public static void TestExceptionThrownForUnclippedAlignment()
{
var refseq = "ACAATATA";
var queryseq = "ACAATAT-";
var r = new Sequence(DnaAlphabet.Instance, refseq);
var q = new Sequence(DnaAlphabet.Instance, queryseq);
var aln = new PairwiseSequenceAlignment(r, q);
var pas = new PairwiseAlignedSequence();
pas.FirstSequence = r;
pas.SecondSequence = q;
aln.Add(pas);
Assert.Throws <FormatException> (() => VariantCaller.LeftAlignIndelsAndCallVariants(aln, true));
refseq = "AAACAATATA";
queryseq = "AA-CAATATA";
r = new Sequence(DnaAlphabet.Instance, refseq);
q = new Sequence(DnaAlphabet.Instance, queryseq);
aln = new PairwiseSequenceAlignment(r, q);
pas = new PairwiseAlignedSequence();
pas.FirstSequence = r;
pas.SecondSequence = q;
aln.Add(pas);
Assert.Throws <FormatException> (() => VariantCaller.LeftAlignIndelsAndCallVariants(aln, true));
}
public void CallThroughAnEmptyNbhd()
{
var originalVcfVariant = TestHelper.CreateDummyAllele("chr1", 123, "A", "T", 1000, 156);
var originalVcfVariant2 = TestHelper.CreateDummyAllele("chr1", 124, "A", "T", 1000, 156);
var vs1 = new VariantSite(originalVcfVariant);
var vs2 = new VariantSite(originalVcfVariant2);
var caller = new VariantCaller(new VariantCallingParameters(), new BamFilterParameters());
//since there is an alt at position 124 ( a call of 156 alt / 1000 total, that means 844 original ref calls.
//Of which we said, 100 will get sucked up. So that leaves 744 / 1000 calls for a reference.
//So, we can still make a confident ref call. (we will call it 0/., since we know its not a homozygous ref)
var nbhd = new VcfNeighborhood(new VariantCallingParameters(), 0, "chr1", vs1, vs2, "");
nbhd.SetRangeOfInterest();
caller.CallMNVs(nbhd);
caller.CallRefs(nbhd);
var acceptedMNVs = nbhd.CalledVariants;
var acceptedRefs = nbhd.CalledRefs;
Assert.Equal(0, acceptedMNVs.Count);
Assert.Equal(2, acceptedRefs.Count);
Assert.Equal(Genotype.RefAndNoCall, acceptedRefs[123].Genotype);
Assert.Equal(Genotype.RefAndNoCall, acceptedRefs[124].Genotype);
Assert.Equal(123, acceptedRefs[123].ReferencePosition);
Assert.Equal(124, acceptedRefs[124].ReferencePosition);
}
public static void Test1BPDeletionCall()
{
string seq1seq = "ATACCCCTT";
string seq2seq = "ATA-CCCTT".Replace("-", String.Empty);
int[] seq2qual = new int[] { 30, 30, 30, 2, 30, 30, 30, 30 };
var refseq = new Sequence(AmbiguousDnaAlphabet.Instance, seq1seq, false);
var query = new Sequence(AmbiguousDnaAlphabet.Instance, seq2seq, false);
NeedlemanWunschAligner aligner = new NeedlemanWunschAligner();
var aln = aligner.Align(refseq, query).First();
// Need to add in the QV Values.
ConvertAlignedSequenceToQualSeq(aln, seq2qual);
var variants = VariantCaller.CallVariants(aln);
Assert.AreEqual(variants.Count, 1);
var variant = variants.First();
Assert.AreEqual(2, variant.QV);
Assert.AreEqual(2, variant.StartPosition);
Assert.AreEqual(VariantType.INDEL, variant.Type);
var vi = variant as IndelVariant;
Assert.AreEqual("C", vi.InsertedOrDeletedBases);
Assert.AreEqual('C', vi.HomopolymerBase);
Assert.AreEqual(4, vi.HomopolymerLengthInReference);
Assert.AreEqual(true, vi.InHomopolymer);
Assert.AreEqual(vi.InsertionOrDeletion, IndelType.Deletion);
}
public static void TestTrickyQVInversions()
{
// This will be hard because normally flip the QV value for a homopolymer, but in this case we won't.
// Note the whole notion of flipping is poorly defined.
