public static BamAlignment StitchifyBamAlignment(ReadPair pair, Read read, char read1dir, char read2dir)
{
var alignment = new BamAlignment(read.BamAlignment);
alignment.SetIsFirstMate(false);
alignment.SetIsProperPair(false);
var tagUtils = new TagUtils();
if (read.StitchedCigar != null)
{
alignment.CigarData = read.StitchedCigar;
}
if (read.CigarDirections != null)
{
tagUtils.AddStringTag("XD", read.CigarDirections.ToString());
}
// if the original reads had UMIs and were collapsed, they will have XU(Z), XV(i), XW(i)
// these need to be copied to correctly populate some fields in the called variants
if (pair.Read1.TagData != null && pair.Read1.TagData.Length > 0)
{
var xu = pair.Read1.GetStringTag("XU");
if (xu != null)
{
tagUtils.AddStringTag("XU", xu);
}
var xv = pair.Read1.GetIntTag("XV");
if (xv.HasValue)
{
tagUtils.AddIntTag("XV", xv.Value);
}
var xw = pair.Read1.GetIntTag("XW");
if (xw.HasValue)
{
tagUtils.AddIntTag("XW", xw.Value);
}
}
var xr = string.Format("{0}{1}", read1dir, read2dir);
tagUtils.AddStringTag("XR", xr);
var tagData = tagUtils.ToBytes();
var existingTags = alignment.TagData;
if (existingTags == null)
{
alignment.TagData = tagData;
}
else
{
alignment.AppendTagData(tagData);
}
return(alignment);
}
private void SetPairedEndAttributes(BamAlignment read1, BamAlignment read2)
{
read1.SetIsFirstMate(true);
read1.SetIsSecondMate(false);
read2.SetIsFirstMate(false);
read2.SetIsSecondMate(true);
read1.SetIsProperPair(true);
read2.SetIsProperPair(true);
read2.SetIsReverseStrand(true);
read2.MatePosition = read1.Position;
read1.MatePosition = read2.Position;
}
// Copied from AlignmentSourceTests.cs (modified to add custom CigarData)
private Read CreateRead(string chr, string sequence, int position, string name, CigarAlignment cigar = null, bool isMapped = true,
bool isPrimaryAlignment = true, bool isProperPair = true, bool isDuplicate = false, int mapQuality = 10, bool addCigarData = true,
bool read2 = false, int matePosition = 0)
{
var alignment = new BamAlignment()
{
Bases = sequence, Position = position, Name = name, MapQuality = (uint)mapQuality
};
alignment.SetIsUnmapped(!isMapped);
alignment.SetIsSecondaryAlignment(!isPrimaryAlignment);
alignment.SetIsDuplicate(isDuplicate);
alignment.SetIsProperPair(isProperPair);
alignment.SetIsFirstMate(!read2);
alignment.MatePosition = matePosition;
alignment.CigarData = cigar ?? new CigarAlignment(sequence.Length + "M");
return(new Read(chr, alignment));
}
public static BamAlignment CreateBamAlignment(string sequence, int position,
int matePosition, byte qualityForAll, bool isReverseMapped, uint mapQ = 30, byte[] qualities = null, CigarAlignment cigar = null, string name = null, bool isFirstMate = true)
{
var bamAlignment = new BamAlignment
{
Bases = sequence,
Position = position - 1,
CigarData = cigar ?? new CigarAlignment(sequence.Length + "M"),
Qualities = qualities ?? Enumerable.Repeat(qualityForAll, sequence.Length).ToArray(),
MatePosition = matePosition - 1,
TagData = new byte[0],
RefID = 1,
MateRefID = 1,
Name = name ?? "Alignment"
};
bamAlignment.SetIsFirstMate(isFirstMate);
bamAlignment.MapQuality = mapQ;
bamAlignment.SetIsReverseStrand(isReverseMapped);
bamAlignment.SetIsMateReverseStrand(!isReverseMapped);
return(bamAlignment);
}
public void BamAlignmentFlag_Setter_Tests()
{
var alignment = new BamAlignment();
//Set Failed QC
alignment.SetIsFailedQC(true);
Assert.Equal((uint)512, alignment.AlignmentFlag);
alignment.SetIsFailedQC(false);
Assert.Equal((uint)0, alignment.AlignmentFlag);
//Set Mate Unmapped
alignment.SetIsMateUnmapped(true);
Assert.Equal((uint)8, alignment.AlignmentFlag);
alignment.SetIsMateUnmapped(false);
Assert.Equal((uint)0, alignment.AlignmentFlag);
//Set Mate Reverse Strand
alignment.SetIsMateReverseStrand(true);
Assert.Equal((uint)32, alignment.AlignmentFlag);
alignment.SetIsMateReverseStrand(false);
Assert.Equal((uint)0, alignment.AlignmentFlag);
//Set Is Paired
alignment.SetIsPaired(true);
Assert.Equal((uint)1, alignment.AlignmentFlag);
alignment.SetIsPaired(false);
Assert.Equal((uint)0, alignment.AlignmentFlag);
//Set Duplicate
