public static void OGlycoTest_Localization()
{
//Get glycanBox
var glycanBox = OGlycanBoxes[19];
//Get unmodified peptide, products, allPossible modPos and all boxes.
Protein protein = new Protein("TTGSLEPSSGASGPQVSSVK", "P16150");
var peptide = protein.Digest(new DigestionParams(), new List <Modification>(), new List <Modification>()).First();
List <Product> products = new List <Product>();
peptide.Fragment(DissociationType.ETD, FragmentationTerminus.Both, products);
int[] modPos = GlycoSpectralMatch.GetPossibleModSites(peptide, new string[] { "S", "T" }).OrderBy(v => v).ToArray();
var boxes = GlycanBox.BuildChildOGlycanBoxes(3, glycanBox.ModIds).ToArray();
Assert.That(boxes.Count() == 6);
//Get Unlocal Fragment
var unlocalCost = GlycoPeptides.GetUnlocalFragment(products, modPos, glycanBox);
Assert.That(unlocalCost.Count == 4); //Basicly, the unlocal are c/z ions that don't localize glycosylation.
//Get scan
CommonParameters commonParameters = new CommonParameters(dissociationType: DissociationType.EThcD, trimMsMsPeaks: false);
string spectraFile = Path.Combine(TestContext.CurrentContext.TestDirectory, @"GlycoTestData\2019_09_16_StcEmix_35trig_EThcD25_rep1_4565.mgf");
var file = new MyFileManager(true).LoadFile(spectraFile, commonParameters);
var scans = MetaMorpheusTask.GetMs2Scans(file, spectraFile, commonParameters).ToArray();
//Known peptideWithMod match.
var peptideWithMod = GlycoPeptides.OGlyGetTheoreticalPeptide(new int[3] {
10, 2, 3
}, peptide, glycanBox);
Assert.That(peptideWithMod.FullSequence == "T[O-Glycosylation:H1N1 on X]T[O-Glycosylation:H1N1 on X]GSLEPSS[O-Glycosylation:N1 on X]GASGPQVSSVK");
List <Product> knownProducts = GlycoPeptides.OGlyGetTheoreticalFragments(DissociationType.EThcD, peptide, peptideWithMod);
var matchedKnownFragmentIons = MetaMorpheusEngine.MatchFragmentIons(scans.First(), knownProducts, commonParameters);
//Graph Localization
LocalizationGraph localizationGraph = new LocalizationGraph(modPos, glycanBox, boxes, -1);
LocalizationGraph.LocalizeOGlycan(localizationGraph, scans.First(), commonParameters.ProductMassTolerance, products);
var allPaths = LocalizationGraph.GetAllHighestScorePaths(localizationGraph.array, localizationGraph.ChildModBoxes);
var knowPath = new int[8] {
2, 4, 4, 4, 5, 5, 5, 5
};
Assert.That(Enumerable.SequenceEqual(knowPath, allPaths[0]));
//Get localized Route
var local = LocalizationGraph.GetLocalizedPath(localizationGraph, allPaths.First());
Assert.That(Enumerable.SequenceEqual(local.Mods.Select(v => v.Item1), new List <int> {
2, 3, 10
}));
Assert.That(Enumerable.SequenceEqual(local.Mods.Select(v => v.Item2), new List <int> {
1, 1, 0
}));
//Get all paths, calculate PScore and calculate position probability.
var p = scans.First().TheScan.MassSpectrum.Size *commonParameters.ProductMassTolerance.GetRange(1000).Width / scans.First().TheScan.MassSpectrum.Range.Width;
var n = knownProducts.Where(v => v.ProductType == ProductType.c || v.ProductType == ProductType.zDot).Count();
var allPathWithWeights = LocalizationGraph.GetAllPaths_CalP(localizationGraph, p, n);
Assert.That(allPathWithWeights.Count == 168);
//Calculate Site Specific Localization Probability
var y = LocalizationGraph.CalSiteSpecificLocalizationProbability(allPathWithWeights, localizationGraph.ModPos);
Assert.That(y.Count == 8);
Assert.That(y.First().Value[1].Item2 > 0.99);
}
public static void OGlycoTest_Localization2()
{
//There may have a bug that MM cannot identify Peptide modified with (HexNAc), This is to test and find the bug.
