public void TryStitch_SoftclipScenarios()
{
//Migrated from old Pisces: Originally called Pisces_MergeRead2First
var sequence =
"GG" + "GGCCACGCGGGGAGCAGCCTCTGGCATTCTGGGAGCTTCATCTGGACCTGGGTCTTCAGTGAACCATTGTTCAATATCGTCCGGGGACAGCATCAAATCATCCATTGCTTGGGACGGCAAGG" + "GGGACTGTAGATGGGTGAAAAGAGCA";
var read1 = ReadTestHelper.CreateRead("chr1",
sequence,
7579464,
new CigarAlignment("2S122M26S"),
Enumerable.Repeat((byte)30, sequence.Length).ToArray(),
7579464);
StitcherTestHelpers.SetReadDirections(read1, DirectionType.Forward);
sequence =
"GTGTAGGAGCTGCTGGTGCAGG" + "GGCCACGCGGGGAGCAGCCTCTGGCATTCTGGGAGCTTCATCTGGACCTGGGTCTTCAGTGAACAATTGTTCAATATCGTCCGGGGCCAGCATCAAATCATCCATTGCTTGGGACGGCAAGG" + "GGGACT";
var read2 = ReadTestHelper.CreateRead("chr1",
sequence,
7579464,
new CigarAlignment("22S122M6S"),
Enumerable.Repeat((byte)30, sequence.Length).ToArray(),
7579464);
StitcherTestHelpers.SetReadDirections(read2, DirectionType.Reverse);
string expectedWithNify =
"GTGTAGGAGCTGCTGGTGCAGG" + "GGCCACGCGGGGAGCAGCCTCTGGCATTCTGGGAGCTTCATCTGGACCTGGGTCTTCAGTGAACNATTGTTCAATATCGTCCGGGGNCAGCATCAAATCATCCATTGCTTGGGACGGCAAGG" + "GGGACTGTAGATGGGTGAAAAGAGCA";
string expectedWithNoNify =
"GTGTAGGAGCTGCTGGTGCAGG" + "GGCCACGCGGGGAGCAGCCTCTGGCATTCTGGGAGCTTCATCTGGACCTGGGTCTTCAGTGAACCATTGTTCAATATCGTCCGGGGACAGCATCAAATCATCCATTGCTTGGGACGGCAAGG" + "GGGACTGTAGATGGGTGAAAAGAGCA";
// both reads have the same reference position, but read2 really starts earlier
// make sure we behave properly
TestSuccesfullyStitchedRead(read1, read2, 0, "22S122M26S", (mergedRead) =>
{
Assert.NotNull(mergedRead);
Assert.Equal("22R122S26F", mergedRead.CigarDirections.ToString());
Assert.Equal(expectedWithNify, mergedRead.Sequence);
}, nifyDisagreements: true);
// Test with allowing probe softclips to merge
TestSuccesfullyStitchedRead(read1, read2, 0, "22S122M26S", (mergedRead) =>
{
Assert.NotNull(mergedRead);
Assert.Equal("20R130S20F", mergedRead.CigarDirections.ToString());
Assert.Equal(expectedWithNify, mergedRead.Sequence);
}, nifyDisagreements: true, ignoreProbeSoftclips: false);
//here, the Q scores where we disagree are equal (30) so we give R1 the preference (R1 usually being better than R2, if we are forced to pick one)
read1.BamAlignment.SetIsFirstMate(true);
read2.BamAlignment.SetIsFirstMate(false);
TestSuccesfullyStitchedRead(read1, read2, 0, "22S122M26S", (mergedRead) =>
{
Assert.NotNull(mergedRead);
Assert.Equal(expectedWithNoNify, mergedRead.Sequence);
}, nifyDisagreements: false);
}
public void TryStitch_NoXC_Stitchable()
{
//Reads without XC tags that do overlap should be added as one merged read in basic stitcher
var basicStitcher = StitcherTestHelpers.GetStitcher(10);
var alignmentSet = StitcherTestHelpers.GetOverlappingReadSet();
basicStitcher.TryStitch(alignmentSet);
Assert.Equal(1, alignmentSet.ReadsForProcessing.Count);
}
public void GenerateNifiedMergedRead()
{
var read1 = DomainTestHelper.CreateRead("chr1", "AAAAA", 2,
new CigarAlignment("1S4M"));
var read2 = DomainTestHelper.CreateRead("chr1", "AAAAA", 2,
new CigarAlignment("4M1S"));
StitcherTestHelpers.SetReadDirections(read2, DirectionType.Reverse);
var alignmentSet = new AlignmentSet(read1, read2);
var stitchedRead = GetStitchedRead(alignmentSet);
Assert.Equal("1S4M1S", stitchedRead.StitchedCigar.ToString());
Assert.Equal("NNNNNN", stitchedRead.Sequence);
Assert.Equal("1F4S1R", stitchedRead.CigarDirections.ToString());
StitcherTestHelpers.SetReadDirections(read1, DirectionType.Reverse);
