Decoy on the fly And Searching Match Ions with All Charges for decoy library generation

CMD/CMD.csproj

@@ -23,7 +23,7 @@
     <PackageReference Include="Microsoft.ML" Version="1.3.1" />
     <PackageReference Include="Microsoft.ML.FastTree" Version="1.3.1" />
     <PackageReference Include="Microsoft.NETCore.App" Version="2.2.8" />
-    <PackageReference Include="mzLib" Version="1.0.494" />
+    <PackageReference Include="mzLib" Version="1.0.496" />
     <PackageReference Include="Nett" Version="0.13.0" />
   </ItemGroup>
 

Dockerfile

@@ -11,4 +11,4 @@ RUN apk add --no-cache bash
 RUN apk add --no-cache procps
 
 ## Set the entrypoint of the Docker image to CMD.dll
-ENTRYPOINT ["dotnet", "/metamorpheus/CMD.dll"]
+ENTRYPOINT ["dotnet", "/metamorpheus/CMD.dll"]

Dockerfile_WindowsInstaller

@@ -5,4 +5,4 @@ FROM mcr.microsoft.com/dotnet/runtime:5.0-nanoserver-1809
 ADD /InstalledFiles/ /metamorpheus/
 
 ## Set the entrypoint of the Docker image to CMD.exe
-ENTRYPOINT ["/metamorpheus/CMD.exe"]
+ENTRYPOINT ["/metamorpheus/CMD.exe"]

EngineLayer/ClassicSearch/ClassicSearchEngine.cs

@@ -22,11 +22,12 @@ public class ClassicSearchEngine : MetaMorpheusEngine
         private readonly PeptideSpectralMatch[] PeptideSpectralMatches;
         private readonly Ms2ScanWithSpecificMass[] ArrayOfSortedMS2Scans;
         private readonly double[] MyScanPrecursorMasses;
+        private readonly bool WriteSpectralLibrary;
 
         public ClassicSearchEngine(PeptideSpectralMatch[] globalPsms, Ms2ScanWithSpecificMass[] arrayOfSortedMS2Scans,
             List<Modification> variableModifications, List<Modification> fixedModifications, List<SilacLabel> silacLabels, SilacLabel startLabel, SilacLabel endLabel,
             List<Protein> proteinList, MassDiffAcceptor searchMode, CommonParameters commonParameters, List<(string FileName, CommonParameters Parameters)> fileSpecificParameters,
-            SpectralLibrary spectralLibrary, List<string> nestedIds)
+            SpectralLibrary spectralLibrary, List<string> nestedIds, bool writeSpectralLibrary)
             : base(commonParameters, fileSpecificParameters, nestedIds)
         {
             PeptideSpectralMatches = globalPsms;
@@ -39,9 +40,16 @@ public ClassicSearchEngine(PeptideSpectralMatch[] globalPsms, Ms2ScanWithSpecifi
             {
                 TurnoverLabels = (startLabel, endLabel);
             }
-            Proteins = proteinList;
+
             SearchMode = searchMode;
             SpectralLibrary = spectralLibrary;
+            WriteSpectralLibrary = writeSpectralLibrary;
+
+            // if we're doing a spectral library search, we can skip the reverse protein decoys generated by metamorpheus.
+            // we will generate reverse peptide decoys w/ in the code just below this (and calculate spectral angles for them later).
+            // we have to generate the reverse peptides instead of the usual reverse proteins because we generate decoy spectral
+            // library spectra from their corresponding paired target peptides
+            Proteins = proteinList.Where(p => !p.IsDecoy || spectralLibrary == null).ToList();
         }
 
         protected override MetaMorpheusEngineResults RunSpecific()
@@ -66,19 +74,22 @@ protected override MetaMorpheusEngineResults RunSpecific()
                 int[] threads = Enumerable.Range(0, maxThreadsPerFile).ToArray();
                 Parallel.ForEach(threads, (i) =>
                 {
-                    var fragmentsForDissociationTypes = new Dictionary<DissociationType, List<Product>>();
+                    var targetFragmentsForEachDissociationType = new Dictionary<DissociationType, List<Product>>();
+                    var decoyFragmentsForEachDissociationType = new Dictionary<DissociationType, List<Product>>();
 