string seq1seq = "ATTGC";
string seq2seq = "ATAGC";
int[] seq2qual = new int[] { 30, 30, 2, 30, 30 };
var refseq = new Sequence(DnaAlphabet.Instance, seq1seq);
var query = new Sequence(DnaAlphabet.Instance, seq2seq);
var s1rc = refseq.GetReverseComplementedSequence();
var s2rc = query.GetReverseComplementedSequence();
NeedlemanWunschAligner aligner = new NeedlemanWunschAligner();
var aln = aligner.Align(s1rc, s2rc).First();
VariantCallTests.ConvertAlignedSequenceToQualSeq(aln, seq2qual.Reverse().ToArray());
aln.PairwiseAlignedSequences [0].Sequences [1].MarkAsReverseComplement();
var variants = VariantCaller.CallVariants(aln);
Assert.AreEqual(1, variants.Count);
var variant = variants.First();
Assert.AreEqual(VariantType.SNP, variant.Type);
Assert.AreEqual(2, variant.QV);
var vs = variant as SNPVariant;
Assert.AreEqual('T', vs.AltBP);
Assert.AreEqual('A', vs.RefBP);
}
public static void TestSNPCallAtStart()
{
string seq1seq = "CTCCCCCTT";
string seq2seq = "TTCCCCCTT";
int[] seq2qual = new int[] { 10, 30, 30, 30, 5, 3, 30, 30, 10 };
var refseq = new Sequence(DnaAlphabet.Instance, seq1seq);
var query = new Sequence(DnaAlphabet.Instance, seq2seq);
NeedlemanWunschAligner aligner = new NeedlemanWunschAligner();
var aln = aligner.Align(refseq, query).First();
ConvertAlignedSequenceToQualSeq(aln, seq2qual);
var variants = VariantCaller.CallVariants(aln);
Assert.AreEqual(variants.Count, 1);
var variant = variants.First();
Assert.AreEqual(10, variant.QV);
Assert.AreEqual(0, variant.StartPosition);
Assert.AreEqual(variant.Type, VariantType.SNP);
var vi = variant as SNPVariant;
Assert.AreEqual(1, vi.Length);
Assert.AreEqual('T', vi.AltBP);
Assert.AreEqual('C', vi.RefBP);
Assert.AreEqual(VariantType.SNP, vi.Type);
Assert.AreEqual(true, vi.AtEndOfAlignment);
}
public void CheckAddingFilters()
{
var originalVcfVariant = TestHelper.CreateDummyAllele("chr1", 123, "A", "T", 1000, 156);
var originalVcfVariant2 = TestHelper.CreateDummyAllele("chr1", 124, "A", "T", 1000, 156);
var vs1 = new VariantSite(originalVcfVariant);
var vs2 = new VariantSite(originalVcfVariant2);
var variantCallingParameters = new VariantCallingParameters();
//Set up filters so calls are sure to trigger them.
variantCallingParameters.LowDepthFilter = 2000;
variantCallingParameters.MinimumFrequencyFilter = 0.80F;
variantCallingParameters.MinimumVariantQScoreFilter = 300;
var caller = new VariantCaller(variantCallingParameters, new BamFilterParameters());
var nbhd = new VcfNeighborhood(new VariantCallingParameters(), 0, "chr1", vs1, vs2, "");
nbhd.SetRangeOfInterest();
nbhd.AddAcceptedPhasedVariant(
new CalledAllele(AlleleCategory.Snv)
{
Chromosome = "chr1",
ReferencePosition = 123,
ReferenceAllele = "A",
AlternateAllele = "T",
VariantQscore = 100,
TotalCoverage = 1000,
AlleleSupport = 500
});
nbhd.UsedRefCountsLookup = new Dictionary <int, SuckedUpRefRecord>()
{
};
caller.CallMNVs(nbhd);
caller.CallRefs(nbhd);
var acceptedMNVs = nbhd.CalledVariants;
var acceptedRefs = nbhd.CalledRefs;
Assert.Equal(1, acceptedMNVs.Count);
Assert.Equal(1, acceptedMNVs[123].Count);
Assert.True(acceptedMNVs[123][0].Filters.Contains(FilterType.LowDepth));
Assert.True(acceptedMNVs[123][0].Filters.Contains(FilterType.LowVariantFrequency));
Assert.True(acceptedMNVs[123][0].Filters.Contains(FilterType.LowVariantQscore));
Assert.Equal(2, acceptedRefs.Count);
Assert.True(acceptedRefs[123].Filters.Contains(FilterType.LowDepth));
Assert.True(acceptedRefs[123].Filters.Contains(FilterType.LowVariantQscore));
//note reference calls dont win the "LowVariantFrequency" flag.