//Get glycanBox
var glycanBox = OGlycanBoxes[0];
//Get unmodified peptide, products, allPossible modPos and all boxes.
Protein protein = new Protein("AATVGSLAGQPLQER", "P16150");
var peptide = protein.Digest(new DigestionParams(), new List <Modification>(), new List <Modification>()).First();
List <Product> products = new List <Product>();
peptide.Fragment(DissociationType.ETD, FragmentationTerminus.Both, products);
int[] modPos = GlycoSpectralMatch.GetPossibleModSites(peptide, new string[] { "S", "T" }).OrderBy(p => p).ToArray();
var boxes = GlycanBox.BuildChildOGlycanBoxes(glycanBox.NumberOfMods, glycanBox.ModIds).ToArray();
//Load scan.
CommonParameters commonParameters = new CommonParameters(dissociationType: DissociationType.ETD, trimMsMsPeaks: false);
string spectraFile = Path.Combine(TestContext.CurrentContext.TestDirectory, @"GlycoTestData\181217_Fusion_(LC2)_NewObj_Serum_deSA_Jacalin_HRM_4h_ETD_HCD_DDA_mz(400_1200)_21707.mgf");
var file = new MyFileManager(true).LoadFile(spectraFile, commonParameters);
var scans = MetaMorpheusTask.GetMs2Scans(file, spectraFile, commonParameters).ToArray();
//Known peptideWithMod match.
var peptideWithMod = GlycoPeptides.OGlyGetTheoreticalPeptide(new int[1] {
4
}, peptide, glycanBox);
Assert.That(peptideWithMod.FullSequence == "AAT[O-Glycosylation:N1 on X]VGSLAGQPLQER");
//List<Product> knownProducts = peptideWithMod.Fragment(DissociationType.EThcD, FragmentationTerminus.Both).ToList();
List <Product> knownProducts = GlycoPeptides.OGlyGetTheoreticalFragments(DissociationType.ETD, peptide, peptideWithMod);
var matchedKnownFragmentIons = MetaMorpheusEngine.MatchFragmentIons(scans.First(), knownProducts, commonParameters);
//Get hashset int
int obsPreviousFragmentCeilingMz = 0;
List <int> binsToSearch = new List <int>();
foreach (var envelope in scans.First().ExperimentalFragments)
{
// assume charge state 1 to calculate mass tolerance
double experimentalFragmentMass = envelope.MonoisotopicMass;
// get theoretical fragment bins within mass tolerance
int obsFragmentFloorMass = (int)Math.Floor((commonParameters.ProductMassTolerance.GetMinimumValue(experimentalFragmentMass)) * 1000);
int obsFragmentCeilingMass = (int)Math.Ceiling((commonParameters.ProductMassTolerance.GetMaximumValue(experimentalFragmentMass)) * 1000);
// prevents double-counting peaks close in m/z and lower-bound out of range exceptions
if (obsFragmentFloorMass < obsPreviousFragmentCeilingMz)
{
obsFragmentFloorMass = obsPreviousFragmentCeilingMz;
}
obsPreviousFragmentCeilingMz = obsFragmentCeilingMass + 1;
// search mass bins within a tolerance
for (int fragmentBin = obsFragmentFloorMass; fragmentBin <= obsFragmentCeilingMass; fragmentBin++)
{
binsToSearch.Add(fragmentBin);
}
}
HashSet <int> allPeaks = new HashSet <int>(binsToSearch);
//Graph Localization
LocalizationGraph localizationGraph = new LocalizationGraph(modPos, glycanBox, boxes, -1);
LocalizationGraph.LocalizeOGlycan(localizationGraph, scans.First(), commonParameters.ProductMassTolerance, products);
var allPaths = LocalizationGraph.GetAllHighestScorePaths(localizationGraph.array, localizationGraph.ChildModBoxes);
var knowPath = new int[2] {
1, 1
};
Assert.That(Enumerable.SequenceEqual(knowPath, allPaths[0]));
var local = LocalizationGraph.GetLocalizedPath(localizationGraph, allPaths.First());
Assert.That(Enumerable.SequenceEqual(local.Mods.Select(p => p.Item1), new List <int> {
4
}));
Assert.That(Enumerable.SequenceEqual(local.Mods.Select(p => p.Item2), new List <int> {
0
}));
}
protected override MyTaskResults RunSpecific(string OutputFolder, List <DbForTask> dbFilenameList, List <string> currentRawFileList, string taskId, FileSpecificParameters[] fileSettingsList)
{
MyTaskResults = new MyTaskResults(this);
List <List <GlycoSpectralMatch> > ListOfGsmsPerMS2Scan = new List <List <GlycoSpectralMatch> >();
