protected override bool ShouldSkipRead(BamAlignment alignment)
{
if (!_filterPairLowMapQ && alignment.MapQuality > 0 && alignment.MapQuality < _minMapQuality)
{
_statusCounter.AddDebugStatusCount("Skipped read below mapQ");
return(true);
}
if (alignment.IsSupplementaryAlignment())
{
_statusCounter.AddDebugStatusCount("Skipped supplementary");
return(true);
}
if (alignment.IsSecondary())
{
_statusCounter.AddDebugStatusCount("Skipped secondary");
return(true);
}
if (_filterForProperPairs && !alignment.IsProperPair())
{
_statusCounter.AddDebugStatusCount("Skipped improper pair");
return(true);
}
return(false);
}
public BamAlignment GetFinalAlignment(BamAlignment origBamAlignment, out bool changed, out bool forcedSoftclip, out bool confirmed, out bool sketchy,
List <PreIndel> selectedIndels = null, List <PreIndel> existingIndels = null,
bool assumeImperfect = true, List <HashableIndel> confirmedAccepteds = null, List <PreIndel> mateIndels = null)
{
sketchy = false;
forcedSoftclip = false;
bool forcedAlignment = false;
var presumeStartPositionForForcedAlignment = 0;
if (origBamAlignment.CigarData.Count == 0)
{
// This was something weird that came up in the halo dataset... mapq is 0 but is still mapped, no cigar
if (origBamAlignment.Position <= 0 && origBamAlignment.FragmentLength != 0) // No sense trying to fiddle with the position otherwise
{
// TODO does this really even move the needle? Is it helping enough to outweigh its weirdness?
var presumedEndPosition = origBamAlignment.MatePosition < origBamAlignment.Position
? origBamAlignment.MatePosition - origBamAlignment.FragmentLength
: origBamAlignment.MatePosition + origBamAlignment.FragmentLength;
presumeStartPositionForForcedAlignment = presumedEndPosition - origBamAlignment.Bases.Length;
forcedAlignment = true;
}
else
{
presumeStartPositionForForcedAlignment = origBamAlignment.Position;
forcedAlignment = true;
}
}
var anyIndelsAtAll = _regionFilterer.AnyIndelsNearby(origBamAlignment.Position);
bool isRealignable = true;
if (anyIndelsAtAll)
{
var isImperfectRead = false || ((origBamAlignment.ContainsDisallowedCigarOps(_suspectCigarOps) ||
origBamAlignment.GetIntTag("NM") > 0 || forcedAlignment));
var isReadWorthCaringAbout = !origBamAlignment.IsDuplicate() && !origBamAlignment.IsSecondary();
isRealignable = isImperfectRead && isReadWorthCaringAbout && origBamAlignment.Bases.Distinct().Count() > 1;
}
else
{
_statusCounter.AddStatusCount("No indels nearby at all");
isRealignable = false;
}
if (!isRealignable)
{
confirmed = false;
changed = false;
sketchy = false;
return(origBamAlignment);
}
// TODO maybe flag (or return all) if there's a lot or high quality stuff that we're missing! Esp with pair specific
var indels = _indelSource.GetRelevantIndels(forcedAlignment ? presumeStartPositionForForcedAlignment : origBamAlignment.Position,
mateIndels, confirmedAccepteds);
// Don't realign around single indels if we already have them
bool hasExistingUnsanctionedIndels = false;
bool existingSanctionedIndelIsBest = false;
bool hasVeryGoodIndel = false;
bool hasHardToCallIndel = false;
var existingMatches = new List <PreIndel>();
HashableIndel existingConfirmedIndel = new HashableIndel();
var existingMatchHashables = new List <HashableIndel>();
if (indels.Any() && existingIndels != null && existingIndels.Any())
{
var topScore = (float)(indels.Max(x => x.Key.Score));
var matchesFound = 0;
var nonPreExistingIndels = new List <KeyValuePair <HashableIndel, GenomeSnippet> >();
var index = 0;
foreach (var kvp in indels)
{
var indel = kvp.Key;
var matches = existingIndels.Where(e => Helper.IsMatch(e, indel));
var isMatch = matches.Any();
if (isMatch)
{
matchesFound++;
if (!indel.InMulti && index == 0)
{
existingSanctionedIndelIsBest = true;
existingConfirmedIndel = indel;
}
var proportionOfTopScore = indel.Score / (float)topScore;
if (proportionOfTopScore >= 0.75)
{
hasVeryGoodIndel = true;
}
if (indel.HardToCall)
{
hasHardToCallIndel = true;
}
existingMatches.AddRange(matches);
// TODO do we need special handling of multis?