}
public static void TestLeftAlignmentStep()
{
var refseq = "ACAATAAAAGCGCGCGCGCGTTACGTATAT--ATGGATAT";
var queryseq = "ACAATAA-AGC--GCGC--GTTACGTATATATATGGATAT";
var r = new Sequence(DnaAlphabet.Instance, refseq);
var q = new Sequence(DnaAlphabet.Instance, queryseq);
var aln = new PairwiseSequenceAlignment(r, q);
var pas = new PairwiseAlignedSequence();
pas.FirstSequence = r;
pas.SecondSequence = q;
aln.Add(pas);
var tpl = VariantCaller.LeftAlignIndelsAndCallVariants(aln, true);
// Check the left alignment
aln = tpl.Item1 as PairwiseSequenceAlignment;
var lar = aln.PairwiseAlignedSequences [0].FirstSequence.ConvertToString();
var laq = aln.PairwiseAlignedSequences [0].SecondSequence.ConvertToString();
var exprefseq = "ACAATAAAAGCGCGCGCGCGTTACG--TATATATGGATAT";
var expqueryseq = "ACAAT-AAA----GCGCGCGTTACGTATATATATGGATAT";
Assert.AreEqual(exprefseq, lar);
Assert.AreEqual(expqueryseq, laq);
// And it's hard, so we might as well check the variants
var variants = tpl.Item2;
Assert.AreEqual(3, variants.Count);
string[] bases = new string[] { "A", "GCGC", "TA" };
char[] hpbases = new char[] { 'A', 'G', 'T' };
bool[] inHp = new bool[] { true, false, false };
int[] lengths = new int[] { 1, 4, 2 };
int[] starts = new int[] { 4, 8, 24 };
IndelType[] types = new IndelType[] { IndelType.Deletion, IndelType.Deletion, IndelType.Insertion };
for (int i = 0; i < 3; i++)
{
Assert.AreEqual(VariantType.INDEL, variants [i].Type);
var vi = variants [i] as IndelVariant;
Assert.AreEqual(hpbases[i], vi.HomopolymerBase);
Assert.AreEqual(starts [i], vi.StartPosition);
Assert.AreEqual(lengths [i], vi.Length);
Assert.AreEqual(bases [i], vi.InsertedOrDeletedBases);
Assert.AreEqual(inHp [i], vi.InHomopolymer);
Assert.AreEqual(types [i], vi.InsertionOrDeletion);
}
}
public void TestGetGtLogLikelihoodScore()
{
var copyNumberModelFactory = new HaplotypeCopyNumberModelFactory();
var copyNumberModel = copyNumberModelFactory.CreateModel(numCnStates: 5, maxCoverage: 200,
meanCoverage: 100, diploidAlleleMeanCounts: 50.0);
var simulatedCn = 3;
var gtModelCounts = PedigreeInfo.GeneratePhasedGenotype(numCnStates: 5).Where(gt => gt.TotalCopyNumber == simulatedCn)
.Select(gt => gt.PhasedGenotype).ToList();
var gtObservedCounts = new Balleles(new List <Ballele>
{
new Ballele(1, 1, 73),
new Ballele(100, 2, 74),
new Ballele(200, 1, 76),
new Ballele(300, 0, 74),
new Ballele(400, 1, 75),
new Ballele(500, 2, 74)
});
var gt0_3 = new PhasedGenotype(3, 0);
int?selectedGtState = null;
// variant caller only calls MCC, only upper triangle of CN genotypes is selected - i.e. CNa=3,CNb=0 from [CNa=3,CNb=0,CNa=0,CNb=3]
double logLikelihoodScore =
VariantCaller.GetGtLogLikelihoodScore(gtObservedCounts, gtModelCounts, ref selectedGtState,
copyNumberModel);
Assert.Equal(gtModelCounts.IndexOf(gt0_3), selectedGtState);
gtObservedCounts = new Balleles(new List <Ballele>
{
new Ballele(1, 23, 53),
new Ballele(100, 22, 54),
new Ballele(200, 25, 46),
new Ballele(300, 24, 50),
new Ballele(400, 26, 51),
new Ballele(500, 24, 51)
});
var gt1_2 = new PhasedGenotype(2, 1);
selectedGtState = null;
// variant caller only calls MCC, only upper triangle of CN genotypes is selected - i.e. CNa=3,CNb=0 from [CNa=3,CNb=0,CNa=0,CNb=3]
logLikelihoodScore =
VariantCaller.GetGtLogLikelihoodScore(gtObservedCounts, gtModelCounts, ref selectedGtState,
copyNumberModel);
Assert.Equal(gtModelCounts.IndexOf(gt1_2), selectedGtState);
}
public static void TestReverseComplement1BPIndelCall()
{
string seq1seq = "ATACCCCTTGCGC";
string seq2seq = "ATA-CCCTTGCGC".Replace("-", String.Empty);
int[] seq2qual = new int[] { 30, 30, 30, 2, 30, 30, 30, 30, 30, 30, 30, 30 };
var refseq = new Sequence(DnaAlphabet.Instance, seq1seq);
var query = new Sequence(DnaAlphabet.Instance, seq2seq);
var s1rc = refseq.GetReverseComplementedSequence();
var s2rc = query.GetReverseComplementedSequence();
NeedlemanWunschAligner aligner = new NeedlemanWunschAligner();
var aln = aligner.Align(s1rc, s2rc).First();
VariantCallTests.ConvertAlignedSequenceToQualSeq(aln, seq2qual.Reverse().ToArray());
aln.PairwiseAlignedSequences [0].Sequences [1].MarkAsReverseComplement();
var variants = VariantCaller.CallVariants(aln);
Assert.AreEqual(variants.Count, 1);
var variant = variants.First();
Assert.AreEqual(2, variant.QV);
Assert.AreEqual(5, variant.StartPosition);
Assert.AreEqual(VariantType.INDEL, variant.Type);
var vi = variant as IndelVariant;
Assert.AreEqual(IndelType.Deletion, vi.InsertionOrDeletion);
Assert.AreEqual('G', vi.HomopolymerBase);
Assert.AreEqual(1, vi.Length);
Assert.AreEqual(4, vi.HomopolymerLengthInReference);
Assert.AreEqual(true, vi.InHomopolymer);
Assert.AreEqual("G", vi.InsertedOrDeletedBases);
Assert.AreEqual(false, vi.AtEndOfAlignment);
Assert.AreEqual(6, vi.EndPosition);
}
public void VarCallsBecomeRefsAndNulls()
{
var originalVcfVariant = TestHelper.CreateDummyAllele("chr1", 123, "A", "T", 1000, 156);
var originalVcfVariant2 = TestHelper.CreateDummyAllele("chr1", 124, "A", "T", 1000, 156);
var vs1 = new VariantSite(originalVcfVariant);
var vs2 = new VariantSite(originalVcfVariant2);
var vcParams = new VariantCallingParameters();
vcParams.Validate();
var caller = new VariantCaller(vcParams, new BamFilterParameters());
//since there is an alt at position 124 ( a call of 156 alt / 1000 total, that means 844 original ref calls.