LoadModifications(taskId, out var variableModifications, out var fixedModifications, out var localizeableModificationTypes);
// load proteins
List <Protein> proteinList = LoadProteins(taskId, dbFilenameList, true, _glycoSearchParameters.DecoyType, localizeableModificationTypes, CommonParameters);
MyFileManager myFileManager = new MyFileManager(true);
int completedFiles = 0;
Status("Searching files...", taskId);
ProseCreatedWhileRunning.Append("\n");
ProseCreatedWhileRunning.Append("protease = " + CommonParameters.DigestionParams.Protease + "; \n");
ProseCreatedWhileRunning.Append("maximum missed cleavages = " + CommonParameters.DigestionParams.MaxMissedCleavages + "; \n");
ProseCreatedWhileRunning.Append("minimum peptide length = " + CommonParameters.DigestionParams.MinPeptideLength + "; \n");
ProseCreatedWhileRunning.Append(CommonParameters.DigestionParams.MaxPeptideLength == int.MaxValue ?
"maximum peptide length = unspecified; " :
"maximum peptide length = " + CommonParameters.DigestionParams.MaxPeptideLength + "; \n");
ProseCreatedWhileRunning.Append("initiator methionine behavior = " + CommonParameters.DigestionParams.InitiatorMethionineBehavior + "; \n");
ProseCreatedWhileRunning.Append("max modification isoforms = " + CommonParameters.DigestionParams.MaxModificationIsoforms + "; \n");
ProseCreatedWhileRunning.Append("fixed modifications = " + string.Join(", ", fixedModifications.Select(m => m.IdWithMotif)) + "; \n");
ProseCreatedWhileRunning.Append("variable modifications = " + string.Join(", ", variableModifications.Select(m => m.IdWithMotif)) + "; \n");
ProseCreatedWhileRunning.Append("parent mass tolerance(s) = " + CommonParameters.PrecursorMassTolerance + "; \n");
ProseCreatedWhileRunning.Append("product mass tolerance = " + CommonParameters.ProductMassTolerance + "; \n");
ProseCreatedWhileRunning.Append("The combined search database contained " + proteinList.Count + " total entries including " + proteinList.Where(p => p.IsContaminant).Count() + " contaminant sequences. \n");
if (_glycoSearchParameters.GlycoSearchType == GlycoSearchType.OGlycanSearch)
{
ProseCreatedWhileRunning.Append("The O-glycan database: " + _glycoSearchParameters.OGlycanDatabasefile + "\n");
}
else if (_glycoSearchParameters.GlycoSearchType == GlycoSearchType.NGlycanSearch)
{
ProseCreatedWhileRunning.Append("The N-glycan database: " + _glycoSearchParameters.OGlycanDatabasefile + "\n");
}
else
{
ProseCreatedWhileRunning.Append("The O-glycan database: " + _glycoSearchParameters.OGlycanDatabasefile + "\n");
ProseCreatedWhileRunning.Append("The N-glycan database: " + _glycoSearchParameters.NGlycanDatabasefile + "\n");
}
ProseCreatedWhileRunning.Append("\n");
for (int spectraFileIndex = 0; spectraFileIndex < currentRawFileList.Count; spectraFileIndex++)
{
var origDataFile = currentRawFileList[spectraFileIndex];
CommonParameters combinedParams = SetAllFileSpecificCommonParams(CommonParameters, fileSettingsList[spectraFileIndex]);
var thisId = new List <string> {
taskId, "Individual Spectra Files", origDataFile
};
NewCollection(Path.GetFileName(origDataFile), thisId);
Status("Loading spectra file...", thisId);
MsDataFile myMsDataFile = myFileManager.LoadFile(origDataFile, combinedParams);
Status("Getting ms2 scans...", thisId);
Ms2ScanWithSpecificMass[] arrayOfMs2ScansSortedByMass = GetMs2Scans(myMsDataFile, origDataFile, combinedParams).OrderBy(b => b.PrecursorMass).ToArray();
List <GlycoSpectralMatch>[] newCsmsPerMS2ScanPerFile = new List <GlycoSpectralMatch> [arrayOfMs2ScansSortedByMass.Length];
myFileManager.DoneWithFile(origDataFile);
for (int currentPartition = 0; currentPartition < CommonParameters.TotalPartitions; currentPartition++)
{
List <PeptideWithSetModifications> peptideIndex = null;
//When partition, the proteinList will be split for each Thread.
