public static void ParseSearchResults(this QcDataContainer qcData, RawDataCollection rawData, IRawDataPlus rawFile, QcParameters qcParameters)
{
XElement results = LoadSearchResults(qcParameters, rawData);
PsmDataCollection Psms = ExtractPsmData(results, qcParameters.searchParameters.SearchAlgorithm);
qcData.ParsePSMs(Psms, qcParameters);
}
public static void UpdateQcCollection(QcDataCollection qcDataCollection, QcDataContainer newQcData, MethodDataContainer methodData, string rawFileName)
{
qcDataCollection.QcData.Add(methodData.CreationDate, newQcData);
qcDataCollection.ProcessedRawFiles.Add(Path.GetFileName(rawFileName));
qcDataCollection.WriteQcToTable();
Console.WriteLine("QC data written to csv file.");
try
{
XmlSerialization.WriteToXmlFile <QcDataCollection>(qcDataCollection.QcFile, qcDataCollection);
Log.Information("QC file saved successfully");
Console.WriteLine("QC file saved successfully");
}
catch (Exception e)
{
Log.Error(e, "Failed during serialization of QC data");
Console.WriteLine("ERROR: failure during serialization of QC data.");
Environment.Exit(1);
}
}
public static void ChromIntMetrics(this QcDataContainer qcData, RawDataCollection rawData, MetricsData metrics)
{
double firstRtToExceed10 = 0;
double lastRtToExceed10 = 0;
double proportionCovered;
var scans = rawData.scanIndex.ScanEnumerators[MSOrderType.Ms];
var reversedScans = scans.Reverse();
var totalIntList = (from x in scans select rawData.metaData[x].SummedIntensity).ToArray();
// get Q1 of total intensity from all scans
double threshold = totalIntList.Max() / 10;
// get first RT which exceeds Q1
for (int i = 0; i < scans.Length; i++)
{
int scan = scans[i];
if (totalIntList.MovingAverage(i, 20) > threshold)
{
firstRtToExceed10 = rawData.retentionTimes[scan];
break;
}
}
for (int i = scans.Length - 1; i >= 0; i--)
{
int scan = scans[i];
if (totalIntList.MovingAverage(i, 20) > threshold)
{
lastRtToExceed10 = rawData.retentionTimes[scan];
break;
}
}
// get proportion of run encompassed by these times
//proportionCovered = (lastRtToExceedQ1 - firstRtToExceedQ1) / metrics.TotalAnalysisTime;
proportionCovered = (lastRtToExceed10 - firstRtToExceed10) / rawData.retentionTimes[rawData.scanIndex.ScanEnumerators[MSOrderType.Ms].Last()];
qcData.TimeBeforeFirstScanToExceedPoint1MaxIntensity = firstRtToExceed10;// - rawData.retentionTimes[1];
qcData.TimeAfterLastScanToExceedPoint1MaxIntensity = rawData.retentionTimes[rawData.scanIndex.ScanEnumerators[MSOrderType.Ms].Last()] - lastRtToExceed10;
qcData.FractionOfRunAbovePoint1MaxIntensity = proportionCovered;
}
public static void GetModificationFrequency(this QcDataContainer qcData, IEnumerable <PsmData> psms, SearchParameters searchParameters)
{
string nmod = searchParameters.NMod;
string kmod = searchParameters.KMod;
string xmod = searchParameters.XMod;
Dictionary <string, string> Modifications = new Dictionary <string, string>();
Dictionary <string, int> TotalLabelingSites = new Dictionary <string, int>();
Dictionary <string, int> LabelingSitesHit = new Dictionary <string, int>();
Dictionary <string, double> LabelingEfficiency = new Dictionary <string, double>();
List <Modification> mods;
List <string> AminosOfInterest = new List <string>();
string[] Mods = new string[] { nmod, kmod, xmod };
// "Prime" the dictionaries
foreach (var item in Mods)
{
if (item == null)
{
continue;
}
var splitString = item.Split('@');
// add the key: value pairs as mass@AA:AA
Modifications.Add(item, splitString.Last());
// and AA:int
TotalLabelingSites.Add(splitString.Last(), 0);
LabelingSitesHit.Add(splitString.Last(), 0);
AminosOfInterest.Add(splitString.Last());
}
// now we need to get at labeling efficiency
int KTotalSites = 0;
int KTotalMissed = 0;
int NTotalSites = 0;
int NTotalMissed = 0;
int XTotalSites = 0;
int XTotalMissed = 0;
// define a function to return the number of labeling sites
int NumberOfSites(string aa, string seq)
{
if (aa == "[" | aa == "]")
{
return(1);
}
else
{
return(seq.Count(x => x.ToString() == aa));
}
}
foreach (PsmData psm in psms)
{
mods = psm.Mods;
bool skipNterm = false;
// check the sequence in two steps. First the n-terminus, then remove the n-terminus and check the rest of it.