StitcherTestHelpers.SetReadDirections(read2, DirectionType.Forward);
stitchedRead = GetStitchedRead(alignmentSet);
Assert.Equal("1S4M1S", stitchedRead.StitchedCigar.ToString());
Assert.Equal("NNNNNN", stitchedRead.Sequence);
Assert.Equal("1R4S1F", stitchedRead.CigarDirections.ToString());
StitcherTestHelpers.SetReadDirections(read1, DirectionType.Forward);
StitcherTestHelpers.SetReadDirections(read2, DirectionType.Reverse);
// Insertion that we don't know what to do with -> Nified match
read1 = DomainTestHelper.CreateRead("chr1", "AAAAA", 2,
new CigarAlignment("1S3M1I"));
alignmentSet = new AlignmentSet(read1, read2);
stitchedRead = GetStitchedRead(alignmentSet);
Assert.Equal("1S4M1S", stitchedRead.StitchedCigar.ToString());
Assert.Equal("NNNNNN", stitchedRead.Sequence);
Assert.Equal("1F4S1R", stitchedRead.CigarDirections.ToString());
// Read 1 goes to end of read 2
read1 = DomainTestHelper.CreateRead("chr1", "AAAAAA", 2,
new CigarAlignment("1S3M2I"));
alignmentSet = new AlignmentSet(read1, read2);
stitchedRead = GetStitchedRead(alignmentSet);
Assert.Equal("1S5M", stitchedRead.StitchedCigar.ToString());
Assert.Equal("NNNNNN", stitchedRead.Sequence);
Assert.Equal("1F5S", stitchedRead.CigarDirections.ToString());
// Read 1 goes past read 2
read1 = DomainTestHelper.CreateRead("chr1", "AAAAAAA", 2,
new CigarAlignment("1S3M3I"));
alignmentSet = new AlignmentSet(read1, read2);
stitchedRead = GetStitchedRead(alignmentSet);
Assert.Equal("1S6M", stitchedRead.StitchedCigar.ToString());
Assert.Equal("NNNNNNN", stitchedRead.Sequence);
Assert.Equal("1F5S1F", stitchedRead.CigarDirections.ToString());
}
private void TestMerge(int pos1, string cigar1, int pos2, string cigar2, int posStitch = 0, string cigarStitch = "", string stitchDirections = "", bool shouldMerge = true, bool ignoreProbeSoftclips = true, int?maxReadLength = null)
{
var r1Bases = new string('A', (int)new CigarAlignment(cigar1).GetReadSpan());
var r2Bases = new string('A', (int)new CigarAlignment(cigar2).GetReadSpan());
var read1 = DomainTestHelper.CreateRead("chr1", r1Bases, pos1,
new CigarAlignment(cigar1));
var read2 = DomainTestHelper.CreateRead("chr1", r2Bases, pos2,
new CigarAlignment(cigar2));
StitcherTestHelpers.SetReadDirections(read2, DirectionType.Reverse);
BasicStitcher stitcher;
if (maxReadLength != null)
{
stitcher = new BasicStitcher(10, ignoreProbeSoftclips: ignoreProbeSoftclips,
maxReadLength: maxReadLength.Value);
}
else
{
// Use the default
stitcher = new BasicStitcher(10, ignoreProbeSoftclips: ignoreProbeSoftclips);
}
if (!shouldMerge)
{
var alignmentSet = new AlignmentSet(read1, read2);
Assert.False(stitcher.TryStitch(alignmentSet));
//StitcherTestHelpers.TestUnstitchableReads(read1, read2, 0, null);
}
else
{
var alignmentSet = new AlignmentSet(read1, read2);
var didStitch = stitcher.TryStitch(alignmentSet);
Assert.True(didStitch);
var mergedRead = StitcherTestHelpers.GetMergedRead(alignmentSet);
Console.WriteLine(mergedRead.Position + " " + mergedRead.CigarData);
Console.WriteLine("---------------");
if (cigarStitch != "")
{
Assert.Equal(posStitch, mergedRead.Position);
Assert.Equal(cigarStitch, mergedRead.CigarData.ToString());
}
if (stitchDirections != "")
{
Assert.Equal(stitchDirections, mergedRead.CigarDirections.ToString());
}
}
}
public static void TestSuccesfullyStitchedRead(Read read1, Read read2, int minQscore, string xcTag, Action <Read> assertions, bool nifyDisagreements = false, bool useSoftclippedBases = true, bool ignoreProbeSoftclips = true)
{