List <Protein> proteinListSubset = proteinList.GetRange(currentPartition * proteinList.Count() / combinedParams.TotalPartitions, ((currentPartition + 1) * proteinList.Count() / combinedParams.TotalPartitions) - (currentPartition * proteinList.Count() / combinedParams.TotalPartitions));
Status("Getting fragment dictionary...", new List <string> {
taskId
});
//Only reverse Decoy for glyco search has been tested and are set as fixed parameter.
var indexEngine = new IndexingEngine(proteinListSubset, variableModifications, fixedModifications, null, null, null, currentPartition, _glycoSearchParameters.DecoyType, combinedParams, this.FileSpecificParameters, 30000.0, false, dbFilenameList.Select(p => new FileInfo(p.FilePath)).ToList(), TargetContaminantAmbiguity.RemoveContaminant, new List <string> {
taskId
});
List <int>[] fragmentIndex = null;
List <int>[] precursorIndex = null;
GenerateIndexes(indexEngine, dbFilenameList, ref peptideIndex, ref fragmentIndex, ref precursorIndex, proteinList, taskId);
//The second Fragment index is for 'MS1-HCD_MS1-ETD_MS2s' type of data. If LowCID is used for MS1, ion-index is not allowed to use.
List <int>[] secondFragmentIndex = null;
//if (combinedParams.MS2ChildScanDissociationType != DissociationType.LowCID
//&& !CrosslinkSearchEngine.DissociationTypeGenerateSameTypeOfIons(combinedParams.DissociationType, combinedParams.MS2ChildScanDissociationType))
//{
// //Becuase two different type of dissociation methods are used, the parameters are changed with different dissociation type.
// var secondCombinedParams = CommonParameters.CloneWithNewDissociationType(combinedParams.MS2ChildScanDissociationType);
// var secondIndexEngine = new IndexingEngine(proteinListSubset, variableModifications, fixedModifications, null, null, null, currentPartition, _glycoSearchParameters.DecoyType, secondCombinedParams, this.FileSpecificParameters, 30000.0, false, dbFilenameList.Select(p => new FileInfo(p.FilePath)).ToList(), new List<string> { taskId });
// GenerateSecondIndexes(indexEngine, secondIndexEngine, dbFilenameList, ref secondFragmentIndex, proteinList, taskId);
//}
Status("Searching files...", taskId);
new GlycoSearchEngine(newCsmsPerMS2ScanPerFile, arrayOfMs2ScansSortedByMass, peptideIndex, fragmentIndex, secondFragmentIndex, currentPartition, combinedParams, this.FileSpecificParameters,
_glycoSearchParameters.OGlycanDatabasefile, _glycoSearchParameters.NGlycanDatabasefile, _glycoSearchParameters.GlycoSearchType, _glycoSearchParameters.GlycoSearchTopNum, _glycoSearchParameters.MaximumOGlycanAllowed, _glycoSearchParameters.OxoniumIonFilt, thisId).Run();
ReportProgress(new ProgressEventArgs(100, "Done with search " + (currentPartition + 1) + "/" + CommonParameters.TotalPartitions + "!", thisId));
if (GlobalVariables.StopLoops)
{
break;
}
}
ListOfGsmsPerMS2Scan.AddRange(newCsmsPerMS2ScanPerFile.Where(p => p != null).ToList());
completedFiles++;
ReportProgress(new ProgressEventArgs(completedFiles / currentRawFileList.Count, "Searching...", new List <string> {
taskId, "Individual Spectra Files"
}));
}
ReportProgress(new ProgressEventArgs(100, "Done with all searches!", new List <string> {
taskId, "Individual Spectra Files"
}));
//For every Ms2Scans, each have a list of candidates psms. The allPsms from GlycoSearchEngine is the list (all ms2scans) of list (each ms2scan) of psm (all candidate psm).