alignment.SetIsDuplicate(true);
Assert.Equal((uint)1024, alignment.AlignmentFlag);
alignment.SetIsDuplicate(false);
Assert.Equal((uint)0, alignment.AlignmentFlag);
//Set First Mate
alignment.SetIsFirstMate(true);
Assert.Equal((uint)64, alignment.AlignmentFlag);
alignment.SetIsFirstMate(false);
Assert.Equal((uint)0, alignment.AlignmentFlag);
//Set Proper Pair
alignment.SetIsProperPair(true);
Assert.Equal((uint)2, alignment.AlignmentFlag);
alignment.SetIsProperPair(false);
Assert.Equal((uint)0, alignment.AlignmentFlag);
//Set Reverse Strand
alignment.SetIsReverseStrand(true);
Assert.Equal((uint)16, alignment.AlignmentFlag);
alignment.SetIsReverseStrand(false);
Assert.Equal((uint)0, alignment.AlignmentFlag);
//Set Secondary Alignment
alignment.SetIsSecondaryAlignment(true);
Assert.Equal((uint)256, alignment.AlignmentFlag);
alignment.SetIsSecondaryAlignment(false);
Assert.Equal((uint)0, alignment.AlignmentFlag);
//Set Second Mate
alignment.SetIsSecondMate(true);
Assert.Equal((uint)128, alignment.AlignmentFlag);
alignment.SetIsSecondMate(false);
Assert.Equal((uint)0, alignment.AlignmentFlag);
//Set Unmapped
alignment.SetIsUnmapped(true);
Assert.Equal((uint)4, alignment.AlignmentFlag);
alignment.SetIsUnmapped(false);
Assert.Equal((uint)0, alignment.AlignmentFlag);
}
private void ExtractReadsStrand(bool r1Reverse, bool r2Reverse, string expectedXRTag)
{
var refIdMapping = new Dictionary <int, string>()
{
{ 1, "chr1" }
};
var stitcher = StitcherTestHelpers.GetStitcher(10, false);
var readStatusCounter = new ReadStatusCounter();
var pairHandler = new PairHandler(refIdMapping, stitcher, readStatusCounter, filterUnstitchablePairs: true);
var alignment1 = new BamAlignment()
{
AlignmentFlag = 0,
Bases = "ABCF",
Bin = 4,
CigarData = new CigarAlignment("2S2M"),
FragmentLength = 42,
MapQuality = 30,
MatePosition = 2,
MateRefID = 43,
Name = "Read1",
Position = 1,
Qualities = new byte[4],
RefID = 1,
TagData = new byte[0]
};
alignment1.SetIsFirstMate(true);
alignment1.SetIsReverseStrand(r1Reverse);
var alignment2 = new BamAlignment()
{
AlignmentFlag = 0,
Bases = "ABCF",
Bin = 4,
CigarData = new CigarAlignment("2S2M"),
FragmentLength = 42,
MapQuality = 30,
MatePosition = 2,
MateRefID = 43,
Name = "Read1",
Position = 1,
Qualities = new byte[4],
RefID = 1,
TagData = new byte[0]
};
alignment2.SetIsSecondMate(true);
alignment2.SetIsReverseStrand(r2Reverse);
var readPair = new ReadPair(alignment1);
readPair.AddAlignment(alignment2);
var alignmentResults = pairHandler.ExtractReads(readPair);
Assert.Equal(1, alignmentResults.Count);
var alignment = alignmentResults[0];
var XD = alignment.GetStringTag("XR");
Assert.Equal(expectedXRTag, alignment.GetStringTag("XR"));
}
public List <BamAlignment> ExtractReads(ReadPair pair)
{
var reads = new List <BamAlignment>();
var chrom1 = _refIdMapping[pair.Read1.RefID];
var chrom2 = _refIdMapping[pair.Read2.RefID];
var alignmentSet = new AlignmentSet(
new Read(chrom1, pair.Read1),
new Read(chrom2, pair.Read2),
false);
var stitched = _stitcher.TryStitch(alignmentSet);
if (stitched)
{
if (alignmentSet.ReadsForProcessing.Count > 1)
{
throw new Exception("AlignmentSets for stitched reads should only have one ReadsForProcessing.");
}
foreach (var read in alignmentSet.ReadsForProcessing)
{
var alignment = new BamAlignment(read.BamAlignment);
alignment.SetIsFirstMate(false);
alignment.SetIsProperPair(false);
var tagUtils = new TagUtils();
if (read.StitchedCigar != null)
{
alignment.CigarData = read.StitchedCigar;
}
if (read.CigarDirections != null)
{
tagUtils.AddStringTag("XD", read.CigarDirections.ToString());
}
var tagData = tagUtils.ToBytes();
var existingTags = alignment.TagData;
if (existingTags == null)
{
alignment.TagData = tagData;
}
else
{
alignment.AppendTagData(tagData);
}
reads.Add(alignment);
}
}
else
{
if (!_filterUnstitchablePairs)
{
_statusCounter.AddStatusCount("Unstitchable Pairs Kept");
reads.Add(new BamAlignment(alignmentSet.PartnerRead1.BamAlignment));
reads.Add(new BamAlignment(alignmentSet.PartnerRead2.BamAlignment));
}
else
{
_statusCounter.AddStatusCount("Unstitchable Pairs Filtered");
}
}
return(reads);
}