existingMatchHashables.Add(indel);
}
if (!isMatch || indel.InMulti)
{
nonPreExistingIndels.Add(kvp);
}
index++;
}
// TODO do we actually want to replace indels with non-pre-existing only?
indels = nonPreExistingIndels;
if (matchesFound == 0)
{
hasExistingUnsanctionedIndels = true;
}
}
// TODO this precludes us from having good multis
if (existingSanctionedIndelIsBest)
{
// If it already had the top ranked indel, there's not really any point in trying to realign around others (here we assume that it's also the best fitting indel for the read, hence why it was originally called by the regular aligner).
_statusCounter.AddStatusCount("Existing indel is already the best available");
changed = false;
confirmed = true;
UpdateOutcomeForConfirmed(existingConfirmedIndel);
if (confirmedAccepteds == null)
{
confirmedAccepteds = new List <HashableIndel>();
}
confirmedAccepteds.Add(existingConfirmedIndel);
return(origBamAlignment);
}
if (!indels.Any() || origBamAlignment.EndPosition - origBamAlignment.Position > 500)
{
if (!indels.Any())
{
// TODO maybe do the forced softclip here if the read did have indels?
_statusCounter.AddStatusCount("No indels to realign to");
_statusCounter.AppendStatusStringTag("RX", $"{origBamAlignment.GetStringTag("RX")},No indels to realign to", origBamAlignment);
}
else
{
_statusCounter.AddStatusCount("Alignment reference span longer than we can realign to");
}
changed = false;
confirmed = false;
return(origBamAlignment);
}
// TODO this should relate to cap on indel size... introducing too large of an indel will make us go beyond this context.
var context = indels.First().Value;
var orderedIndels = indels.Select(x => x.Key).ToList();
var numIndels = orderedIndels.Count;
_statusCounter.AddStatusCount("Realigning to " + numIndels);
var bamAlignment = new BamAlignment(origBamAlignment);
if (forcedAlignment)
{
bamAlignment.CigarData = new CigarAlignment(origBamAlignment.Bases.Length + "M");
bamAlignment.Position = presumeStartPositionForForcedAlignment;
}
var realignResult = _readRealigner.Realign(new Read(_chromosome, bamAlignment),
orderedIndels, indels.ToDictionary(x => x.Key, x => x.Value), confirmedAccepteds != null && confirmedAccepteds.Any());
var acceptedIndels = realignResult?.AcceptedIndels;
var hasAnyIndels = acceptedIndels != null && acceptedIndels.Any();
if (realignResult != null)
{
_statusCounter.AddStatusCount("Able to realign at all (may still be worse than original)");
_statusCounter.AppendStatusStringTag("RX", "Able to realign at all(may still be worse than original)", bamAlignment);
}
else
{
_statusCounter.AddStatusCount("Not able to realign at all");
_statusCounter.AppendStatusStringTag("RX", "Not able to realign at all", origBamAlignment);
}
AlignmentSummary originalAlignmentSummary = null;
var realignmentUnchanged = true;
if (realignResult != null)
{
originalAlignmentSummary =
Extensions.GetAlignmentSummary((new Read(_chromosome, origBamAlignment)), context.Sequence,
_trackActualMismatches, _checkSoftclipsForMismatches, context.StartPosition);
realignmentUnchanged = _judger.RealignmentIsUnchanged(realignResult, origBamAlignment);
if (originalAlignmentSummary.NumMismatches > 0)
{
// TODO PERF do we still want to use this ever?