var alignmentSet = new AlignmentSet(read1, read2);
var stitcher = StitcherTestHelpers.GetStitcher(minQscore,
nifyDisagreements: nifyDisagreements, useSoftclippedBases: useSoftclippedBases, ignoreProbeSoftclips: ignoreProbeSoftclips);
stitcher.TryStitch(alignmentSet);
// -----------------------------------------------
// Basic Stitcher, No XC tag
// -----------------------------------------------
CheckMergedRead(xcTag, assertions, alignmentSet);
}
public void TryStitch_CalculateStitchedCigar()
{
// -----------------------------------------------
// Read position maps disagree
// -----------------------------------------------
// Should throw out the pair
var read1 = DomainTestHelper.CreateRead("chr1", "ATCGATCG", 12345,
new CigarAlignment("2M2D3M1D3M"), qualityForAll: 30); //Within the overlap, we have a deletion so there will be a shifting of positions from that point on
var read2 = DomainTestHelper.CreateRead("chr1", "ATCGATCG", 12349,
new CigarAlignment("8M"), qualityForAll: 30);
var stitcher = StitcherTestHelpers.GetStitcher(10);
var alignmentSet = new AlignmentSet(read1, read2);
Assert.True(!alignmentSet.ReadsForProcessing.Any());
// -----------------------------------------------
// When calculating stitched cigar, stitched cigar should have
// - everything from read1 before the overlap
// - everything from read2 starting from the overlap
// But since we ensure that the position maps agree in the overlap region, it's really not a matter of one taking precedence over the other
// 1234... 1 - - 2 3 4 5 6 - - 7 8 9 0
// Read1 X X X X X X X X - - - - -
// Read1 M I I M M M M M - - - - -
// Read2 - - - X X X X X X X X - -
// Read2 - - - M M M M M I M M - -
// -----------------------------------------------
// Stitched cigar should have R1's insertion from before the overlap and R2's insertion from after the overlap
read1 = DomainTestHelper.CreateRead("chr1", "ATCGATCG", 12341,
new CigarAlignment("1M2I5M"), qualityForAll: 30);
read2 = DomainTestHelper.CreateRead("chr1", "ATCGATCG", 12342,
new CigarAlignment("5M1I2M"), qualityForAll: 30);
stitcher = StitcherTestHelpers.GetStitcher(10);
alignmentSet = new AlignmentSet(read1, read2);
stitcher.TryStitch(alignmentSet);
Assert.Equal("1M2I5M1I2M", StitcherTestHelpers.GetMergedRead(alignmentSet).CigarData.ToString());
}
public void TryStitch_RealExamples()
{
// Real example from Kristina's problematic variant #73
var read1Bases =
"GAAGCCACACTGACGTGCCTCTCCCTCCCTCCAGGAAGCCTTCCAGGAAGCCTACGTGATGGCCAGCGTGGACAACCCCCACGTGTGCCGCCTGCTGGGCATCTGCCTCACCTCCACCGTGCAGCTCATCACGCAGCTCATGCCCTTCGG";
var read2Bases =
"AGGAAGCCTTCCAGGAAGCCTACGTGATGGCCAGCGTGGACAACCCCCACGTGTGCCGCCTGCTGGGCATCTGCCTCACCTCCACCGTGCAGCTCATCACGCAGCTCATGCCCTTCGGCTGCCTCCTGGACTATGTCCGGGAACACAAAG";
var read1 = DomainTestHelper.CreateRead("chr7", read1Bases, 55248972, new CigarAlignment("20S9M12I109M"));
var read2 = DomainTestHelper.CreateRead("chr7", read2Bases, 55248981, new CigarAlignment("9S120M21S"));
StitcherTestHelpers.SetReadDirections(read2, DirectionType.Reverse);
var stitcher = new BasicStitcher(10, useSoftclippedBases: false);
var alignmentSet = new AlignmentSet(read1, read2);
stitcher.TryStitch(alignmentSet);
var mergedRead = StitcherTestHelpers.GetMergedRead(alignmentSet);
Assert.Equal("20S9M12I120M21S", mergedRead.CigarData.ToString());
Assert.Equal("41F109S32R", mergedRead.CigarDirections.ToString());
// Shouldn't stitch - problem Yu was having (tried to merge and then said the base at position 158 is null).