//Of which we said, 100 will get sucked up. So that leaves 744 / 1000 calls for a reference.
//So, we can still make a confident ref call.
var nbhd = new VcfNeighborhood(vcParams, 0, "chr1", vs1, vs2, "");
nbhd.SetRangeOfInterest();
nbhd.AddAcceptedPhasedVariant(
new CalledAllele(AlleleCategory.Snv)
{
Chromosome = "chr1",
ReferencePosition = 123,
ReferenceAllele = "A",
AlternateAllele = "T",
VariantQscore = 100,
TotalCoverage = 1000,
AlleleSupport = 500
});
nbhd.UsedRefCountsLookup = new Dictionary <int, SuckedUpRefRecord>()
{
};
caller.CallMNVs(nbhd);
caller.CallRefs(nbhd);
var acceptedMNVs = nbhd.CalledVariants;
var acceptedRefs = nbhd.CalledRefs;
Assert.Equal(1, acceptedMNVs.Count);
Assert.Equal(1, acceptedMNVs[123].Count);
Assert.Equal(2, acceptedRefs.Count);
var vcfVariant2asRef = new VcfVariant()
{
ReferenceName = "chr1",
ReferencePosition = 124,
ReferenceAllele = "A",
VariantAlleles = new[] { "." },
Genotypes = new List <Dictionary <string, string> >()
{
new Dictionary <string, string>()
{
{ "GT", "0/." }, { "DP", "1000" }, { "AD", "844" }
}
},
};
VcfMergerTests.CheckVariantsMatch(originalVcfVariant, acceptedMNVs[123][0]);
VcfMergerTests.CheckVariantsMatch(vcfVariant2asRef, acceptedRefs[124]);
// If one has been sucked up and there are refs remaining, we should output it as a ref.
var suckedUpRefRecord100 = new SuckedUpRefRecord()
{
Counts = 100, AlleleThatClaimedIt = new CalledAllele()
};
nbhd.UsedRefCountsLookup = new Dictionary <int, SuckedUpRefRecord>()
{
{ 124, suckedUpRefRecord100 }
};
caller.CallMNVs(nbhd);
caller.CallRefs(nbhd);
acceptedMNVs = nbhd.CalledVariants;
acceptedRefs = nbhd.CalledRefs;
Assert.Equal(1, acceptedMNVs.Count);
Assert.Equal(1, acceptedMNVs[123].Count);
Assert.Equal(2, acceptedRefs.Count);
vcfVariant2asRef = new VcfVariant()
{
ReferenceName = "chr1",
ReferencePosition = 124,
ReferenceAllele = "A",
VariantAlleles = new[] { "." },
Genotypes = new List <Dictionary <string, string> >()
{
new Dictionary <string, string>()
{
{ "GT", "0/." }, { "DP", "1000" }, { "AD", "744" }
}
},
};
VcfMergerTests.CheckVariantsMatch(originalVcfVariant, acceptedMNVs[123][0]);
VcfMergerTests.CheckVariantsMatch(vcfVariant2asRef, acceptedRefs[124]);
// If one has been sucked up all the way
// we should output it as a null.
var suckedUpRefRecord1000 = new SuckedUpRefRecord()
{
Counts = 1000, AlleleThatClaimedIt = new CalledAllele()
};
nbhd.UsedRefCountsLookup = new Dictionary <int, SuckedUpRefRecord>()
{
{ 124, suckedUpRefRecord1000 }
};
caller.CallMNVs(nbhd);
caller.CallRefs(nbhd);
acceptedMNVs = nbhd.CalledVariants;
acceptedRefs = nbhd.CalledRefs;
Assert.Equal(1, acceptedMNVs.Count);
Assert.Equal(1, acceptedMNVs[123].Count);
Assert.Equal(2, acceptedRefs.Count);
var vcfVariant2asNull = new VcfVariant()
{
ReferenceName = "chr1",
ReferencePosition = 124,
ReferenceAllele = "A",
VariantAlleles = new[] { "." },
Genotypes = new List <Dictionary <string, string> >()
{
new Dictionary <string, string>()
{
{ "GT", "./." }, { "DP", "1000" }, { "AD", "0" }
}
},
};
VcfMergerTests.CheckVariantsMatch(originalVcfVariant, acceptedMNVs[123][0]);
VcfMergerTests.CheckVariantsMatch(vcfVariant2asNull, acceptedRefs[124]);
}
public void CallAVariantInANewLocation()
{
//set up the original variants
var originalVcfVariant1 = TestHelper.CreateDummyAllele("chr1", 123, "A", "T", 1000, 156);
var originalVcfVariant2 = TestHelper.CreateDummyAllele("chr1", 124, "A", "T", 1000, 156);
var originalVcfVariant3 = TestHelper.CreateDummyAllele("chr1", 234, "A", "T", 1000, 156);
var originalVcfVariant4 = TestHelper.CreateDummyAllele("chr1", 234, "A", "T", 1000, 156);
var vs1 = new VariantSite(originalVcfVariant1);
var vs2 = new VariantSite(originalVcfVariant2);
var vs3 = new VariantSite(originalVcfVariant3);
var vs4 = new VariantSite(originalVcfVariant4);
var vcParams = new VariantCallingParameters();
vcParams.Validate();
var caller = new VariantCaller(vcParams, new BamFilterParameters());
var nbhd = new VcfNeighborhood(vcParams, 0, "chr1", vs1, vs2, "");
nbhd.AddVariantSite(vs3, "RRRRR"); //note, we do not add vs4, that is not going to get used for phasing. Sps it is a variant that failed filters.