var sumMismatch = Helper.GetSumOfMismatchQualities(origBamAlignment.Qualities,
origBamAlignment.Bases, new Read(_chromosome, origBamAlignment).PositionMap, context.Sequence,
context.StartPosition);
originalAlignmentSummary.SumOfMismatchingQualities = sumMismatch;
}
// Within this logic also checking the same as "!realignmentUnchanged" above.. consolidate this.
if (selectedIndels != null &&
(_judger.RealignmentBetterOrEqual(realignResult, originalAlignmentSummary, confirmedAccepteds != null && confirmedAccepteds.Any())) ||
ResultIsGoodEnough(realignResult, origBamAlignment, originalAlignmentSummary,
realignmentUnchanged, confirmedAccepteds != null && confirmedAccepteds.Any()))
{
UpdateIndelOutcomes(numIndels, orderedIndels, hasAnyIndels, acceptedIndels, confirmedAccepteds, true, realignResult);
if (realignResult.IsSketchy)
{
sketchy = true;
}
return(AcceptRealignment(origBamAlignment, out changed, selectedIndels, existingIndels, realignResult, originalAlignmentSummary, bamAlignment, hasExistingUnsanctionedIndels, out confirmed));
}
}
// At this point, any good realignment would have been returned. If it's realigned and changed now, it's an unaccepted (not good enough) realignment.
// If it had an indel to begin with, it's basically a vote that we don't trust that indel. Optionally softclip it out.
if (!realignmentUnchanged)
{
changed = false;
confirmed = false;
HandleFailedRealignment(origBamAlignment, ref forcedSoftclip, existingIndels, realignResult, hasExistingUnsanctionedIndels, existingMatches);
if ((hasVeryGoodIndel || (hasHardToCallIndel && _judger.IsVeryConfident(originalAlignmentSummary))) && !hasExistingUnsanctionedIndels && existingMatchHashables.Any())
{
// It didn't have the tip-top indel, but it had one that was very close, and we tried realigning around the top guys and failed - this one looks better. Give it credit.
confirmed = true;
foreach (var indel in existingMatchHashables)
{
UpdateOutcomeForConfirmed(indel);
if (confirmedAccepteds != null)
{
confirmedAccepteds.Add(indel);
}
}
}
UpdateIndelOutcomes(numIndels, orderedIndels, hasAnyIndels, acceptedIndels, confirmedAccepteds, false, realignResult);
}
else
{
if (acceptedIndels != null)
{
foreach (var indelNum in acceptedIndels)
{
var indel = orderedIndels[indelNum];
UpdateOutcomeForConfirmed(indel);
}
}
_statusCounter.AddStatusCount("INDEL STATUS\tUnchanged\t" + realignResult?.Indels);
_statusCounter.AppendStatusStringTag("RX", "Unchanged: " + realignResult?.Indels, origBamAlignment);
confirmed = true;
changed = false;
return(origBamAlignment);
}
if (realignResult == null)
{
if (_softclipUnknownIndels && hasExistingUnsanctionedIndels)
{
var unsanctioned = existingIndels.Where(x => !existingMatches.Contains(x));
foreach (var preIndel in unsanctioned.OrderBy(x => x.ReferencePosition))
{
var reverseClip = false;
var clipLength = preIndel.RightAnchor;
if (preIndel.LeftAnchor < preIndel.RightAnchor)
{
reverseClip = true;
clipLength = preIndel.LeftAnchor;
}
// TODO arbitrary number here...
// If it's pretty well-anchored, don't remove the indel
if (clipLength > 20)
{
continue;
}
forcedSoftclip = true;
_statusCounter.AddStatusCount("Softclipped out bad indel");
_statusCounter.AppendStatusStringTag("RX",
$"Softclipped out bad indel({origBamAlignment.CigarData},{string.Join(",", existingIndels)}... No realignment",
origBamAlignment);
_statusCounter.AddStatusCount("INDEL STATUS\tRemoved\t" + string.Join("|", existingIndels));
OverlappingIndelHelpers.SoftclipAfterIndel(origBamAlignment,
reverseClip, preIndel.ReferencePosition);
}
}
}
_statusCounter.AppendStatusStringTag("RX", "Realignment failed", origBamAlignment);
_statusCounter.AddStatusCount("Realignment failed");
return(origBamAlignment);
}