private PairStatus SingleReadStatus(BamAlignment alignment)
{
if ((alignment.RefID != alignment.MateRefID && alignment.IsPaired()))
{
return(PairStatus.SplitChromosomes); // Stitched reads will have split ref ids too but not the same thing
}
if (((!alignment.IsMateMapped() && alignment.RefID == -1) || (!alignment.IsMapped() && alignment.MateRefID == -1)))
{
return(PairStatus.MateUnmapped);
}
if (alignment.IsDuplicate())
{
return(PairStatus.Duplicate);
}
if (_considerInsertSize)
{
if (alignment.IsPaired() && !OverlapsMate(alignment))
{
return(PairStatus.LongFragment);
}
}
return(PairStatus.Unknown);
}
/// <summary>
/// Bins the fragment identified by alignment. Increases bin count if the first read of a pair passes all the filters.
/// Decreases bin count if the second read of a pair does not pass all the filters.
/// </summary>
/// <param name="alignment"></param>
/// <param name="qualityThreshold">minimum mapping quality</param>
/// <param name="readNameToBinIndex">Dictionary of read name to bin index</param>
/// <param name="usableFragmentCount">number of usable fragments</param>
/// <param name="bins">predefined bins</param>
/// <param name="binIndexStart">bin index from which to start searching for the best bin</param>
public static void BinOneAlignment(BamAlignment alignment, uint qualityThreshold, Dictionary <string, int> readNameToBinIndex,
HashSet <string> samePositionReadNames, ref long usableFragmentCount, List <SampleGenomicBin> bins, ref int binIndexStart)
{
if (!alignment.IsMapped())
{
return;
}
if (!alignment.IsMateMapped())
{
return;
}
if (!alignment.IsPrimaryAlignment())
{
return;
}
if (!(alignment.IsPaired() && alignment.IsProperPair()))
{
return;
}
bool duplicateFailedQCLowQuality = IsDuplicateFailedQCLowQuality(alignment, qualityThreshold);
// Check whether we have binned the fragment using the mate
if (readNameToBinIndex.ContainsKey(alignment.Name))
{
// Undo binning when one of the reads is a duplicate, fails QC or has low mapping quality
if (duplicateFailedQCLowQuality)
{
usableFragmentCount--;
bins[readNameToBinIndex[alignment.Name]].Count--;
}
readNameToBinIndex.Remove(alignment.Name); // clean up
return;
}
if (duplicateFailedQCLowQuality)
{
return;
}
if (alignment.RefID != alignment.MateRefID)
{
return;
} // does this ever happen?
if (IsRightMostInPair(alignment))
{
return;
} // look at only one read of the pair
// handle the case where alignment.Position == alignment.MatePosition
if (alignment.Position == alignment.MatePosition)
{
if (samePositionReadNames.Contains(alignment.Name))
{
samePositionReadNames.Remove(alignment.Name);
return;
}
samePositionReadNames.Add(alignment.Name);
}
if (alignment.FragmentLength == 0)
{
return;
} // Janus-SRS-190: 0 when the information is unavailable
// Try to bin the fragment
int fragmentStart = alignment.Position; // 0-based, inclusive
int fragmentStop = alignment.Position + alignment.FragmentLength; // 0-based, exclusive
while (binIndexStart < bins.Count && bins[binIndexStart].Stop <= fragmentStart) // Bins[binIndexStart] on the left of the fragment
{
binIndexStart++;
}
if (binIndexStart >= bins.Count)
{
return;
} // all the remaining fragments are on the right of the last bin
// now Bins[binIndexStart].Stop > fragmentStart
int bestBinIndex = FindBestBin(bins, binIndexStart, fragmentStart, fragmentStop);
if (bestBinIndex >= 0) // Bin the fragment
{
usableFragmentCount++;
bins[bestBinIndex].Count++;
readNameToBinIndex[alignment.Name] = bestBinIndex;
}
}
/// <summary>
/// Bins fragments.
