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);
}
public void AppendTagData_Test()
{
var alignment = new BamAlignment()
{
TagData = new byte[] { 10, 10, 10 }
};
alignment.AppendTagData(new byte[] { 10, 10, 10 });
Assert.Equal(6, alignment.TagData.Count());
foreach (var item in alignment.TagData)
{
Assert.Equal(item, (byte)10);
}
}
private void ExtractReadsStrandXRValidation()
{
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 = 99,
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]
};
var tagUtils = new TagUtils();
tagUtils.AddStringTag("XR", "BLABLA"); //start with random XR tag and confirm whether it is overwritten by read direction
alignment1.AppendTagData(tagUtils.ToBytes());
var alignment2 = new BamAlignment()
{
AlignmentFlag = 144,
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]
};
var tagUtils2 = new TagUtils();
tagUtils.AddStringTag("XR", "BLABLA"); //start with random XR tag and confirm whether it is overwritten by read direction
alignment2.AppendTagData(tagUtils.ToBytes());
var readPair = new ReadPair(alignment1);
readPair.AddAlignment(alignment2);
var alignmentResults = pairHandler.ExtractReads(readPair);
Assert.Equal(1, alignmentResults.Count);
var alignment = alignmentResults[0];
Assert.Equal("FR", alignment.GetStringTag("XR"));
}
private void ExtractReads(bool addUmiTags)
{
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]
};
var tagUtils = new TagUtils();
tagUtils.AddStringTag("BC", "14");
tagUtils.AddIntTag("SM", 40);
if (addUmiTags)
{
tagUtils.AddIntTag("XV", 1);
tagUtils.AddIntTag("XW", 2);
tagUtils.AddStringTag("XU", "ABBA-ZABBA");
}
alignment1.AppendTagData(tagUtils.ToBytes());
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]
};
var tagUtils2 = new TagUtils();
tagUtils2.AddIntTag("NM", 5);
tagUtils2.AddStringTag("BC", "14");
tagUtils2.AddIntTag("SM", 20);
if (addUmiTags)
{
tagUtils2.AddIntTag("XV", 1);
tagUtils2.AddIntTag("XW", 2);
tagUtils2.AddStringTag("XU", "ABBA-ZABBA");
}
alignment2.AppendTagData(tagUtils2.ToBytes());
var readPair = new ReadPair(alignment1);
readPair.AddAlignment(alignment2);
var alignmentResults = pairHandler.ExtractReads(readPair);
Assert.Equal(1, alignmentResults.Count);
var alignment = alignmentResults[0];
Assert.NotNull(alignment.GetStringTag("XD"));
Assert.Null(alignment.GetIntTag("NM"));
Assert.Null(alignment.GetStringTag("BC"));
Assert.Null(alignment.GetIntTag("SM"));
if (addUmiTags)
{
Assert.Equal("ABBA-ZABBA", alignment.GetStringTag("XU"));
Assert.Equal(1, alignment.GetIntTag("XV"));
Assert.Equal(2, alignment.GetIntTag("XW"));
}
}
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);
}