// FIRST STEP: N-TERMINUS
if (nmod != null)
{
// check if the first residue is lysine
if (psm.Seq[0] == 'K')
{
// if so, we need to see if it was only labeled once. Skip the psm if that is the case because it is ambiguous
IEnumerable <Modification> nMods = from x in mods
where x.Loc == 0
select x;
int numMods = nMods.Count();
if (numMods == 1)
{
// we can't know which reactive site is modified, so don't include this peptide
continue;
}
if (numMods == 0)
{
// nothing is labeled
TotalLabelingSites["["] += 1;
if (AminosOfInterest.Contains("K"))
{
TotalLabelingSites["K"] += 1;
}
}
if (numMods == 2)
{
TotalLabelingSites["["] += 1;
LabelingSitesHit["["] += 1;
if (AminosOfInterest.Contains("K"))
{
TotalLabelingSites["K"] += 1;
LabelingSitesHit["K"] += 1;
}
}
}
// If the first residue is not lysine
else
{
IEnumerable <Modification> nMods = from x in mods
where x.Loc == 0
select x;
// add 1 to total n-termini, because it is always there
TotalLabelingSites["["] += 1;
// get the aa residue letter
string residue = psm.Seq[0].ToString();
//see if it is of interest
if (AminosOfInterest.Contains(residue))
{
// if so, add 1 to total sites for it
TotalLabelingSites[residue] += 1;
}
// now go through each detected modification
foreach (Modification mod in nMods)
{
if (nmod.Contains(mod.Mass.ToString()))
{
LabelingSitesHit["["] += 1;
}
else
{
if (AminosOfInterest.Contains(mod.AA))
{
LabelingSitesHit[mod.AA] += 1;
}
}
}
}
}
int start;
if (nmod != null)
{
start = 1;
}
else
{
start = 0;
}
// now continue with the rest
for (int i = start; i < psm.Seq.Length; i++)
{
// check if we care about this amino acid
string aa = psm.Seq[i].ToString();
if (AminosOfInterest.Contains(aa))
{
// add one to potential labeling sites
TotalLabelingSites[aa] += 1;
// There should only ever be one modification for each of the rest of the residues, so we can reference it by location to see if it exists
bool hit = (from x in mods
where x.Loc == i
select 1).Count() == 1;
if (hit)
{
LabelingSitesHit[aa] += 1;
}
}
else
{
continue;
}
}
}
// spit out some metrics to the console
foreach (string aa in AminosOfInterest)
{
if (aa == "[")
{
Console.WriteLine("Total N-term sites: {0}", TotalLabelingSites["["]);
}
else
{
Console.WriteLine("Total {0} sites: {1}", aa, TotalLabelingSites[aa]);
}
}
foreach (string aa in AminosOfInterest)
{
if (aa == "[")
{
Console.WriteLine("Missed modifications at N-term: {0}", TotalLabelingSites["["] - LabelingSitesHit["["]);
}
else
{
Console.WriteLine("Missed modifications at {0}: {1}", aa, TotalLabelingSites[aa] - LabelingSitesHit[aa]);
}
}
// calculate labelling efficiency for each site
foreach (var aa in AminosOfInterest)
{
double efficiency = (double)LabelingSitesHit[aa] / TotalLabelingSites[aa];
LabelingEfficiency.Add(aa, efficiency);
if (aa == "[")
{
Console.WriteLine("Modification frequency at N-term: {0}", efficiency);
}
else
{
Console.WriteLine("Modification frequency at {0}: {1}", aa, efficiency);
}
// if the sites are n-term or K add them to their own attributes
if (aa == "[")
{
qcData.LabelingEfficiencyAtNTerm = efficiency;