/// </summary>
private void binFragments()
{
// Sanity check: The BAM index file must exist, in order for us to seek to our target chromosome!
if (!Bam.Index.Exists)
{
throw new Exception(string.Format("Fatal error: Bam index not found at {0}", Bam.Index.FullName));
}
long pairedAlignmentCount = 0; // keep track of paired alignments
usableFragmentCount = 0;
using (BamReader reader = new BamReader(Bam.BamFile.FullName))
{
int desiredRefIndex = -1;
desiredRefIndex = reader.GetReferenceIndex(Chromosome);
if (desiredRefIndex == -1)
{
throw new ApplicationException(
string.Format("Unable to retrieve the reference sequence index for {0} in {1}.", Chromosome, Bam.BamFile.FullName));
}
bool result = reader.Jump(desiredRefIndex, 0);
if (!result)
{
// Note: This is not necessarily an error, it just means that there *are* no reads for this chromosome in this
// .bam file. That is not uncommon e.g. for truseq amplicon.
return;
}
Dictionary<string, int> readNameToBinIndex = new Dictionary<string, int>();
HashSet<string> samePositionReadNames = new HashSet<string>();
int binIndexStart = 0;
int prevPosition = -1;
BamAlignment alignment = new BamAlignment();
while (reader.GetNextAlignment(ref alignment, true))
{
int refID = alignment.RefID;
// quit if the current reference index is different from the desired reference index
if (refID != desiredRefIndex)
break;
if (refID == -1)
continue;
if (alignment.Position < prevPosition) // Make sure the BAM is properly sorted
{
throw new ApplicationException(
string.Format("The alignment on {0} are not properly sorted in {1}: {2}", Chromosome, Bam.BamFile.FullName, alignment.Name));
}
prevPosition = alignment.Position;
if (alignment.IsPaired()) { pairedAlignmentCount++; }
BinOneAlignment(alignment, FragmentBinnerConstants.MappingQualityThreshold, readNameToBinIndex,
samePositionReadNames, ref usableFragmentCount, Bins, ref binIndexStart);
}
}
if (pairedAlignmentCount == 0)
{
throw new ApplicationException(string.Format("No paired alignments found for {0} in {1}", Chromosome, Bam.BamFile.FullName));
}
}
/// <summary>
/// Bins fragments.
/// </summary>
private void binFragments()
{
// Sanity check: The BAM index file must exist, in order for us to seek to our target chromosome!
if (!Bam.Index.Exists)
{
throw new Exception(string.Format("Fatal error: Bam index not found at {0}", Bam.Index.FullName));
}
long pairedAlignmentCount = 0; // keep track of paired alignments
usableFragmentCount = 0;
using (BamReader reader = new BamReader(Bam.BamFile.FullName))
{
int desiredRefIndex = -1;
desiredRefIndex = reader.GetReferenceIndex(Chromosome);
if (desiredRefIndex == -1)
{
throw new Illumina.Common.IlluminaException(
string.Format("Unable to retrieve the reference sequence index for {0} in {1}.", Chromosome, Bam.BamFile.FullName));
}
bool result = reader.Jump(desiredRefIndex, 0);
if (!result)
{
// Note: This is not necessarily an error, it just means that there *are* no reads for this chromosome in this
// .bam file. That is not uncommon e.g. for truseq amplicon.