                     // check if we're supposed to autodetect dissociation type from the scan header or not
                     if (CommonParameters.DissociationType == DissociationType.Autodetect)
                     {
                         foreach (var item in GlobalVariables.AllSupportedDissociationTypes.Where(p => p.Value != DissociationType.Autodetect))
                         {
-                            fragmentsForDissociationTypes.Add(item.Value, new List<Product>());
+                            targetFragmentsForEachDissociationType.Add(item.Value, new List<Product>());
+                            decoyFragmentsForEachDissociationType.Add(item.Value, new List<Product>());
                         }
                     }
                     else
                     {
-                        fragmentsForDissociationTypes.Add(CommonParameters.DissociationType, new List<Product>());
+                        targetFragmentsForEachDissociationType.Add(CommonParameters.DissociationType, new List<Product>());
+                        decoyFragmentsForEachDissociationType.Add(CommonParameters.DissociationType, new List<Product>());
                     }
 
                     for (; i < Proteins.Count; i += maxThreadsPerFile)
@@ -89,22 +100,28 @@ protected override MetaMorpheusEngineResults RunSpecific()
                         // digest each protein into peptides and search for each peptide in all spectra within precursor mass tolerance
                         foreach (PeptideWithSetModifications peptide in Proteins[i].Digest(CommonParameters.DigestionParams, FixedModifications, VariableModifications, SilacLabels, TurnoverLabels))
                         {
-                            foreach (var fragmentSet in fragmentsForDissociationTypes)
+                            PeptideWithSetModifications reversedOnTheFlyDecoy = null;
+
+                            if (SpectralLibrary != null)
+                            {
+                                int[] newAAlocations = new int[peptide.BaseSequence.Length];
+                                reversedOnTheFlyDecoy = peptide.GetReverseDecoyFromTarget(newAAlocations);
+                            }
+
+                            // clear fragments from the last peptide
+                            foreach (var fragmentSet in targetFragmentsForEachDissociationType)
                             {
                                 fragmentSet.Value.Clear();
+                                decoyFragmentsForEachDissociationType[fragmentSet.Key].Clear();
                             }
 
+                            // score each scan that has an acceptable precursor mass
                             foreach (ScanWithIndexAndNotchInfo scan in GetAcceptableScans(peptide.MonoisotopicMass, SearchMode))
                             {
-                                if (SpectralLibrary != null && !SpectralLibrary.ContainsSpectrum(peptide.FullSequence, scan.TheScan.PrecursorCharge))
-                                {
-                                    continue;
-                                }
-
                                 var dissociationType = CommonParameters.DissociationType == DissociationType.Autodetect ?
                                     scan.TheScan.TheScan.DissociationType.Value : CommonParameters.DissociationType;
 
-                                if (!fragmentsForDissociationTypes.TryGetValue(dissociationType, out var peptideTheorProducts))
+                                if (!targetFragmentsForEachDissociationType.TryGetValue(dissociationType, out var peptideTheorProducts))
                                 {
                                     //TODO: print some kind of warning here. the scan header dissociation type was unknown
                                     continue;
@@ -116,32 +133,32 @@ protected override MetaMorpheusEngineResults RunSpecific()
                                     peptide.Fragment(dissociationType, CommonParameters.DigestionParams.FragmentationTerminus, peptideTheorProducts);
                                 }
 
-                                List<MatchedFragmentIon> matchedIons = MatchFragmentIons(scan.TheScan, peptideTheorProducts, CommonParameters);
+                                // match theoretical target ions to spectrum
+                                List<MatchedFragmentIon> matchedIons = MatchFragmentIons(scan.TheScan, peptideTheorProducts, CommonParameters,
+                                        matchAllCharges: WriteSpectralLibrary);
 
-                                double thisScore = CalculatePeptideScore(scan.TheScan.TheScan, matchedIons);
-                                bool meetsScoreCutoff = thisScore >= CommonParameters.ScoreCutoff;
+                                // calculate the peptide's score
+                                double thisScore = CalculatePeptideScore(scan.TheScan.TheScan, matchedIons, fragmentsCanHaveDifferentCharges: WriteSpectralLibrary);
 