read1Bases =
"CGACGCTCTTGCGATCTTCAAAGCAATAGGATGGGTGATCAGGGATGTTGCTTACAAGAAAAGAACTGCCATACAGCTTCAACAACAACTTCTTCCACCCACCCCTAAAATGATGCTAAAAAGTAAGTCATCTCTGGTTCTCCCCCGATT";
read2Bases =
"TCAAAGCAATAGGATGGATGATCAGAGATGTTGCTTACAAGAAAAGAACTGCCATACAGCTTCAACAACAACTTCTTCCACTCCCCCCTAAAGTGATGCTAAAAAGTAAATCATCCCTGTTTCTCCCCCGTTCGCGAATTTCTACGATCG";
read1 = DomainTestHelper.CreateRead("chr7", read1Bases, 109465121, new CigarAlignment("44S56M1I23M26S"));
read2 = DomainTestHelper.CreateRead("chr7", read2Bases, 109465121, new CigarAlignment("27S55M1I24M43S"));
StitcherTestHelpers.SetReadDirections(read2, DirectionType.Reverse);
stitcher = new BasicStitcher(10, useSoftclippedBases: true);
alignmentSet = new AlignmentSet(read1, read2);
Assert.False(stitcher.TryStitch(alignmentSet));
}
public void TryStitch_MergeReadsSmall()
{
//Migrated from old Pisces: Originally called Pisces_MergeReadsSmall
//test1: happy path
//0 1 2 3 4 5 6 7 8 9
//- C A T A T
//- - - - A T A G G
var read1 = ReadTestHelper.CreateRead("chr1", "CATAT", 1, new CigarAlignment("5M"), new byte[] { 1, 2, 3, 4, 5 }, 4);
StitcherTestHelpers.SetReadDirections(read1, DirectionType.Forward);
var read2 = ReadTestHelper.CreateRead("chr1", "ATAGG", 4, new CigarAlignment("5M"), new byte[] { 1, 20, 30, 40, 50 }, 1);
StitcherTestHelpers.SetReadDirections(read2, DirectionType.Reverse);
var alignmentSet = new AlignmentSet(read1, read2);
var stitcher = StitcherTestHelpers.GetStitcher(10, nifyDisagreements: false);
stitcher.TryStitch(alignmentSet);
TestSuccesfullyStitchedRead(read1, read2, 0, "8M", (mergedRead) =>
{
Assert.Equal(mergedRead.Sequence, "CATATAGG");
StitcherTestHelpers.CompareQuality(new byte[] { 1, 2, 3, 5, 25, 30, 40, 50 }, mergedRead.Qualities);
var expectedDirections = StitcherTestHelpers.BuildDirectionMap(new List <IEnumerable <DirectionType> >
{
StitcherTestHelpers.BuildDirectionSegment(DirectionType.Forward, 3),
StitcherTestHelpers.BuildDirectionSegment(DirectionType.Stitched, 2),
StitcherTestHelpers.BuildDirectionSegment(DirectionType.Reverse, 3)
});
StitcherTestHelpers.VerifyDirectionType(expectedDirections, mergedRead.CigarDirections.Expand().ToArray());
});
//test2: different bases, one with low Q
//0 1 2 3 4 5 6 7 8 9
//- C A T A G
//- - - - A T A G G
read1 = ReadTestHelper.CreateRead("chr1", "CATAG", 1, new CigarAlignment("5M"), new byte[] { 1, 2, 3, 4, 5 }, 4);
StitcherTestHelpers.SetReadDirections(read1, DirectionType.Reverse);
read2 = ReadTestHelper.CreateRead("chr1", "ATAGG", 4, new CigarAlignment("5M"), new byte[] { 1, 20, 30, 40, 50 },
1);
StitcherTestHelpers.SetReadDirections(read2, DirectionType.Forward);
TestSuccesfullyStitchedRead(read1, read2, 10, "8M", (mergedRead) =>
{
Assert.NotNull(mergedRead);
Assert.Equal("CATATAGG", mergedRead.Sequence);
StitcherTestHelpers.CompareQuality(new byte[] { 1, 2, 3, 5, 20, 30, 40, 50 }, mergedRead.Qualities);
var expectedDirections = StitcherTestHelpers.BuildDirectionMap(new List <IEnumerable <DirectionType> >
{
StitcherTestHelpers.BuildDirectionSegment(DirectionType.Reverse, 3),
StitcherTestHelpers.BuildDirectionSegment(DirectionType.Stitched, 2),
StitcherTestHelpers.BuildDirectionSegment(DirectionType.Forward, 3)
});
StitcherTestHelpers.VerifyDirectionType(expectedDirections, mergedRead.CigarDirections.Expand().ToArray());
});
//test3: different bases, both with high Q
//0 1 2 3 4 5 6 7 8 9
//- C A T A G
//- - - - A T A G G
read1 = ReadTestHelper.CreateRead("chr1", "CATAG", 1, new CigarAlignment("5M"), new byte[] { 50, 92, 92, 92, 92 }, 4);
read2 = ReadTestHelper.CreateRead("chr1", "ATAGG", 4, new CigarAlignment("5M"), new byte[] { 2, 20, 30, 40, 50 }, 1);
StitcherTestHelpers.SetReadDirections(read1, DirectionType.Forward);
StitcherTestHelpers.SetReadDirections(read2, DirectionType.Reverse);
TestSuccesfullyStitchedRead(read1, read2, 10, "8M", (mergedRead) =>
{
Assert.NotNull(mergedRead);
Assert.Equal("CATANAGG", mergedRead.Sequence);
StitcherTestHelpers.CompareQuality(new byte[] { 50, 92, 92, 93, 0, 30, 40, 50 }, mergedRead.Qualities);
var expectedDirections = StitcherTestHelpers.BuildDirectionMap(new List <IEnumerable <DirectionType> >
{
StitcherTestHelpers.BuildDirectionSegment(DirectionType.Forward, 3),
StitcherTestHelpers.BuildDirectionSegment(DirectionType.Stitched, 2),