//Currently, only keep the first scan for consideration.
List <GlycoSpectralMatch> GsmPerScans = ListOfGsmsPerMS2Scan.Select(p => p.First()).ToList();
var filteredAllPsms = new List <GlycoSpectralMatch>();
//For each ms2scan, try to find the best candidate psm from the psms list. Do the localizaiton analysis. Add it into filteredAllPsms.
foreach (var gsmsPerScan in GsmPerScans.GroupBy(p => p.ScanNumber))
{
var glycos = RemoveSimilarSequenceDuplicates(gsmsPerScan.OrderByDescending(p => p.Score).ToList());
foreach (var glycoSpectralMatch in glycos)
{
if (glycoSpectralMatch.LocalizationGraphs != null)
{
List <Route> localizationCandidates = new List <Route>();
for (int i = 0; i < glycoSpectralMatch.LocalizationGraphs.Count; i++)
{
var allPathWithMaxScore = LocalizationGraph.GetAllHighestScorePaths(glycoSpectralMatch.LocalizationGraphs[i].array, glycoSpectralMatch.LocalizationGraphs[i].ChildModBoxes);
foreach (var path in allPathWithMaxScore)
{
var local = LocalizationGraph.GetLocalizedPath(glycoSpectralMatch.LocalizationGraphs[i], path);
local.ModBoxId = glycoSpectralMatch.LocalizationGraphs[i].ModBoxId;
localizationCandidates.Add(local);
}
}
glycoSpectralMatch.Routes = localizationCandidates;
}
if (glycoSpectralMatch.Routes != null)
{
LocalizationLevel localLevel;
glycoSpectralMatch.LocalizedGlycan = GlycoSpectralMatch.GetLocalizedGlycan(glycoSpectralMatch.Routes, out localLevel);
glycoSpectralMatch.LocalizationLevel = localLevel;
//Localization PValue.
if (localLevel == LocalizationLevel.Level1 || localLevel == LocalizationLevel.Level2)
{
List <Route> allRoutes = new List <Route>();
foreach (var graph in glycoSpectralMatch.LocalizationGraphs)
{
allRoutes.AddRange(LocalizationGraph.GetAllPaths_CalP(graph, glycoSpectralMatch.ScanInfo_p, glycoSpectralMatch.Thero_n));
}
glycoSpectralMatch.SiteSpeciLocalProb = LocalizationGraph.CalSiteSpecificLocalizationProbability(allRoutes, glycoSpectralMatch.LocalizationGraphs.First().ModPos);
}
}
filteredAllPsms.Add(glycoSpectralMatch);
}
}
PostGlycoSearchAnalysisTask postGlycoSearchAnalysisTask = new PostGlycoSearchAnalysisTask();
postGlycoSearchAnalysisTask.FileSpecificParameters = this.FileSpecificParameters;
return(postGlycoSearchAnalysisTask.Run(OutputFolder, dbFilenameList, currentRawFileList, taskId, fileSettingsList, filteredAllPsms.OrderByDescending(p => p.Score).ToList(), CommonParameters, _glycoSearchParameters, proteinList, variableModifications, fixedModifications, localizeableModificationTypes, MyTaskResults));
}