nbhd.SetRangeOfInterest();
//now stage one candidate MNV:
var newMNV = new CalledAllele(AlleleCategory.Snv)
{
Chromosome = "chr1",
ReferencePosition = 129,
ReferenceAllele = "A",
AlternateAllele = "TT",
VariantQscore = 100,
TotalCoverage = 1000,
AlleleSupport = 500
};
nbhd.AddAcceptedPhasedVariant(newMNV);
var suckedUpRefRecord1000 = new SuckedUpRefRecord()
{
Counts = 1000, AlleleThatClaimedIt = new CalledAllele()
};
nbhd.UsedRefCountsLookup = new Dictionary <int, SuckedUpRefRecord>()
{
{ 124, suckedUpRefRecord1000 }
};
caller.CallMNVs(nbhd);
caller.CallRefs(nbhd);
var acceptedMNVs = nbhd.CalledVariants;
var acceptedRefs = nbhd.CalledRefs;
var vcfVariant0asRef = new VcfVariant()
{
ReferenceName = "chr1",
ReferencePosition = 123,
ReferenceAllele = "A",
VariantAlleles = new[] { "." },
Genotypes = new List <Dictionary <string, string> >()
{
new Dictionary <string, string>()
{
{ "GT", "0/." }
}
},
};
var vcfVariant3asRef = new VcfVariant()
{
ReferenceName = "chr1",
ReferencePosition = 234,
ReferenceAllele = "A",
VariantAlleles = new[] { "." },
Genotypes = new List <Dictionary <string, string> >()
{
new Dictionary <string, string>()
{
{ "GT", "0/." }
}
},
};
var vcfVariant2asNull = new VcfVariant()
{
ReferenceName = "chr1",
ReferencePosition = 124,
ReferenceAllele = "A",
VariantAlleles = new[] { "." },
Genotypes = new List <Dictionary <string, string> >()
{
new Dictionary <string, string>()
{
{ "GT", "./." }
}
},
};
Assert.Equal(1, acceptedMNVs.Count);
Assert.Equal(1, acceptedMNVs[129].Count);
Assert.Equal(3, acceptedRefs.Count);
VcfMergerTests.CheckVariantsMatch(vcfVariant0asRef, acceptedRefs[123]);
VcfMergerTests.CheckVariantsMatch(vcfVariant2asNull, acceptedRefs[124]);
VcfMergerTests.CheckVariantsMatch(newMNV, acceptedMNVs[129][0]);
VcfMergerTests.CheckVariantsMatch(vcfVariant3asRef, acceptedRefs[234]);
}
//this unit test was made after we found bug ScyllaLoosingRefCalls_PICS-723.
//We had a 1/. GT reported when it should be 1/0.
//The reason for this is that all the refs (the "0"s) got incorrectly sucked up.
//Ie, MNV ACG-> AG claimed 50 refs, so we (incorrectly) subtracted 50 refs from it.
//The bug is that the ref counts got subtractedfrom the exact same mnv that claimed them.
// This should never happen, and was not the intent of the alg.
