variant... but...
if (Case != VariantComparisonCase.AgreedOnReference)
{
// ifcombined freq <1% and both per-pool freq <3% -> 0/0
// ifcombined freq <1% and a per-pool freq >3% -> ./.
// ifcombined freq >1% and <3% -> ./.
//if combined freq <1%
if (VarFrequency < variantCallingParameters.MinimumFrequency)
{
//if its < 3% in both pools but still <1% overall
if ((VarFrequencyA < variantCallingParameters.MinimumFrequencyFilter) &&
(VarFrequencyB < variantCallingParameters.MinimumFrequencyFilter))
{
tempGT = Genotype.HomozygousRef;
}
else //if its > 3% in at least one pool but still <1% overall
{
tempGT = Genotype.AltLikeNoCall;
}
}
else if (VarFrequency < variantCallingParameters.MinimumFrequencyFilter)
{//if combined freq more than 1% but still < 3%
tempGT = Genotype.AltLikeNoCall;
}
//next - we have to clean up any multiple allelic sites.
}
// also, dont call it a variant *or* a reference
// if the combined Depth is less than the minimum.
// (this case is defensive programing. The SVC should already call
// each pool variant as ".\." , due to indiviudal low depth,
// so the combined results
// shoud already be ".\." by default. )
else if (TotalDepth < variantCallingParameters.MinimumCoverage)
{
// note, this could happen even though your input variants are one 'no call' and one 'var',
//or even two variants-failing-filters.
tempGT = Genotype.RefLikeNoCall;
}
return(tempGT);
}
private static bool PushThroughRamificationsOfGTChange(CalledAllele VariantA, CalledAllele VariantB,
CalledAllele Consensus, int RefCountA, int RefCountB, int DepthA, int DepthB,
Genotype GT, int MaxQScore, VariantComparisonCase Case)
{
int NoiseLevel = Consensus.NoiseLevelApplied;
if (((GT == Genotype.HomozygousRef) || (GT == Genotype.RefLikeNoCall)) && (Case == VariantComparisonCase.OneReferenceOneAlternate))
{
Consensus.AlternateAllele = ".";
Consensus.ReferenceAllele = Consensus.ReferenceAllele.Substring(0, 1);
if (VariantA != null)
{
VariantA.VariantQscore = VariantQualityCalculator.AssignPoissonQScore(
RefCountA, DepthA, NoiseLevel, MaxQScore);
}
if (VariantB != null)
{
VariantB.VariantQscore = VariantQualityCalculator.AssignPoissonQScore(
RefCountB, DepthB, NoiseLevel, MaxQScore);
}
Consensus.AlleleSupport = Consensus.ReferenceSupport;
return(true);
}
return(false);
}
private static void RecalculateScoring(CalledAllele VariantA, CalledAllele VariantB,
VariantComparisonCase Case,
AggregateAllele ConsensusAllele, SampleAggregationParameters SampleAggregationOptions, VariantCallingParameters variantCallingParameters)
{
int RefCountB = 0, RefCountA = 0;
int AltCountB = 0, AltCountA = 0;
int DepthA = 0;
int DepthB = 0;
//1) first, calculate all the component values (variant frequency, etc...)
if (VariantA != null)
{
RefCountA = VariantA.ReferenceSupport;
AltCountA = (VariantA.IsRefType) ? 0 : VariantA.AlleleSupport;
DepthA = VariantA.TotalCoverage;
}
if (VariantB != null)
{
RefCountB = VariantB.ReferenceSupport;
AltCountB = (VariantB.IsRefType) ? 0 : VariantB.AlleleSupport;
DepthB = VariantB.TotalCoverage;
}
int TotalDepth = DepthA + DepthB;
int ReferenceDepth = RefCountA + RefCountB;
int AltDepth = AltCountA + AltCountB;
double VarFrequency = ((AltDepth == 0) || (TotalDepth == 0)) ? 0.0 : ((double)AltDepth) / ((double)(TotalDepth));
double VarFrequencyA = ((AltCountA == 0) || (DepthA == 0)) ? 0.0 : ((double)AltCountA) / ((double)(DepthA));
double VarFrequencyB = ((AltCountB == 0) || (DepthB == 0)) ? 0.0 : ((double)AltCountB) / ((double)(DepthB));
ConsensusAllele.TotalCoverage = TotalDepth;
ConsensusAllele.AlleleSupport = AltDepth;
ConsensusAllele.ReferenceSupport = ReferenceDepth;
var GT = GetGenotype(VariantA, VariantB, Case,
TotalDepth, VarFrequency, VarFrequencyA, VarFrequencyB, SampleAggregationOptions, variantCallingParameters);
ConsensusAllele.NoiseLevelApplied = GetCombinedNLValue(VariantA, VariantB, SampleAggregationOptions);
ConsensusAllele.StrandBiasResults = GetCombinedSBValue(VariantA, VariantB, SampleAggregationOptions);
//its possible the GTString went from var -> ref when we combined the results.