StitcherTestHelpers.BuildDirectionSegment(DirectionType.Reverse, 3)
});
StitcherTestHelpers.VerifyDirectionType(expectedDirections, mergedRead.CigarDirections.Expand().ToArray());
}, nifyDisagreements: true);
}
private void ExecuteConsensusTests(bool nifyDisagreements)
{
// 1234... 1 - - 2 3 4 5 6 - - 7 8 9 0 //Reference Positions
// Read1 X X X X X X X X - - - - -
// Read1 M I I M M M M M - - - - -
// Read1 T T T T T T T T - - - - -
// Read2 - - - X X X X X X X X - -
// Read2 - - - M M M M M I M M - -
// Read2 - - - A A A A A A A A - -
var r1qualities = 30;
var r2qualities = 20;
var read1 = DomainTestHelper.CreateRead("chr1", "TTTTTTTT", 12341,
new CigarAlignment("1M2I5M"), qualityForAll: (byte)r1qualities);
var read2 = DomainTestHelper.CreateRead("chr1", "AAAAAAAA", 12342,
new CigarAlignment("5M1I2M"), qualityForAll: (byte)r2qualities);
var stitcher = StitcherTestHelpers.GetStitcher(10, false, nifyDisagreements: nifyDisagreements);
var alignmentSet = new AlignmentSet(read1, read2);
stitcher.TryStitch(alignmentSet);
// Merged A T C ? ? ? ? ? T C G - -
// Merged M I I M M M M M I M M - -
// Merged 0 1 2 3 4 5 6 7 8 9 0 1 2
var overlapStart = 3;
var overlapEnd = 8;
var overlapLength = 5;
//Consensus sequence should have everything from read1 for positions before overlap
var mergedRead = StitcherTestHelpers.GetMergedRead(alignmentSet);
Assert.Equal("TTT", mergedRead.Sequence.Substring(0, overlapStart));
//Consensus sequence should have everything from read2 for positions after overlap
Assert.Equal("AAA", mergedRead.Sequence.Substring(overlapEnd, 3));
//Consensus sequence should have an N where we have two high-quality (both above min) disagreeing bases
Assert.Equal(nifyDisagreements? "NNNNN":"TTTTT", mergedRead.Sequence.Substring(overlapStart, 5));
//Consensus sequence should have 0 quality where we have two high-quality (both above min) disagreeing bases
Assert.True(mergedRead.Qualities.Take(overlapStart).All(q => q == r1qualities));
Assert.True(mergedRead.Qualities.Skip(overlapStart).Take(overlapLength).All(q => q == 0));
Assert.True(mergedRead.Qualities.Skip(overlapEnd).Take(mergedRead.Sequence.Length - overlapEnd).All(q => q == r2qualities));
//Consensus sequence should take higher quality base if one or more of the bases is below min quality
//Read 2 trumps whole overlap
read1.BamAlignment.Qualities = new byte[] { 30, 30, 30, 5, 5, 5, 5, 5 };
read2.BamAlignment.Qualities = new byte[] { 40, 40, 40, 40, 40, 20, 19, 18 };
alignmentSet = new AlignmentSet(read1, read2);
stitcher.TryStitch(alignmentSet);
mergedRead = StitcherTestHelpers.GetMergedRead(alignmentSet);
Assert.Equal(nifyDisagreements ? "NNNNN" : read2.Sequence.Substring(0, 5), mergedRead.Sequence.Substring(overlapStart, 5));
Assert.Equal(nifyDisagreements ? "TTTNNNNNAAA" : "TTTAAAAAAAA", mergedRead.Sequence);
StitcherTestHelpers.CompareQuality(nifyDisagreements ? new byte[] { 30, 30, 30, 0, 0, 0, 0, 0, 20, 19, 18 } : new byte[] { 30, 30, 30, 40, 40, 40, 40, 40, 20, 19, 18 }, mergedRead.Qualities);
//Read 1 trumps whole overlap
read1.BamAlignment.Qualities = new byte[] { 30, 30, 30, 40, 40, 40, 40, 40 };
read2.BamAlignment.Qualities = new byte[] { 5, 5, 5, 5, 5, 20, 19, 18 };
alignmentSet = new AlignmentSet(read1, read2);
stitcher.TryStitch(alignmentSet);
mergedRead = StitcherTestHelpers.GetMergedRead(alignmentSet);
Assert.Equal(nifyDisagreements ? "NNNNN" : read1.Sequence.Substring(3, 5), mergedRead.Sequence.Substring(overlapStart, 5));
Assert.Equal(nifyDisagreements ? "TTTNNNNNAAA" : "TTTTTTTTAAA", mergedRead.Sequence);
StitcherTestHelpers.CompareQuality(nifyDisagreements ? new byte[] { 30, 30, 30, 0, 0, 0, 0, 0, 20, 19, 18 } : new byte[] { 30, 30, 30, 40, 40, 40, 40, 40, 20, 19, 18 }, mergedRead.Qualities);
//Little bit of each
read1.BamAlignment.Qualities = new byte[] { 30, 30, 30, 5, 45, 5, 45, 5 };
read2.BamAlignment.Qualities = new byte[] { 40, 5, 40, 5, 40, 20, 19, 18 };
alignmentSet = new AlignmentSet(read1, read2);
stitcher.TryStitch(alignmentSet);
mergedRead = StitcherTestHelpers.GetMergedRead(alignmentSet);
Assert.Equal(nifyDisagreements ? "TTTNNNNNAAA" : "TTTATATAAAA", mergedRead.Sequence);
StitcherTestHelpers.CompareQuality(nifyDisagreements ? new byte[] { 30, 30, 30, 0, 0, 0, 0, 0, 20, 19, 18 } : new byte[] { 30, 30, 30, 40, 45, 40, 45, 40, 20, 19, 18 }, mergedRead.Qualities);
