Only quantify peptides that clear the q-value threshold

MetaMorpheus/EngineLayer/FdrAnalysis/FdrAnalysisEngine.cs

@@ -39,6 +39,10 @@ private void AddPsmAndPeptideFdrInfoIfNotPresent()
             foreach (var psm in AllPsms.Where(p => p.PeptideFdrInfo == null))
             {
                 psm.PeptideFdrInfo = new FdrInfo();
+                // If peptide level Q-values default to zero, it is hard to determine which PSMs have a legitimate peptide-level Q-value of zero and 
+                // which just weren't included in peptide-level calculations. Hence, we set q values to one here. 
+                psm.PeptideFdrInfo.QValue = 1;
+                psm.PeptideFdrInfo.PEP_QValue = 1;
             }
         }
 

MetaMorpheus/GUI/TaskWindows/SearchTaskWindow.xaml

@@ -544,6 +544,24 @@
                                     <ToolTip Content="Use peptide sequences shared between proteins for protein quantification" ToolTipService.ShowDuration="999999" ToolTipService.InitialShowDelay="500" />
                                 </ToolTipService.ToolTip>
                             </CheckBox>
+                            <CheckBox x:Name="CheckBoxOnlyQuantifyConfidentPeptides" Content="Only quantify confident peptides" ToolTipService.ShowDuration="999999" ToolTipService.InitialShowDelay="500">
+                                <CheckBox.IsEnabled>
+                                    <MultiBinding Converter="{StaticResource boolOrConverter}">
+                                        <Binding  ElementName="CheckBoxLFQ" Path ="IsChecked"/>
+                                        <Binding  ElementName="CheckBoxLFQwSpectralRecovery" Path ="IsChecked"/>
+                                    </MultiBinding>
+                                </CheckBox.IsEnabled>
+                                <CheckBox.Visibility>
+                                    <MultiBinding Converter="{StaticResource boolOr2VisConverter}">
+                                        <Binding  ElementName="CheckBoxLFQ" Path ="IsChecked"/>
+                                        <Binding  ElementName="CheckBoxLFQwSpectralRecovery" Path ="IsChecked"/>
+                                    </MultiBinding>
+                                </CheckBox.Visibility>
+                                <ToolTipService.ToolTip>
+                                    <ToolTip Content="Only quantify peptides that meet the Q-Value or PEP Q-Value threshold.&#x0a;Unchecking this option results in low-confidence peptides being reported in the AllQuantifiedPeptides output" ToolTipService.ShowDuration="999999" ToolTipService.InitialShowDelay="500" />
+                                </ToolTipService.ToolTip>
+                            </CheckBox>
+
 
                             <!-- Multiplex Options -->
                             <StackPanel Orientation="Horizontal" Margin="5 5 5 0" Grid.Column="0" Grid.Row="0">

MetaMorpheus/GUI/TaskWindows/SearchTaskWindow.xaml.cs

@@ -190,6 +190,7 @@ private void UpdateFieldsFromTask(SearchTask task)
             CheckBoxNoQuant.IsChecked = !task.SearchParameters.DoLabelFreeQuantification;
             CheckBoxLFQ.IsChecked = task.SearchParameters.DoLabelFreeQuantification;
             CheckBoxUseSharedPeptidesForQuant.IsChecked = task.SearchParameters.UseSharedPeptidesForLFQ;
+            CheckBoxOnlyQuantifyConfidentPeptides.IsChecked = task.SearchParameters.OnlyQuantifyConfidentPeptides;
             CheckBoxMultiplex.IsChecked = task.SearchParameters.DoMultiplexQuantification;
             MultiplexComboBox.SelectedItem = task.SearchParameters.MultiplexModId ?? _defaultMultiplexType;
             // If Spectral Recovery is enabled
@@ -628,6 +629,7 @@ private void SaveButton_Click(object sender, RoutedEventArgs e)
             TheTask.SearchParameters.NoOneHitWonders = CheckBoxNoOneHitWonders.IsChecked.Value;
             TheTask.SearchParameters.DoLabelFreeQuantification = !CheckBoxNoQuant.IsChecked.Value;
             TheTask.SearchParameters.UseSharedPeptidesForLFQ = CheckBoxUseSharedPeptidesForQuant.IsChecked.Value;
+            TheTask.SearchParameters.OnlyQuantifyConfidentPeptides = CheckBoxOnlyQuantifyConfidentPeptides.IsChecked.Value;
             TheTask.SearchParameters.DoSpectralRecovery = CheckBoxLFQwSpectralRecovery.IsChecked.Value;
             TheTask.SearchParameters.DoMultiplexQuantification = CheckBoxMultiplex.IsChecked.Value;
             TheTask.SearchParameters.MultiplexModId = (string)MultiplexComboBox.SelectedItem;