return;
}
Dictionary <string, int> readNameToBinIndex = new Dictionary <string, int>();
HashSet <string> samePositionReadNames = new HashSet <string>();
int binIndexStart = 0;
int prevPosition = -1;
BamAlignment alignment = new BamAlignment();
while (reader.GetNextAlignment(ref alignment, true))
{
int refID = alignment.RefID;
// quit if the current reference index is different from the desired reference index
if (refID != desiredRefIndex)
{
break;
}
if (refID == -1)
{
continue;
}
if (alignment.Position < prevPosition) // Make sure the BAM is properly sorted
{
throw new Illumina.Common.IlluminaException(
string.Format("The alignment on {0} are not properly sorted in {1}: {2}", Chromosome, Bam.BamFile.FullName, alignment.Name));
}
prevPosition = alignment.Position;
if (alignment.IsPaired())
{
pairedAlignmentCount++;
}
BinOneAlignment(alignment, FragmentBinnerConstants.MappingQualityThreshold, readNameToBinIndex,
samePositionReadNames, ref usableFragmentCount, Bins, ref binIndexStart);
}
}
if (pairedAlignmentCount == 0)
{
throw new Illumina.Common.IlluminaException(string.Format("No paired alignments found for {0} in {1}", Chromosome, Bam.BamFile.FullName));
}
}
/// <summary>
/// Bins the fragment identified by alignment. Increases bin count if the first read of a pair passes all the filters.
/// Decreases bin count if the second read of a pair does not pass all the filters.
/// </summary>
/// <param name="alignment"></param>
/// <param name="qualityThreshold">minimum mapping quality</param>
/// <param name="readNameToBinIndex">Dictionary of read name to bin index</param>
/// <param name="usableFragmentCount">number of usable fragments</param>
/// <param name="bins">predefined bins</param>
/// <param name="binIndexStart">bin index from which to start searching for the best bin</param>
public static void BinOneAlignment(BamAlignment alignment, uint qualityThreshold, Dictionary<string, int> readNameToBinIndex,
HashSet<string> samePositionReadNames, ref long usableFragmentCount, List<GenomicBin> bins, ref int binIndexStart)
{
if (!alignment.IsMapped()) { return; }
if (!alignment.IsMateMapped()) { return; }
if (!alignment.IsPrimaryAlignment()) { return; }
if (!(alignment.IsPaired() && alignment.IsProperPair())) { return; }
bool duplicateFailedQCLowQuality = IsDuplicateFailedQCLowQuality(alignment, qualityThreshold);
// Check whether we have binned the fragment using the mate
if (readNameToBinIndex.ContainsKey(alignment.Name))
{
// Undo binning when one of the reads is a duplicate, fails QC or has low mapping quality
if (duplicateFailedQCLowQuality)
{
usableFragmentCount--;
bins[readNameToBinIndex[alignment.Name]].Count--;
}
readNameToBinIndex.Remove(alignment.Name); // clean up
return;
}
if (duplicateFailedQCLowQuality) { return; }
if (alignment.RefID != alignment.MateRefID) { return; } // does this ever happen?
if (IsRightMostInPair(alignment)) { return; } // look at only one read of the pair
// handle the case where alignment.Position == alignment.MatePosition
if (alignment.Position == alignment.MatePosition)
{
if (samePositionReadNames.Contains(alignment.Name))
{
samePositionReadNames.Remove(alignment.Name);
return;
}
samePositionReadNames.Add(alignment.Name);
}
if (alignment.FragmentLength == 0) { return; } // Janus-SRS-190: 0 when the information is unavailable
// Try to bin the fragment
int fragmentStart = alignment.Position; // 0-based, inclusive
int fragmentStop = alignment.Position + alignment.FragmentLength; // 0-based, exclusive
while (binIndexStart < bins.Count && bins[binIndexStart].Stop <= fragmentStart) // Bins[binIndexStart] on the left of the fragment
{
binIndexStart++;
}
if (binIndexStart >= bins.Count) { return; } // all the remaining fragments are on the right of the last bin
// now Bins[binIndexStart].Stop > fragmentStart
int bestBinIndex = FindBestBin(bins, binIndexStart, fragmentStart, fragmentStop);
if (bestBinIndex >= 0) // Bin the fragment
{
usableFragmentCount++;
bins[bestBinIndex].Count++;
readNameToBinIndex[alignment.Name] = bestBinIndex;
}
}