//
//The affected mehtod is: CreateMnvsFromClusters in VcfNbhd
public void CreateMnvsFromClusters_TakeUpRefCount()
{
var originalVcfVariant1 = TestHelper.CreateDummyAllele("chr1", 123, "ACG", "AT", 1000, 156);
var originalVcfVariant2 = TestHelper.CreateDummyAllele("chr1", 123, "A", "TTTTTT", 1000, 200);
var originalVcfVariant3 = TestHelper.CreateDummyAllele("chr1", 123, "AC", "TT", 1000, 100);
var vs1 = new VariantSite(originalVcfVariant1);
var vs2 = new VariantSite(originalVcfVariant2);
var caller = new VariantCaller(new VariantCallingParameters(), new BamFilterParameters());
var nbhd = new VcfNeighborhood(new VariantCallingParameters(), 0, "chr1", vs1, vs2, "");
nbhd.SetRangeOfInterest();
nbhd.AddAcceptedPhasedVariant(
new CalledAllele(AlleleCategory.Snv)
{
Chromosome = "chr1",
ReferencePosition = 123,
ReferenceAllele = "A",
AlternateAllele = "T",
VariantQscore = 100,
TotalCoverage = 1000,
AlleleSupport = 200
});
nbhd.AddAcceptedPhasedVariant(
new CalledAllele(AlleleCategory.Mnv)
{
Chromosome = "chr1",
ReferencePosition = 123,
ReferenceAllele = "ACG",
AlternateAllele = "AT",
VariantQscore = 100,
TotalCoverage = 1000,
AlleleSupport = 300
});
nbhd.AddAcceptedPhasedVariant(
new CalledAllele(AlleleCategory.Insertion)
{
Chromosome = "chr1",
ReferencePosition = 123,
ReferenceAllele = "A",
AlternateAllele = "AAAAA",
VariantQscore = 100,
TotalCoverage = 1000,
AlleleSupport = 250
});
//default behavior, nothing gets sucked up
nbhd.UsedRefCountsLookup = new Dictionary <int, SuckedUpRefRecord>()
{
};
vs1.VcfReferencePosition = 123;
var vead = new Vead("dummy", new VariantSite[] { vs1 });
var vg = new VeadGroup(vead);
var fakeCluster = new Cluster("test", new List <VeadGroup>()
{
vg
});
fakeCluster.ResetConsensus();
nbhd.CreateMnvsFromClusters(new List <Cluster> {
fakeCluster
},
20, 100);
caller.CallMNVs(nbhd);
caller.CallRefs(nbhd);
var acceptedMNVs = nbhd.CalledVariants;
var acceptedRefs = nbhd.CalledRefs;
Assert.Equal(2, acceptedMNVs.Count);
Assert.Equal(3, acceptedMNVs[123].Count);
Assert.Equal(1, acceptedRefs.Count);
//check the ref counts on all the MNVs. Nothing should be sucked up.
Assert.Equal(1000 - 200, acceptedMNVs[123][0].ReferenceSupport); //total depth - allele suport. overly simple for now)
Assert.Equal(1000 - 300, acceptedMNVs[123][1].ReferenceSupport); //total depth - allele suport. overly simple for now)
Assert.Equal(1000 - 250, acceptedMNVs[123][2].ReferenceSupport); //total depth - allele suport. overly simple for now)
// now variant 0 will suck up 100 ref calls:
var suckedUpRefRecord100 = new SuckedUpRefRecord()
{
Counts = 100, AlleleThatClaimedIt = nbhd.CandidateVariants[0]
};
nbhd.UsedRefCountsLookup = new Dictionary <int, SuckedUpRefRecord>()
{
{ 123, suckedUpRefRecord100 }
};
nbhd.CreateMnvsFromClusters(new List <Cluster> {
fakeCluster
},
20, 100);
caller.CallMNVs(nbhd);
caller.CallRefs(nbhd);
acceptedMNVs = nbhd.CalledVariants;
acceptedRefs = nbhd.CalledRefs;
//check the ref counts on all the MNVs. refs should only be taken up by the first one
Assert.Equal(1000 - 200, acceptedMNVs[123][0].ReferenceSupport); //total depth - allele suport. overly simple for now)
//old result - has bug
//Assert.Equal(1000 - 300, acceptedMNVs[123][1].ReferenceSupport); //total depth - allele suport - sucked up ref)
//Assert.Equal(1000 - 250, acceptedMNVs[123][2].ReferenceSupport); //total depth - allele suport - sucked up ref)
//new result, fixed
Assert.Equal(1000 - 300 - 100, acceptedMNVs[123][1].ReferenceSupport); //total depth - allele suport - sucked up ref)
Assert.Equal(1000 - 250 - 100, acceptedMNVs[123][2].ReferenceSupport); //total depth - allele suport - sucked up ref)
}
//this unit test was made after we found bug ScyllaShouldMergeClusters_PICS-1122.
//We had an output vcf with the following lines
//chr11 64577365 . C . 100 PASS DP=1429 GT:GQ:AD:DP:VF:NL:SB:NC:US 0/0:1:1429:1429:0.00000:65:-100.0000:0.0592:0,0,0,0,0,0,0,0,0,0,0,0
//chr11 64577366 . A T 78 PASS DP = 559 GT:GQ:AD:DP:VF:NL:SB:NC:US 0/1:78:538,2:559:0.00358:65:-100.0000:0.7509:0,0,0,0,0,0,0,0,0,0,0,0
//chr11 64577366 . A T 78 PASS DP = 559 GT:GQ:AD:DP:VF:NL:SB:NC:US 0/1:78:538,2:559:0.00358:65:-100.0000:0.7509:0,0,0,0,0,0,0,0,0,0,0,0
//chr11 64577367 . G. 100 PASS DP = 1411 GT:GQ:AD:DP:VF:NL:SB:NC:US 0/0:1:1411:1411:0.00000:65:-100.0000:0.0741:0,0,0,0,0,0,0,0,0,0,0,0
//The affected methods are "AddAcceptedPhasedVariant" and "AddRejectedPhasedVariant"
//the new fix will merge the added variant, if its the same as a varaint that already exists
public void AddAcceptedAndRejectedPhasedVariantTests()
{
//for this test we take three SNPs, two of which can be combined and 1 that cannot, and
//we take three ref calls, two of which can be combined and 1 that cannot.