MetaMorpheus/TaskLayer/FilteredPsms.cs

@@ -95,7 +95,7 @@ public static FilteredPsms Filter(IEnumerable<SpectralMatch> psms,
             pepQValueThreshold ??= commonParams.PepQValueThreshold;
             double filterThreshold = Math.Min((double)qValueThreshold, (double)pepQValueThreshold);
             bool filteringNotPerformed = false;
-            List<SpectralMatch> filteredPsms = new List<SpectralMatch>();
+            List<SpectralMatch> filteredPsms = new();
 
             // set the filter type
             FilterType filterType = FilterType.QValue;

MetaMorpheus/TaskLayer/SearchTask/PostSearchAnalysisTask.cs

@@ -501,16 +501,32 @@ private void QuantificationAnalysis()
                 }
             }
 
+            // Determine if only select, high confidence peptides will be used for quant
+            List<string> peptideSequencesForQuantification = null;
+            if (Parameters.SearchParameters.SilacLabels == null && Parameters.SearchParameters.OnlyQuantifyConfidentPeptides)
+            {
+                peptideSequencesForQuantification = FilteredPsms.Filter(Parameters.AllPsms,
+                                CommonParameters,
+                                includeDecoys: false,
+                                includeContaminants: true,
+                                includeAmbiguous: false,
+                                includeAmbiguousMods: false,
+                                includeHighQValuePsms: false,
+                                filterAtPeptideLevel: true)
+                    .Select(pep => pep.FullSequence).Distinct().ToList();
+            }
+
             // run FlashLFQ
-            var flashLfqEngine = new FlashLfqEngine(
+                var flashLfqEngine = new FlashLfqEngine(
                 allIdentifications: flashLFQIdentifications,
                 normalize: Parameters.SearchParameters.Normalize,
                 ppmTolerance: Parameters.SearchParameters.QuantifyPpmTol,
                 matchBetweenRunsPpmTolerance: Parameters.SearchParameters.QuantifyPpmTol,  // If these tolerances are not equivalent, then MBR will falsely classify peptides found in the initial search as MBR peaks
                 matchBetweenRuns: Parameters.SearchParameters.MatchBetweenRuns,
                 useSharedPeptidesForProteinQuant: Parameters.SearchParameters.UseSharedPeptidesForLFQ,
                 silent: true,
-                maxThreads: CommonParameters.MaxThreadsToUsePerFile);
+                maxThreads: CommonParameters.MaxThreadsToUsePerFile,
+                peptideSequencesToUse: peptideSequencesForQuantification);
 
             if (flashLFQIdentifications.Any())
             {

MetaMorpheus/TaskLayer/SearchTask/SearchParameters.cs

@@ -17,6 +17,7 @@ public SearchParameters()
             ModPeptidesAreDifferent = false;
             DoLabelFreeQuantification = true;
             UseSharedPeptidesForLFQ = false;
+            OnlyQuantifyConfidentPeptides = true;
             DoSpectralRecovery = false;
             QuantifyPpmTol = 5;
             SearchTarget = true;
@@ -69,6 +70,7 @@ public SearchParameters()
         public bool MatchBetweenRuns { get; set; }
         public bool Normalize { get; set; }
         public double QuantifyPpmTol { get; set; }
+        public bool OnlyQuantifyConfidentPeptides { get; set; }
         public bool DoHistogramAnalysis { get; set; }
         public bool SearchTarget { get; set; }
         public DecoyType DecoyType { get; set; }

