protected void OutputGraphicAndFindDeletion(MitochondrialAssembly attemptedAssembly)
{
if (attemptedAssembly.SuccessfulAssembly)
{
MitochondrialAssemblyPlotMaker plotMaker = new MitochondrialAssemblyPlotMaker(attemptedAssembly);
#if !NO_R
if (OutputIntermediateGraphSteps)
{
plotMaker.Render(rInt, DiagnosticFileOutputPrefix + "AssemblyView.pdf");
}
#endif
DecidedAssemblyTotalLength = plotMaker.Assembly.AssemblyLength;
//Output all possible assemblies and deletions if possible
RaiseMessage("Graph contains " + plotMaker.Assembly.AllNodesInGraph.Count.ToString() + " Contained Nodes");
if (plotMaker.Assembly.AllNodesInGraph.Count < 10)
{
DeletionSearchAttempted = true;
LargeDeletionFinder ldf = new LargeDeletionFinder();
var deletions = ldf.FindAllDeletions(this.Graph, plotMaker.Assembly);
// Check to see if any sections
DeletionsFoundInAssemblyGraph = deletions.Where(z => z.HasDeletion).Count();
PossibleAssemblyCount = ldf.PossibleDeletionPaths.Count;
RaiseMessage("Found a total of: " + deletions.Count + " possible mutations in " + ldf.PossibleDeletionPaths.Count.ToString() + " possible assembly paths");
//throw error as not finalized here
ldf.OutputReport(this.DiagnosticFileOutputPrefix + "DeletionReport.csv");
}
else
{
PossibleAssemblyCount = 999;
DeletionSearchAttempted = false;
}
}
}
public List <DeletionAnalysis> FindAllDeletions(DeBruijnGraph graph, MitochondrialAssembly assembly)
{
LargeDeletionFinder.graph = graph;
KmerLength = graph.KmerLength;
//set all edges in the graph to not be visited
graph.GetNodes().AsParallel().ForAll(x => x.ResetVisitState());
foreach (DeBruijnNode node in graph.GetNodes())
{
//starting from any unused edges in the network, make any/all paths one can
//take
try
{
PossibleDeletionPaths.AddRange(ExtendFromStartNode(node));
}
catch (Exception thrown) {
Console.WriteLine(thrown.Message);
}
}
DeletionReports = PossibleDeletionPaths.Select(x => new DeletionAnalysis(x)).ToList();
return(DeletionReports);
}
public override PadenaAssembly Assemble(IEnumerable <ISequence> inputSequences)
{
if (inputSequences == null)
{
throw new ArgumentNullException("inputSequences");
}
// Step 0: Load the reference genome as a fasta file.
// Remove ambiguous reads and set up fields for assembler process
this.Initialize();
// Step 1, 2: Create k-mers from reads and build de bruijn graph and paint them with the reference
System.Diagnostics.Stopwatch sw = System.Diagnostics.Stopwatch.StartNew();
this.CreateGraphStarted();
this.CreateGraph(inputSequences);
this.CreateGraphEnded();
sw.Stop();
this.NodeCountReport();
this.TaskTimeSpanReport(sw.Elapsed);
int count = this.Graph.GetNodes().Where(x => x.IsInReference).Count();
ReferenceNodeCountAfterCreation = count;
TotalSequencingBP = Graph.GetNodes().Sum(x => x.KmerCount * KmerLength);
RaiseMessage("A total of: " + count.ToString() + " nodes remain from the reference");
RaiseMessage("A total of: " + this.Graph.NodeCount + " nodes are in the graph");
NodeCountAfterCreation = Graph.NodeCount;
SkippedReadsAfterQCCount = Graph.SkippedSequencesCount;
ReadCount = Graph.ProcessedSequencesCount;
if (NodeCountAfterCreation < 100)
{
return(null);
}
//Step 2.1, Remove nodes are below coverage cutoff
sw.Reset();
sw.Start();
// Estimate and set default value for erosion and coverage thresholds
this.EstimateDefaultThresholds();
int sqrtCoverageCutOff = this.CalculateSqrtOfMedianCoverageCutoff();
var coverageCutOff = ForceSqrtThreshold ? sqrtCoverageCutOff : Math.Min(sqrtCoverageCutOff, AlternateMinimumNodeCount);
if (MTAssembleArguments.MinNodeCountSet)
{
coverageCutOff = AlternateMinimumNodeCount;
}
//var coverageCutOff = sqrtCoverageCutOff;
KmerCutOff = coverageCutOff;
if (OutputIntermediateGraphSteps)
{
OutputNodeCountHistograms("PreFiltered", coverageCutOff);
}
long originalNodes = this.Graph.NodeCount;
ThresholdCoverageNodePurger snr = new ThresholdCoverageNodePurger(coverageCutOff);
snr.RemoveLowCoverageNodes(Graph);
PercentNodesRemovedByLowCoverageOrThreshold = originalNodes / (double)this.Graph.NodeCount;
sw.Stop();
TaskTimeSpanReport(sw.Elapsed);
RaiseMessage("Finished removing nodes with less than " + snr.CoverageCutOff.ToString() + " counts");
NodeCountReport();
NodeCountAfterCoveragePurge = Graph.NodeCount;
sw.Reset();
sw.Start();
RaiseMessage("Start removing unconnected nodes");
// Remove pathological nodes
var badNodes = PathologicalSequencePurger.RemovePathologicalNodes(Graph);
if (badNodes > 0)
{
RaiseMessage("WARNING!!!! Found and removed " + badNodes.ToString() + " pathological nodes. These were removed.", false);
}
//Step 2.2 Remove nodes that are not connected to the reference genome
UnlinkedToReferencePurger remover = new UnlinkedToReferencePurger();
remover.RemoveUnconnectedNodes(Graph, referenceNodes);
RaiseMessage("Finished removing unconnected nodes");
this.NodeCountReport();
NodeCountAfterUndangle = Graph.NodeCount;
//outputVisualization ("PostUnconnectedFilter");
// Step 2.3: Remove dangling links from graph
///NIGEL: This also removes the low coverage nodes
sw.Reset();
sw.Restart();
this.UndangleGraphStarted();
this.UnDangleGraph();
this.UndangleGraphEnded();
sw.Stop();
this.TaskTimeSpanReport(sw.Elapsed);
this.NodeCountReport();
if (OutputIntermediateGraphSteps)
{
outputVisualization("PostUndangleFilter");
}
// Step 4: Remove redundant SNP and indel paths from graph
RaiseMessage(string.Format(CultureInfo.CurrentCulture, "Starting to remove redundant paths", DateTime.Now));
this.RemoveRedundancyStarted();
RaiseMessage(string.Format(CultureInfo.CurrentCulture, "Starting to remove SNPs", DateTime.Now));
this.RemoveRedundancy();
RaiseMessage(string.Format(CultureInfo.CurrentCulture, "Finished removing SNPs", DateTime.Now));
this.NodeCountReport();
//Now remove redundant indel paths as well
//TODO: For historic reasons this is largely similar to the snp remover, which isn't so great...