//Consensus sequence should take base and assign the higher quality if both bases agree
var read2_agreeingBases = DomainTestHelper.CreateRead("chr1", "TTTTTTTT", 12342,
new CigarAlignment("5M1I2M"), new byte[] { 40, 5, 40, 5, 40, 20, 19, 18 });
read1.BamAlignment.Qualities = new byte[] { 30, 30, 30, 5, 45, 5, 45, 5 };
alignmentSet = new AlignmentSet(read1, read2_agreeingBases);
stitcher.TryStitch(alignmentSet);
mergedRead = StitcherTestHelpers.GetMergedRead(alignmentSet);
Assert.Equal("TTTTTTTTTTT", mergedRead.Sequence);
StitcherTestHelpers.CompareQuality(new byte[] { 30, 30, 30, 45, 50, 45, 50, 45, 20, 19, 18 }, mergedRead.Qualities);
//Bases disagree and both are below minimum quality, read1>read2 : take base/q from read1
read1.BamAlignment.Qualities = new byte[] { 30, 30, 30, 8, 8, 8, 8, 8 };
read2.BamAlignment.Qualities = new byte[] { 5, 5, 5, 5, 5, 20, 19, 18 };
alignmentSet = new AlignmentSet(read1, read2);
stitcher.TryStitch(alignmentSet);
mergedRead = StitcherTestHelpers.GetMergedRead(alignmentSet);
Assert.Equal(nifyDisagreements ? "NNNNN" : read1.Sequence.Substring(3, 5), mergedRead.Sequence.Substring(overlapStart, 5));
Assert.Equal(nifyDisagreements ? "TTTNNNNNAAA" : "TTTTTTTTAAA", mergedRead.Sequence);
StitcherTestHelpers.CompareQuality(nifyDisagreements ? new byte[] { 30, 30, 30, 0, 0, 0, 0, 0, 20, 19, 18 } : new byte[] { 30, 30, 30, 8, 8, 8, 8, 8, 20, 19, 18 }, mergedRead.Qualities);
//Bases disagree and both are below minimum quality, read2>read1 : take base/q from read2
read1.BamAlignment.Qualities = new byte[] { 30, 30, 30, 5, 5, 5, 5, 5 };
read2.BamAlignment.Qualities = new byte[] { 8, 8, 8, 8, 8, 20, 19, 18 };
alignmentSet = new AlignmentSet(read1, read2);
stitcher.TryStitch(alignmentSet);
mergedRead = StitcherTestHelpers.GetMergedRead(alignmentSet);
Assert.Equal(nifyDisagreements ? "NNNNN" : read2.Sequence.Substring(0, 5), mergedRead.Sequence.Substring(overlapStart, 5));
Assert.Equal(nifyDisagreements ? "TTTNNNNNAAA" : "TTTAAAAAAAA", mergedRead.Sequence);
StitcherTestHelpers.CompareQuality(nifyDisagreements ? new byte[] { 30, 30, 30, 0, 0, 0, 0, 0, 20, 19, 18 } : new byte[] { 30, 30, 30, 8, 8, 8, 8, 8, 20, 19, 18 }, mergedRead.Qualities);
//Bases disagree and both are below minimum quality, read1==read2 : take base/q from read1
read1.BamAlignment.Qualities = new byte[] { 30, 30, 30, 5, 5, 5, 5, 5 };
read2.BamAlignment.Qualities = new byte[] { 5, 5, 5, 5, 5, 20, 19, 18 };
alignmentSet = new AlignmentSet(read1, read2);
stitcher.TryStitch(alignmentSet);
mergedRead = StitcherTestHelpers.GetMergedRead(alignmentSet);
Assert.Equal(nifyDisagreements ? "NNNNN" : read1.Sequence.Substring(3, 5), mergedRead.Sequence.Substring(overlapStart, 5));
Assert.Equal(nifyDisagreements ? "TTTNNNNNAAA" : "TTTTTTTTAAA", mergedRead.Sequence);
StitcherTestHelpers.CompareQuality(nifyDisagreements ? new byte[] { 30, 30, 30, 0, 0, 0, 0, 0, 20, 19, 18 } : new byte[] { 30, 30, 30, 5, 5, 5, 5, 5, 20, 19, 18 }, mergedRead.Qualities);
}
public void TryStitch_NoXC_Unstitchable()
{
var read1 = DomainTestHelper.CreateRead("chr1", "ATCGATCG", 12345,
new CigarAlignment("8M"), qualityForAll: 30);
var read2_noOverlap = DomainTestHelper.CreateRead("chr1", "A", 2384,
new CigarAlignment("1M"), qualityForAll: 30);
var read2_overlap = DomainTestHelper.CreateRead("chr1", "ATCGTT", 12349,
new CigarAlignment("1I5M"), qualityForAll: 30);
var read2_diffChrom = DomainTestHelper.CreateRead("chr2", "ATCGTT", 12349,
new CigarAlignment("6M"), qualityForAll: 30);
var read2_nonOverlap_border = DomainTestHelper.CreateRead("chr1", "AT", 12343,
new CigarAlignment("2M"), qualityForAll: 30);
var stitcher = StitcherTestHelpers.GetStitcher(10);
;
// -----------------------------------------------
// Either of the partner reads is missing*
// *(only read that could be missing is read 2, if read 1 was missing couldn't create alignment set)
// -----------------------------------------------
// Should throw an exception
var alignmentSet = new AlignmentSet(read1, null);
Assert.Throws <ArgumentException>(() => stitcher.TryStitch(alignmentSet));
// -----------------------------------------------
// No overlap, reads are far away