MetaMorpheus/Test/EverythingRunnerEngineTestCase.cs

@@ -126,6 +126,7 @@ static EverythingRunnerEngineTestCase()
                 @"TestData\TaGe_SA_A549_3_snip_2.mzML");
             string myDatabase = Path.Combine(TestContext.CurrentContext.TestDirectory,
                 @"TestData\TaGe_SA_A549_3_snip.fasta");
+            searchTaskLoaded.SearchParameters.OnlyQuantifyConfidentPeptides = true;
             _cases.Add(EverythingRunnerEngineTestCases.BottomUpQValue,
                 new EverythingRunnerEngineTestCase(EverythingRunnerEngineTestCases.BottomUpQValue,
                     new List<(string, MetaMorpheusTask)> { ("postSearchAnalysisTaskTestOutput", searchTaskLoaded) },

MetaMorpheus/Test/FdrTest.cs

@@ -96,19 +96,19 @@ public static void FdrTestMethod()
             fdr.Run();
 
             Assert.AreEqual(2, searchModes.NumNotches);
-            Assert.AreEqual(0, newPsms[0].FdrInfo.CumulativeDecoyNotch);
-            Assert.AreEqual(1, newPsms[0].FdrInfo.CumulativeTargetNotch);
-            Assert.AreEqual(0, newPsms[1].FdrInfo.CumulativeDecoyNotch);
-            Assert.AreEqual(1, newPsms[1].FdrInfo.CumulativeTargetNotch);
-            Assert.AreEqual(0, newPsms[2].FdrInfo.CumulativeDecoyNotch);
-            Assert.AreEqual(1, newPsms[2].FdrInfo.CumulativeTargetNotch);
-
-            Assert.AreEqual(0, newPsms[0].FdrInfo.CumulativeDecoy);
-            Assert.AreEqual(1, newPsms[0].FdrInfo.CumulativeTarget);
-            Assert.AreEqual(0, newPsms[1].FdrInfo.CumulativeDecoy);
-            Assert.AreEqual(2, newPsms[1].FdrInfo.CumulativeTarget);
-            Assert.AreEqual(0, newPsms[2].FdrInfo.CumulativeDecoy);
-            Assert.AreEqual(3, newPsms[2].FdrInfo.CumulativeTarget);
+            Assert.AreEqual(0, newPsms[0].PsmFdrInfo.CumulativeDecoyNotch);
+            Assert.AreEqual(1, newPsms[0].PsmFdrInfo.CumulativeTargetNotch);
+            Assert.AreEqual(0, newPsms[1].PsmFdrInfo.CumulativeDecoyNotch);
+            Assert.AreEqual(1, newPsms[1].PsmFdrInfo.CumulativeTargetNotch);
+            Assert.AreEqual(0, newPsms[2].PsmFdrInfo.CumulativeDecoyNotch);
+            Assert.AreEqual(1, newPsms[2].PsmFdrInfo.CumulativeTargetNotch);
+
+            Assert.AreEqual(0, newPsms[0].PsmFdrInfo.CumulativeDecoy);
+            Assert.AreEqual(1, newPsms[0].PsmFdrInfo.CumulativeTarget);
+            Assert.AreEqual(0, newPsms[1].PsmFdrInfo.CumulativeDecoy);
+            Assert.AreEqual(2, newPsms[1].PsmFdrInfo.CumulativeTarget);
+            Assert.AreEqual(0, newPsms[2].PsmFdrInfo.CumulativeDecoy);
+            Assert.AreEqual(3, newPsms[2].PsmFdrInfo.CumulativeTarget);
         }
 
         [Test]
@@ -266,7 +266,7 @@ public static void TestComputePEPValue()
 
             foreach (var item in allPsmsArray.Where(p => p != null))
             {
-                var b = item.FdrInfo.PEP;
+                var b = item.PsmFdrInfo.PEP;
                 if (b >= 0.5)
                 {
                     trueCount++;
@@ -349,7 +349,7 @@ public static void TestComputePEPValue()
 
             foreach (var item in psmCopyForCZETest.Where(p => p != null))
             {
-                var b = item.FdrInfo.PEP;
+                var b = item.PsmFdrInfo.PEP;
                 if (b >= 0.5)
                 {
                     trueCount++;