RaiseMessage(string.Format(CultureInfo.CurrentCulture, "Starting to call INDELs", DateTime.Now));
var indels = CallAndRemoveIndels();
RaiseMessage(string.Format(CultureInfo.CurrentCulture, "Finished calling and removing small INDELs paths", DateTime.Now));
this.NodeCountReport();
// Perform dangling link purger step once more.
// This is done to remove any links created by redundant paths purger.
this.UnDangleGraph();
RaiseMessage(string.Format(CultureInfo.CurrentCulture, "Finished removing all redundant paths", DateTime.Now));
this.NodeCountReport();
//STEP 4.2 Rerun the unlinked to reference purger after graph is cleaned
ChangeNodeVisitFlag(false);
remover = new UnlinkedToReferencePurger();
remover.RemoveUnconnectedNodes(Graph, referenceNodes);
this.RemoveRedundancyEnded();
this.NodeCountReport();
NodeCountAfterRedundancyRemoval = Graph.NodeCount;
if (OutputIntermediateGraphSteps)
{
outputVisualization("Post-redundant-path-removal");
}
//Now attempt to assemble and find deletions
var attemptedAssembly = new MitochondrialAssembly(Graph, DiagnosticFileOutputPrefix);
FinalMetaNodeCount = attemptedAssembly.AllNodesInGraph.Count;
SuccessfulAssembly = attemptedAssembly.SuccessfulAssembly;
if (SuccessfulAssembly)
{
SuccessfulAssemblyLength = attemptedAssembly.AssemblyLength;
MinSplitPercentage = attemptedAssembly.MinimumGreedySplit;
if (OutputDiagnosticInformation)
{
var outReport = attemptedAssembly.OutputAssembly(DiagnosticFileOutputPrefix);
if (outReport != null)
{
//TODO: This matching is really crappy, need to find a better way to propogate this on up.
BestHaplotypeScore = outReport.BestHit.Rank;
SecondBestHaplotypeScore = outReport.SecondBestHit.Rank;
BestMatchingHaplotype = outReport.BestHit.node.haplogroup.id;
SecondBestMatchingHaplotype = outReport.SecondBestHit.node.haplogroup.id;
NumberOfEquallyGoodHaplotypes = outReport.NumberOfEquallyGoodBestHits;
PolymorphismsMatchingHaplotype = outReport.BestHit.NumberOfMatchingPolymorphisms;
PolymorphismsMissingFromHaplotype = outReport.BestHit.NumberOfPolymorphismsMissingFromHaplotype;
PolymorphismsMissingFromGenotype = outReport.BestHit.NumberOfPolymorphismsMissingFromGenotype;
}
}
else
{
RaiseMessage("Greedy assembly skipped as assembly failed.");
}
}
else
{
RaiseMessage("Greedy assembly skipped as assembly failed.");
}
//Now find deletions
this.OutputGraphicAndFindDeletion(attemptedAssembly);
PercentageOfScannedReadsUsed = Graph.GetNodes().Sum(x => x.KmerCount * KmerLength) / (double)TotalSequencingBP;
RaiseMessage("Used a total of " + PercentageOfScannedReadsUsed.ToString("p") + " of basepairs in reads for assembly");
// Step 5: Build Contigs - This is essentially independent of deletion finding
this.BuildContigsStarted();
List <ISequence> contigSequences = this.BuildContigs().ToList();
contigSequences.ForEach(x => ReferenceGenome.AssignContigToMTDNA(x));
contigSequences.Sort((x, y) => - x.Count.CompareTo(y.Count));
this.BuildContigsEnded();
PadenaAssembly result = new PadenaAssembly();
result.AddContigs(contigSequences);
long totalLength = contigSequences.Sum(x => x.Count);
RaiseMessage("Assembled " + totalLength.ToString() + " bases of sequence in " + contigSequences.Count.ToString() + " contigs.");
if (contigSequences.Count > 0)
{
N50 = CalculateN50(contigSequences, totalLength);
RaiseMessage("N50: " + N50.ToString());
}
count = this.Graph.GetNodes().Where(x => x.IsInReference).Count();
RaiseMessage("A total of: " + count.ToString() + " nodes remain from the reference");
RaiseMessage("A total of: " + this.Graph.NodeCount + " nodes are in the graph");
return(result);
}