// TODO I strongly advise we remove this logic altogether. It is brittle, incomplete, and hasn't been shown to reliably improve results.
public Read GenerateNifiedMergedRead(AlignmentSet set, bool useSoftclippedBases)
{
var read1InsertionAdjustedEnd = set.PartnerRead1.ClipAdjustedEndPosition +
set.PartnerRead1.CigarData.GetSuffixInsertionLength();
var read2InsertionAdjustedEnd = set.PartnerRead2.ClipAdjustedEndPosition +
set.PartnerRead2.CigarData.GetSuffixInsertionLength();
var read1LongerThanRead2 = read2InsertionAdjustedEnd < read1InsertionAdjustedEnd;
var furthestRight = read1LongerThanRead2 ? read1InsertionAdjustedEnd : read2InsertionAdjustedEnd;
var nifiedStitchedLength = furthestRight + 1 - set.PartnerRead1.ClipAdjustedPosition;
var prefixClip = set.PartnerRead1.CigarData.GetPrefixClip();
var suffixClip = read1LongerThanRead2
? set.PartnerRead1.CigarData.GetSuffixClip()
: set.PartnerRead2.CigarData.GetSuffixClip();
if (read2InsertionAdjustedEnd == read1InsertionAdjustedEnd)
{
suffixClip = Math.Min(set.PartnerRead1.CigarData.GetSuffixClip(),
set.PartnerRead2.CigarData.GetSuffixClip());
}
if (prefixClip + suffixClip >= nifiedStitchedLength)
{
throw new ArgumentException($"Reads cannnot be Nified using this simple algorithm. The prefix and suffix sofctlips overlap for reads: {set.PartnerRead1.Name} {set.PartnerRead1.Position}:{set.PartnerRead1.CigarData} and {set.PartnerRead2.Position}:{set.PartnerRead2.CigarData}");
}
var nifiedStitchedCigar = new CigarAlignment((prefixClip > 0 ? string.Format("{0}S", prefixClip) : "")
+ string.Format("{0}M", nifiedStitchedLength - prefixClip - suffixClip)
+ (suffixClip > 0 ? string.Format("{0}S", suffixClip) : ""));
var beforeOverlap = (useSoftclippedBases ? set.PartnerRead2.ClipAdjustedPosition : set.PartnerRead2.Position) -
set.PartnerRead1.ClipAdjustedPosition;
var afterOverlap = read1LongerThanRead2
? (read1InsertionAdjustedEnd - read2InsertionAdjustedEnd)
: (read2InsertionAdjustedEnd - read1InsertionAdjustedEnd);
var r1Forward = set.PartnerRead1.SequencedBaseDirectionMap.First() == DirectionType.Forward;
var beforeOverlapDirection = r1Forward ? "F" : "R";
var afterOverlapDirection = read1LongerThanRead2 ? (r1Forward ? "F" : "R") : (r1Forward ? "R" : "F");
var nifiedStitchedDirections = (beforeOverlap > 0
? string.Format("{0}{1}", beforeOverlap, beforeOverlapDirection)
: "")
+ string.Format("{0}S", nifiedStitchedLength - beforeOverlap - afterOverlap)
+
(afterOverlap > 0 ? string.Format("{0}{1}", afterOverlap, afterOverlapDirection) : "");
var mergedRead = new Read(set.PartnerRead1.Chromosome, new BamAlignment
{
Name = set.PartnerRead1.Name,
Bases = new string('N', nifiedStitchedLength),
Position = Math.Min(set.PartnerRead1.Position - 1, set.PartnerRead2.Position - 1),
Qualities = Enumerable.Repeat((byte)0, nifiedStitchedLength).ToArray(),
CigarData = nifiedStitchedCigar
})
{
StitchedCigar = nifiedStitchedCigar,
CigarDirections = new CigarDirection(nifiedStitchedDirections)
};
return(mergedRead);
}
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)));
});
}
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());
}