MetaMorpheus/Test/MultiProteaseParsimonyTest.cs

@@ -710,7 +710,7 @@ public static void TestPSMFdrFiltering_Simulated()
             var fdrFilteredPsms = new List<SpectralMatch>();
             foreach (SpectralMatch psm in psms)
             {
-                if (psm != null && psm.FdrInfo.QValue <= 0.0100 && psm.FdrInfo.QValueNotch <= 0.0100)
+                if (psm != null && psm.PsmFdrInfo.QValue <= 0.0100 && psm.PsmFdrInfo.QValueNotch <= 0.0100)
                 {
                     fdrFilteredPsms.Add(psm);
                 }

MetaMorpheus/Test/PostSearchAnalysisTaskTests.cs

@@ -101,6 +101,16 @@ public static void AllResultsAndResultsTxtContainsCorrectValues_PepQValue_Bottom
             Assert.AreEqual("TaGe_SA_A549_3_snip_2 - Target protein groups within 1 % FDR: 140", results[15]);
         }
 
+        [Test]
+        public static void TestPeptideQuantificationResults()
+        {
+            EverythingRunnerEngineTestCase.TryGetTestCase(EverythingRunnerEngineTestCases.BottomUpQValue, out var testCase);
+
+            string peptideQuantFile = Path.Combine(testCase.OutputDirectory, @"postSearchAnalysisTaskTestOutput\AllQuantifiedPeptides.tsv");
+            string[] peptideQuant = File.ReadAllLines(peptideQuantFile);
+            Assert.AreEqual(175, peptideQuant.Length); // 174 peptides + header. Make sure that we're quantifying only those peptides with q-value <= 0.01
+        }
+
         /// <summary>
         /// Ensures that there is the proper ratio of summary and individual lines in the result.txt file and that peptides and proteoforms are distinct
         /// </summary>

MetaMorpheus/Test/SearchEngineTests.cs

@@ -553,13 +553,13 @@ public static void TestModernSearchEngineLowResOneRealSpectrum()
             // Single search mode
             Assert.AreEqual(12, allPsmsArray.Length);
 
-            var goodPsm = nonNullPsms.Where(p => p.FdrInfo.QValue <= 0.01).ToList();
+            var goodPsm = nonNullPsms.Where(p => p.PsmFdrInfo.QValue <= 0.01).ToList();
 
             var myMatchedIons = goodPsm[0].MatchedFragmentIons;
 
             Assert.AreEqual(47, myMatchedIons.Count());
 
-            var goodScore = nonNullPsms.Where(p => p.FdrInfo.QValue <= 0.01).Select(s => s.Score).ToList();
+            var goodScore = nonNullPsms.Where(p => p.PsmFdrInfo.QValue <= 0.01).Select(s => s.Score).ToList();
             goodScore.Sort();
             Assert.AreEqual(2, goodPsm.Count());
         }
@@ -656,7 +656,7 @@ public static void TestClassicSearchEngineLowResSimple()
             // Single search mode
             Assert.AreEqual(535, allPsmsArray.Length);
 
-            var goodScore = nonNullPsms.Where(p => p.FdrInfo.QValue <= 0.01).Select(s => s.Score).ToList();
+            var goodScore = nonNullPsms.Where(p => p.PsmFdrInfo.QValue <= 0.01).Select(s => s.Score).ToList();
 
             Assert.AreEqual(181, goodScore.Count());
             Directory.Delete(outputFolder, true);
@@ -748,7 +748,7 @@ public static void TestModernSearchEngineLowResSimple()
             // Single search mode
             Assert.AreEqual(535, allPsmsArray.Length);
 
-            var goodScore = nonNullPsms.Where(p => p.FdrInfo.QValue <= 0.01).Select(s => s.Score).ToList();
+            var goodScore = nonNullPsms.Where(p => p.PsmFdrInfo.QValue <= 0.01).Select(s => s.Score).ToList();
 
             Assert.AreEqual(181, goodScore.Count());
         }

MetaMorpheus/Test/SpectralRecoveryTest.cs

@@ -167,10 +167,8 @@ public static void SpectralRecoveryPostSearchAnalysisTest()
             List<PsmFromTsv> matches02ng = mbrPsms.Where(p => p.FileNameWithoutExtension == "K13_02ng_1min_frac1").ToList();
             List<string> expectedMatches = mbrPsms.Select(p => p.BaseSeq).Intersect(expectedMbrPsms.Select(p => p.BaseSeq).ToList()).ToList();
 