//So 6 diff alleles go in, but only 4 should come out in the lists.
var originalVcfVariant1 = TestHelper.CreateDummyAllele("chr1", 123, "A", "T", 500, 156);
var originalVcfVariant2 = TestHelper.CreateDummyAllele("chr1", 123, "A", "T", 1000, 200);
var vs1 = new VariantSite(originalVcfVariant1);
var vs2 = new VariantSite(originalVcfVariant2);
var caller = new VariantCaller(new VariantCallingParameters(), new BamFilterParameters());
var nbhd = new VcfNeighborhood(0, "chr1", vs1, vs2);
var callableNeighborhood = new CallableNeighborhood(nbhd, new VariantCallingParameters());
//variants:
callableNeighborhood.AddAcceptedPhasedVariant(
new CalledAllele(AlleleCategory.Snv)
{
Chromosome = "chr1",
ReferencePosition = 123,
ReferenceAllele = "A",
AlternateAllele = "T",
VariantQscore = 100,
TotalCoverage = 1000,
AlleleSupport = 200,
ReferenceSupport = 350,
NoiseLevelApplied = 20
});
callableNeighborhood.AddAcceptedPhasedVariant(
new CalledAllele(AlleleCategory.Snv)
{
Chromosome = "chr1",
ReferencePosition = 123,
ReferenceAllele = "A",
AlternateAllele = "T",
VariantQscore = 20,
TotalCoverage = 500,
AlleleSupport = 300,
ReferenceSupport = 50,
NoiseLevelApplied = 20
});
callableNeighborhood.AddAcceptedPhasedVariant(
new CalledAllele(AlleleCategory.Snv)
{
Chromosome = "chr1",
ReferencePosition = 123,
ReferenceAllele = "A",
AlternateAllele = "G",
VariantQscore = 20,
TotalCoverage = 500,
AlleleSupport = 300,
ReferenceSupport = 50,
NoiseLevelApplied = 20
});
callableNeighborhood.AddAcceptedPhasedVariant(
new CalledAllele(AlleleCategory.Snv)
{
Chromosome = "chr1",
ReferencePosition = 123,
ReferenceAllele = "A",
AlternateAllele = "AG",
VariantQscore = 20,
TotalCoverage = 500,
AlleleSupport = 300,
ReferenceSupport = 50,
NoiseLevelApplied = 20
});
//refs:
callableNeighborhood.AddRejectedPhasedVariant(
new CalledAllele(AlleleCategory.Reference)
{
Chromosome = "chr1",
ReferencePosition = 123,
ReferenceAllele = "A",
AlternateAllele = ".",
VariantQscore = 100,
TotalCoverage = 1000,
AlleleSupport = 200,
ReferenceSupport = 350,
NoiseLevelApplied = 20
});
callableNeighborhood.AddRejectedPhasedVariant(
new CalledAllele(AlleleCategory.Reference)
{
Chromosome = "chr1",
ReferencePosition = 123,
ReferenceAllele = "A",
AlternateAllele = ".",
VariantQscore = 20,
TotalCoverage = 500,
AlleleSupport = 300,
ReferenceSupport = 50,
NoiseLevelApplied = 20
});
callableNeighborhood.AddRejectedPhasedVariant(
new CalledAllele(AlleleCategory.Reference)
{
Chromosome = "chr1",
ReferencePosition = 124,
ReferenceAllele = "A",
AlternateAllele = ".",
VariantQscore = 20,
TotalCoverage = 500,
AlleleSupport = 300,
ReferenceSupport = 50,
NoiseLevelApplied = 20
});
//check results.