//If that is the case we do not want to write "variant" anymore to the .vcf.
//We also have to re-calculate the Q scores for a reference call.
//They need to be based on a reference model, not a variant model.
bool AltChangedToRef = PushThroughRamificationsOfGTChange(VariantA, VariantB,
ConsensusAllele, RefCountA, RefCountB, DepthA, DepthB, GT,
variantCallingParameters.MaximumVariantQScore, Case);
ConsensusAllele.Genotype = GT;
ConsensusAllele.PoolBiasResults = GetProbePoolBiasScore(Case, ConsensusAllele,
SampleAggregationOptions.ProbePoolBiasThreshold, variantCallingParameters, AltCountA, AltCountB, DepthA, DepthB, GT, AltChangedToRef);
if (SampleAggregationOptions.HowToCombineQScore == SampleAggregationParameters.CombineQScoreMethod.TakeMin)
{
ConsensusAllele.VariantQscore = CombineQualitiesByTakingMinValue(VariantA, VariantB);
}
else //VariantCallingCombinePoolSettings.CombineQScoreMethod.CombinePoolsAndReCalculate
{
//where we apply the reference Q model:
if (Case == VariantComparisonCase.AgreedOnReference)
{
ConsensusAllele.VariantQscore = CombineQualitiesByPoolingReads(ReferenceDepth, TotalDepth, ConsensusAllele.NoiseLevelApplied,
variantCallingParameters.MaximumVariantQScore);
}
else if ((Case == VariantComparisonCase.OneReferenceOneAlternate) && (AltChangedToRef))
{
ConsensusAllele.VariantQscore = CombineQualitiesByPoolingReads(ReferenceDepth, TotalDepth, ConsensusAllele.NoiseLevelApplied,
variantCallingParameters.MaximumVariantQScore);
}
else if ((Case == VariantComparisonCase.CanNotCombine) && (AltDepth == 0)) //so the only call we had must have been ref
{
ConsensusAllele.VariantQscore = CombineQualitiesByPoolingReads(ReferenceDepth, TotalDepth, ConsensusAllele.NoiseLevelApplied,
variantCallingParameters.MaximumVariantQScore);
}
//where we apply the variant Q model. this is most cases
else // cases are aggreed on alt, or one alt call. in which case, apply variant Q model.
{
ConsensusAllele.VariantQscore = CombineQualitiesByPoolingReads(AltDepth, TotalDepth, ConsensusAllele.NoiseLevelApplied,
variantCallingParameters.MaximumVariantQScore);
}
}
//assuming this is only used on Somatic...
ConsensusAllele.GenotypeQscore = ConsensusAllele.VariantQscore;
ConsensusAllele.SetType();
if (ConsensusAllele.IsRefType)
{
ConsensusAllele.AlleleSupport = ConsensusAllele.ReferenceSupport;
}
}
private static string CombineVariantAlleles(CalledAllele VariantA, CalledAllele VariantB, VariantComparisonCase ComparisonCase)
{
if (ComparisonCase == VariantComparisonCase.AgreedOnReference)
{
return(".");
}
if (ComparisonCase == VariantComparisonCase.AgreedOnAlternate)
{
return(VariantA.AlternateAllele);
}
if (ComparisonCase == VariantComparisonCase.OneReferenceOneAlternate)
{
CalledAllele NonRef = (VariantA.Type == AlleleCategory.Reference) ? VariantB : VariantA;
return(NonRef.AlternateAllele);
}
if ((ComparisonCase == VariantComparisonCase.CanNotCombine))
{
//if we have two different alleles, then we shoud only be trying to combine one at once.
if (VariantB == null)
{
return(VariantA.AlternateAllele);
}
else
{
return(VariantB.AlternateAllele);
}
}
else
{
throw new ArgumentException("Option not supported");
}
}
