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 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 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);
}
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());
}