-                                // this is thread-safe because even if the score improves from another thread writing to this PSM,
-                                // the lock combined with AddOrReplace method will ensure thread safety
-                                if (meetsScoreCutoff)
+                                AddPeptideCandidateToPsm(scan, myLocks, thisScore, peptide, matchedIons);
+
+                                if (SpectralLibrary != null)
                                 {
-                                    // valid hit (met the cutoff score); lock the scan to prevent other threads from accessing it
-                                    lock (myLocks[scan.ScanIndex])
+                                    // match decoy ions for decoy-on-the-fly
+                                    var decoyTheoreticalFragments = decoyFragmentsForEachDissociationType[dissociationType];
+
+                                    if (decoyTheoreticalFragments.Count == 0)
                                     {
-                                        bool scoreImprovement = PeptideSpectralMatches[scan.ScanIndex] == null || (thisScore - PeptideSpectralMatches[scan.ScanIndex].RunnerUpScore) > -PeptideSpectralMatch.ToleranceForScoreDifferentiation;
-
-                                        if (scoreImprovement)
-                                        {
-                                            if (PeptideSpectralMatches[scan.ScanIndex] == null)
-                                            {
-                                                PeptideSpectralMatches[scan.ScanIndex] = new PeptideSpectralMatch(peptide, scan.Notch, thisScore, scan.ScanIndex, scan.TheScan, CommonParameters, matchedIons, 0);
-                                            }
-                                            else
-                                            {
-                                                PeptideSpectralMatches[scan.ScanIndex].AddOrReplace(peptide, thisScore, scan.Notch, CommonParameters.ReportAllAmbiguity, matchedIons, 0);
-                                            }
-                                        }
+                                        reversedOnTheFlyDecoy.Fragment(dissociationType, CommonParameters.DigestionParams.FragmentationTerminus, decoyTheoreticalFragments);
                                     }
+
+                                    var decoyMatchedIons = MatchFragmentIons(scan.TheScan, decoyTheoreticalFragments, CommonParameters,
+                                        matchAllCharges: WriteSpectralLibrary);
+
+                                    // calculate decoy's score
+                                    var decoyScore = CalculatePeptideScore(scan.TheScan.TheScan, decoyMatchedIons, fragmentsCanHaveDifferentCharges: WriteSpectralLibrary);
+
+                                    AddPeptideCandidateToPsm(scan, myLocks, decoyScore, reversedOnTheFlyDecoy, decoyMatchedIons);
                                 }
                             }
                         }
@@ -167,6 +184,34 @@ protected override MetaMorpheusEngineResults RunSpecific()
             return new MetaMorpheusEngineResults(this);
         }
 
+        private void AddPeptideCandidateToPsm(ScanWithIndexAndNotchInfo scan, object[] myLocks, double thisScore, PeptideWithSetModifications peptide, List<MatchedFragmentIon> matchedIons)
+        {
+            bool meetsScoreCutoff = thisScore >= CommonParameters.ScoreCutoff;
+
+            // this is thread-safe because even if the score improves from another thread writing to this PSM,
+            // the lock combined with AddOrReplace method will ensure thread safety
+            if (meetsScoreCutoff)
+            {
+                // valid hit (met the cutoff score); lock the scan to prevent other threads from accessing it
+                lock (myLocks[scan.ScanIndex])
+                {
+                    bool scoreImprovement = PeptideSpectralMatches[scan.ScanIndex] == null || (thisScore - PeptideSpectralMatches[scan.ScanIndex].RunnerUpScore) > -PeptideSpectralMatch.ToleranceForScoreDifferentiation;
+
+                    if (scoreImprovement)
+                    {
+                        if (PeptideSpectralMatches[scan.ScanIndex] == null)
+                        {
+                            PeptideSpectralMatches[scan.ScanIndex] = new PeptideSpectralMatch(peptide, scan.Notch, thisScore, scan.ScanIndex, scan.TheScan, CommonParameters, matchedIons, 0);
+                        }
+                        else
+                        {
+                            PeptideSpectralMatches[scan.ScanIndex].AddOrReplace(peptide, thisScore, scan.Notch, CommonParameters.ReportAllAmbiguity, matchedIons, 0);
+                        }
+                    }
+                }
+            }
+        }
+
         private IEnumerable<ScanWithIndexAndNotchInfo> GetAcceptableScans(double peptideMonoisotopicMass, MassDiffAcceptor searchMode)
         {
             foreach (AllowedIntervalWithNotch allowedIntervalWithNotch in searchMode.GetAllowedPrecursorMassIntervalsFromTheoreticalMass(peptideMonoisotopicMass).ToList())

EngineLayer/EngineLayer.csproj

@@ -20,7 +20,7 @@
     <PackageReference Include="Microsoft.ML" Version="1.3.1" />
     <PackageReference Include="Microsoft.ML.FastTree" Version="1.3.1" />
     <PackageReference Include="Microsoft.NETCore.App" Version="2.2.8" />
-    <PackageReference Include="mzLib" Version="1.0.494" />
+    <PackageReference Include="mzLib" Version="1.0.496" />
     <PackageReference Include="NETStandard.Library" Version="2.0.3" />
     <PackageReference Include="Nett" Version="0.13.0" />
     <PackageReference Include="Newtonsoft.Json" Version="12.0.3" />