// -----------------------------------------------
// Shouldn't stitch
alignmentSet = new AlignmentSet(read1, read2_noOverlap);
stitcher.TryStitch(alignmentSet);
Assert.Equal(2, alignmentSet.ReadsForProcessing.Count);
StitcherTestHelpers.TestUnstitchableReads(read1, read2_noOverlap, 0, (unStitchableReads) =>
{
Assert.Equal(1, unStitchableReads.Count(x => StitcherTestHelpers.VerifyReadsEqual(read1, x)));
Assert.Equal(1, unStitchableReads.Count(x => StitcherTestHelpers.VerifyReadsEqual(read2_noOverlap, x)));
});
// -----------------------------------------------
// No overlap, reads are directly neighboring
// -----------------------------------------------
// Shouldn't stitch
alignmentSet = new AlignmentSet(read1, read2_nonOverlap_border);
stitcher.TryStitch(alignmentSet);
Assert.Equal(2, alignmentSet.ReadsForProcessing.Count);
StitcherTestHelpers.TestUnstitchableReads(read1, read2_nonOverlap_border, 0, (unStitchableReads) =>
{
Assert.Equal(1, unStitchableReads.Count(x => StitcherTestHelpers.VerifyReadsEqual(read1, x)));
Assert.Equal(1, unStitchableReads.Count(x => StitcherTestHelpers.VerifyReadsEqual(read2_nonOverlap_border, x)));
});
// -----------------------------------------------
// No overlap, reads on diff chromosomes
// -----------------------------------------------
// Shouldn't stitch
alignmentSet = new AlignmentSet(read1, read2_diffChrom);
stitcher.TryStitch(alignmentSet);
Assert.Equal(2, alignmentSet.ReadsForProcessing.Count);
StitcherTestHelpers.TestUnstitchableReads(read1, read2_diffChrom, 0, (unStitchableReads) =>
{
Assert.Equal(1, unStitchableReads.Count(x => StitcherTestHelpers.VerifyReadsEqual(read1, x)));
Assert.Equal(1, unStitchableReads.Count(x => StitcherTestHelpers.VerifyReadsEqual(read2_diffChrom, x)));
});
// -----------------------------------------------
// Has overlap, but cigars are incompatible
// -----------------------------------------------
// Shouldn't stitch
alignmentSet = new AlignmentSet(read1, read2_overlap);
stitcher.TryStitch(alignmentSet);
Assert.Equal(2, alignmentSet.ReadsForProcessing.Count);
StitcherTestHelpers.TestUnstitchableReads(read1, read2_overlap, 0, (unStitchableReads) =>
{
Assert.Equal(1, unStitchableReads.Count(x => StitcherTestHelpers.VerifyReadsEqual(read1, x)));
Assert.Equal(1, unStitchableReads.Count(x => StitcherTestHelpers.VerifyReadsEqual(read2_overlap, x)));
});
// -----------------------------------------------
// Has overlap, but cigars are incompatible, but read 2 starts with SC
// -----------------------------------------------
// Overlap is just S and I - should stitch
// 5678----90123456789
// MMMMIIII
// SSSSMMMM
var read1_withIns = DomainTestHelper.CreateRead("chr1", "ATCGATCG", 12345,
new CigarAlignment("4M4I"), qualityForAll: 30);
var read2_withSC = DomainTestHelper.CreateRead("chr1", "ATCGATCG", 12349,
new CigarAlignment("4S4M"), qualityForAll: 30);
alignmentSet = new AlignmentSet(read1_withIns, read2_withSC);
//stitcher.TryStitch(alignmentSet);
//Assert.Equal(1, alignmentSet.ReadsForProcessing.Count);
//Assert.Equal("4M4I4M", alignmentSet.ReadsForProcessing.First().CigarData.ToString());
// Overlap is S and some disagreeing ops with I - should not stitch
read2_withSC = DomainTestHelper.CreateRead("chr1", "ATCGATCG", 12348,
new CigarAlignment("2S1D6M"), qualityForAll: 30);
alignmentSet = new AlignmentSet(read1_withIns, read2_withSC);
stitcher.TryStitch(alignmentSet);
Assert.Equal(2, alignmentSet.ReadsForProcessing.Count);
StitcherTestHelpers.TestUnstitchableReads(read1_withIns, read2_withSC, 0, (unStitchableReads) =>
{
Assert.Equal(1, unStitchableReads.Count(x => StitcherTestHelpers.VerifyReadsEqual(read1_withIns, x)));
Assert.Equal(1, unStitchableReads.Count(x => StitcherTestHelpers.VerifyReadsEqual(read2_withSC, x)));
});
}
public void TryStitch_ReCo()
{
// Real example from ReCo, was failing to generate the correct stitched cigar
var read1Bases =
"GTACTCCTACAGTCCCACCCCTCCCCTATAAACCTTATGAATCCCCGTTCACTTAGATGCCAGCTTGGCAAGGAAGGGAAGTACACATCTGTTGACAGTAATGAAATATCCTTGATAAGGATTTAAATTTTGGATGTGCTG";
var read2Bases =
"ACCTACAGTCCCACCCCTCCCCTATAAACCTTAGGAATCCCCGTTCACTTAGATGCCAGCTTGGCAAGGAAGGGAAGTACACATCTGTTGACAGTAATGAAATATCCTTGATAAGGATTTAAATTTTGGATGTGCTGAGCT";
// 8 9
// 3 4 5 6 7 8 9 0 1 2
// s s s s s M M M M M ...
