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_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)); }
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_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()); }