//check we got the right number of results
Assert.Equal(3, callableNeighborhood.CandidateVariants.Count);
Assert.Equal(2, callableNeighborhood.Refs.Count);
//check the snps did what we expected
var combinedSnp = callableNeighborhood.CandidateVariants[0];
Assert.Equal(123, combinedSnp.ReferencePosition);
Assert.Equal("chr1", combinedSnp.Chromosome);
Assert.Equal("A", combinedSnp.ReferenceAllele);
Assert.Equal("T", combinedSnp.AlternateAllele);
Assert.Equal(200 + 300, combinedSnp.AlleleSupport);
Assert.Equal(0, combinedSnp.NumNoCalls);
Assert.Equal(100, combinedSnp.VariantQscore);
Assert.Equal((1000 + 500) / 2, combinedSnp.TotalCoverage);
Assert.Equal((350 + 50) / 2, combinedSnp.ReferenceSupport);
Assert.Equal(AlleleCategory.Snv, combinedSnp.Type);
Assert.Equal(20, combinedSnp.NoiseLevelApplied);
//these values should not have changed
var justAddedSnp = callableNeighborhood.CandidateVariants[1];
Assert.Equal(123, justAddedSnp.ReferencePosition);
Assert.Equal("chr1", justAddedSnp.Chromosome);
Assert.Equal("A", justAddedSnp.ReferenceAllele);
Assert.Equal("G", justAddedSnp.AlternateAllele);
Assert.Equal(300, justAddedSnp.AlleleSupport);
Assert.Equal(0, justAddedSnp.NumNoCalls);
Assert.Equal(20, justAddedSnp.VariantQscore);
Assert.Equal(500, justAddedSnp.TotalCoverage);
Assert.Equal(50, justAddedSnp.ReferenceSupport);
Assert.Equal(AlleleCategory.Snv, justAddedSnp.Type);
Assert.Equal(20, justAddedSnp.NoiseLevelApplied);
}
public static void Main(string[] args)
{
try {
PlatformManager.Services.MaxSequenceSize = int.MaxValue;
PlatformManager.Services.DefaultBufferSize = 4096;
PlatformManager.Services.Is64BitProcessType = true;
if (args.Length > 3)
{
Console.WriteLine("Too many arguments");
DisplayHelp();
}
else if (args.Length < 2)
{
Console.WriteLine("Not enough arguments");
DisplayHelp();
}
else if (args [0] == "h" || args [0] == "help" || args [0] == "?" || args [0] == "-h")
{
DisplayHelp();
}
else
{
string bam_name = args [0];
string out_dir = args [1];
string ref_name = args.Length > 2 ? args [2] : null;
if (!File.Exists(bam_name))
{
Console.WriteLine("Can't find file: " + bam_name);
return;
}
if (ref_name != null && !File.Exists(ref_name))
{
Console.WriteLine("Can't find file: " + ref_name);
return;
}
if (Directory.Exists(out_dir))
{
Console.WriteLine("The output directory already exists, please specify a new directory or delete the old one.");
return;
}
Directory.CreateDirectory(out_dir);
List <CCSReadMetricsOutputter> outputters = new List <CCSReadMetricsOutputter> ()
{
new ZmwOutputFile(out_dir),
new ZScoreOutputter(out_dir),
new VariantOutputter(out_dir),
new SNROutputFile(out_dir),
new QVCalibration(out_dir)
};
ISequenceParser reader;
if (bam_name.EndsWith(".fastq", StringComparison.OrdinalIgnoreCase))
{
reader = new FastQCCSReader();
}
else
{
reader = new PacBioCCSBamReader();
}
BWAPairwiseAligner bwa = null;
bool callVariants = ref_name != null;
if (callVariants)
{
bwa = new BWAPairwiseAligner(ref_name, false);
}
// Produce aligned reads with variants called in parallel.
var reads = new BlockingCollection <Tuple <PacBioCCSRead, BWAPairwiseAlignment, List <Variant> > >();
Task producer = Task.Factory.StartNew(() =>
{
try
{
Parallel.ForEach(reader.Parse(bam_name), y => {
var z = y as PacBioCCSRead;
try {
BWAPairwiseAlignment aln = null;
List <Variant> variants = null;
if (callVariants)
{
aln = bwa.AlignRead(z.Sequence) as BWAPairwiseAlignment;
if (aln != null)
{
variants = VariantCaller.CallVariants(aln);
variants.ForEach(p => {
p.StartPosition += aln.AlignedSAMSequence.Pos;
p.RefName = aln.Reference;
});
}
}
var res = new Tuple <PacBioCCSRead, BWAPairwiseAlignment, List <Variant> >(z, aln, variants);
reads.Add(res);
}
catch (Exception thrown) {
Console.WriteLine("CCS READ FAIL: " + z.Sequence.ID);
Console.WriteLine(thrown.Message);
}
});
} catch (Exception thrown) {
Console.WriteLine("Could not parse BAM file: " + thrown.Message);
while (thrown.InnerException != null)
{
Console.WriteLine(thrown.InnerException.Message);
thrown = thrown.InnerException;
}
}
reads.CompleteAdding();
});
// Consume them into output files.
foreach (var r in reads.GetConsumingEnumerable())
{
foreach (var outputter in outputters)
{
outputter.ConsumeCCSRead(r.Item1, r.Item2, r.Item3);
}
}
// throw any exceptions (this should be used after putting the consumer on a separate thread)
producer.Wait();
// Close the files
outputters.ForEach(z => z.Finish());
}
}
catch (DllNotFoundException thrown) {
Console.WriteLine("Error thrown when attempting to generate the CCS results.");
Console.WriteLine("A shared library was not found. To solve this, please add the folder" +
" with the downloaded files libbwasharp and libMonoPosixHelper" +
"to your environmental variables (LD_LIBRARY_PATH on Ubuntu, DYLD_LIBRARY_PATH on Mac OS X).");
Console.WriteLine("Error: " + thrown.Message);
Console.WriteLine(thrown.StackTrace);
}
catch (Exception thrown) {
Console.WriteLine("Error thrown when attempting to generate the CCS results");
Console.WriteLine("Error: " + thrown.Message);
Console.WriteLine(thrown.StackTrace);
while (thrown.InnerException != null)
{
Console.WriteLine("Inner Exception: " + thrown.InnerException.Message);
thrown = thrown.InnerException;
}
}
}