// - - - - M M M M M M ...
// F F F F R S S S S S ... // Stitched directions if we don't allow softclip to contribute
// F F F F S S S S S S ... // Stitched directions if we do allow softclip to contribute
var read1 = DomainTestHelper.CreateRead("chr21", read1Bases, 16685488,
new CigarAlignment("5S136M"));
var read2 = DomainTestHelper.CreateRead("chr21", read2Bases, 16685487,
new CigarAlignment("137M4S"));
StitcherTestHelpers.SetReadDirections(read2, DirectionType.Reverse);
var stitcher = new BasicStitcher(10, useSoftclippedBases: false);
var alignmentSet = new AlignmentSet(read1, read2);
stitcher.TryStitch(alignmentSet);
// Without allowing softclips to count to support, should still get a M at an M/S overlap, but it won't be stitched.
var mergedRead = StitcherTestHelpers.GetMergedRead(alignmentSet);
Assert.Equal("4S137M4S", mergedRead.CigarData.ToString());
var expectedDirections = StitcherTestHelpers.BuildDirectionMap(new List <IEnumerable <DirectionType> >
{
StitcherTestHelpers.BuildDirectionSegment(DirectionType.Forward, 4),
StitcherTestHelpers.BuildDirectionSegment(DirectionType.Reverse, 1),
StitcherTestHelpers.BuildDirectionSegment(DirectionType.Stitched, 136),
StitcherTestHelpers.BuildDirectionSegment(DirectionType.Reverse, 4)
});
StitcherTestHelpers.VerifyDirectionType(expectedDirections, mergedRead.CigarDirections.Expand().ToArray());
stitcher = new BasicStitcher(10, useSoftclippedBases: true);
alignmentSet = new AlignmentSet(read1, read2);
stitcher.TryStitch(alignmentSet);
mergedRead = StitcherTestHelpers.GetMergedRead(alignmentSet);
Assert.Equal("4S137M4S", mergedRead.CigarData.ToString());
expectedDirections = StitcherTestHelpers.BuildDirectionMap(new List <IEnumerable <DirectionType> >
{
StitcherTestHelpers.BuildDirectionSegment(DirectionType.Forward, 4),
StitcherTestHelpers.BuildDirectionSegment(DirectionType.Reverse, 1),
StitcherTestHelpers.BuildDirectionSegment(DirectionType.Stitched, 136),
StitcherTestHelpers.BuildDirectionSegment(DirectionType.Reverse, 4)
});
StitcherTestHelpers.VerifyDirectionType(expectedDirections, mergedRead.CigarDirections.Expand().ToArray());
// If we're not ignoring probe softclips, go back to the original expected directions (1 more stitched from probe)
stitcher = new BasicStitcher(10, useSoftclippedBases: true, ignoreProbeSoftclips: false);
alignmentSet = new AlignmentSet(read1, read2);
stitcher.TryStitch(alignmentSet);
mergedRead = StitcherTestHelpers.GetMergedRead(alignmentSet);
Assert.Equal("4S137M4S", mergedRead.CigarData.ToString());
expectedDirections = StitcherTestHelpers.BuildDirectionMap(new List <IEnumerable <DirectionType> >
{
StitcherTestHelpers.BuildDirectionSegment(DirectionType.Forward, 4),
StitcherTestHelpers.BuildDirectionSegment(DirectionType.Stitched, 137),
StitcherTestHelpers.BuildDirectionSegment(DirectionType.Reverse, 4)
});
StitcherTestHelpers.VerifyDirectionType(expectedDirections, mergedRead.CigarDirections.Expand().ToArray());
}