-            // Changing Q-value calculation methods results in more PSMs being discovered, and so fewer spectra are available to be "recovered"
-            // (as they were identified in the orignal search)
-            Assert.That(matches2ng.Count >= 3);
-            Assert.That(matches02ng.Count >= 10);
+            Assert.That(matches2ng.Count >= 2);
+            Assert.That(matches02ng.Count >= 7); // AS: 6/17/24 - Fewer quantification events (8 -> 7) because one of the peptides doesn't clear peptide level FDR threshold
             Assert.That(expectedMatches.Count >= 2); // FlashLFQ doesn't find all 6 expected peaks, only 3. MbrAnalysis finds these three peaks
 
             //TODO: Add test for recovering fdrInfo from original. Currently, PsmTsvReader doesn't support the new columns, so it's hard to test

MetaMorpheus/Test/Test.csproj

@@ -322,6 +322,12 @@
     <None Update="TestData\oneEmptyEntry.fa">
       <CopyToOutputDirectory>Always</CopyToOutputDirectory>
     </None>
+    <None Update="TestData\PostSearchAnalysisTaskTestData\TaGe_SA_A549_3_snip.mzML">
+      <CopyToOutputDirectory>Always</CopyToOutputDirectory>
+    </None>
+    <None Update="TestData\PostSearchAnalysisTaskTestData\TaGe_SA_A549_3_snip_2.mzML">
+      <CopyToOutputDirectory>Always</CopyToOutputDirectory>
+    </None>
     <None Update="TestData\PrunedDbSpectra.mzml">
       <CopyToOutputDirectory>Always</CopyToOutputDirectory>
     </None>

MetaMorpheus/Test/TestData/Task1-SearchTaskconfig.toml

@@ -15,7 +15,7 @@ MassDiffAcceptorType = "Exact"
 WritePrunedDatabase = false
 KeepAllUniprotMods = true
 DoLocalizationAnalysis = true
-DoQuantification = false
+DoQuantification = true
 SearchType = "Classic"
 LocalFdrCategories = ["FullySpecific"]
 MaxFragmentSize = 30000.0

MetaMorpheus/Test/TestPsm.cs

@@ -305,7 +305,7 @@ public static void TestPsmMatchingToTargetAndDecoyWithDifferentSequences()
             List<(string fileName, CommonParameters fileSpecificParameters)> fsp = new List<(string fileName, CommonParameters fileSpecificParameters)> { ("filename", commonParameters) };
 
             new FdrAnalysisEngine(new List<SpectralMatch> { psm }, 1, new CommonParameters(), fsp, new List<string>()).Run();
-            Assert.AreEqual(0.5, psm.FdrInfo.CumulativeDecoy);
+            Assert.AreEqual(0.5, psm.PsmFdrInfo.CumulativeDecoy);
         }
 
         [Test]

MetaMorpheus/Test/XLTest.cs

@@ -374,7 +374,8 @@ public static void XlTest_MoreComprehensive()
 
             var xlSearchParameters = new XlSearchParameters
             {
-                CrosslinkAtCleavageSite = true
+                CrosslinkAtCleavageSite = true,
+                WritePepXml = true
             };
 
             //Create databases contain two protein.
@@ -579,7 +580,7 @@ public static void XlTest_MoreComprehensive()
             deadendNH2 = 0;
             deadendTris = 0;
 
-            foreach (CrosslinkSpectralMatch csm in firstCsmsFromListsOfCsms.Where(c => c.FdrInfo.QValue <= 0.01).ToList())
+            foreach (CrosslinkSpectralMatch csm in firstCsmsFromListsOfCsms.Where(c => c.PsmFdrInfo.QValue <= 0.01).ToList())
             {
                 switch (csm.CrossType)
                 {
@@ -776,7 +777,7 @@ public static void XlTest_MoreComprehensive()
             deadendNH2 = 0;
             deadendTris = 0;
 
-            foreach (CrosslinkSpectralMatch csm in firstCsmsFromListsOfCsms.Where(c => c.FdrInfo.QValue <= 0.01).ToList())
+            foreach (CrosslinkSpectralMatch csm in firstCsmsFromListsOfCsms.Where(c => c.PsmFdrInfo.QValue <= 0.01).ToList())
             {
                 switch (csm.CrossType)
                 {