}
else
{
if (aa == "K")
{
qcData.LabelingEfficiencyAtK = efficiency;
}
// if not, then add it to xmod attributes
else
{
qcData.LabelingEfficiencyAtX = efficiency;
qcData.LabelX = aa;
}
}
}
}
public static void ParsePSMs(this QcDataContainer qcData, PsmDataCollection psmCollection, QcParameters qcParameters)
{
XElement results, searchSummary;
IEnumerable <XElement> decoyPSMs, search_hits, spectrumQueries;
int numGoodPSMs, totalCleavageSites, pepsWithNoMissedCleavages, peptidesWithNoMissedCleavages;
IEnumerable <int> allMissedCleavages, charges;
double IdRate, chargeRatio3to2, chargeRatio4to2;
double digestionEfficiencyByCleavage, digestionEfficiency, topDecoyScore;
double missedCleavageRate;
Dictionary <int, int> numCharges = new Dictionary <int, int>();
SearchParameters searchParameters = qcParameters.searchParameters;
int numSearched = searchParameters.NumSpectra;
List <PsmData> psms;
IEnumerable <PsmData> goodPsms, nonDecoys;
// convert the dictionary to a list for easy parsing
psms = psmCollection.Values.ToList();
// get the top decoy score
topDecoyScore = (from x in psms
where x.Decoy
select x.Hyperscore)
.ToArray().Percentile(95);
// get the non-decoys
nonDecoys = from x in psms
where !x.Decoy
select x;
// and select the non-decoy hits which are above the top decoy score
goodPsms = from x in psms
where !x.Decoy & x.Hyperscore > topDecoyScore
select x;
Console.WriteLine("Total hits: {0}", psms.Count());
Console.WriteLine("Top decoy score: {0}", topDecoyScore);
Console.WriteLine("Non-decoy hits: {0}", nonDecoys.Count());
Console.WriteLine("Non-decoy hits above top decoy score: {0}", goodPsms.Count());
// parse out the charges
charges = from x in goodPsms
select x.Charge;
// get the number of each charge, add to a dictionary
foreach (int charge in new List <int>()
{
2, 3, 4
})
{
numCharges.Add(charge, (from x in charges where x == charge select 1).Count());
}
// calculate charge ratios
chargeRatio3to2 = Convert.ToDouble(numCharges[3]) / Convert.ToDouble(numCharges[2]);
chargeRatio4to2 = Convert.ToDouble(numCharges[4]) / Convert.ToDouble(numCharges[2]);
// parse out the missed cleavage data
pepsWithNoMissedCleavages = (from x in goodPsms
where x.MissedCleavages == 0
select 1).Sum();
// number of PSMs is the length of this collection
numGoodPSMs = goodPsms.Count();
// missed cleavages per PSM
digestionEfficiency = (double)pepsWithNoMissedCleavages / numGoodPSMs;
Console.WriteLine("Digestion efficiency: {0}", digestionEfficiency);
// get missed cleavage rate, i.e. number of missed cleavages per psm
missedCleavageRate = (double)(from x in goodPsms select x.MissedCleavages).Sum() / numGoodPSMs;
Console.WriteLine("Missed cleavage rate (/PSM): {0}", missedCleavageRate);
// calculate ID rate
IdRate = (double)numGoodPSMs / numSearched;
Console.WriteLine("IDrate: {0}", IdRate);
// get labeling efficiency metrics
if ((searchParameters.NMod != null) | (searchParameters.KMod != null) | (searchParameters.XMod != null))
{
qcData.GetModificationFrequency(goodPsms, searchParameters);
}
// get median mass drift
qcData.MedianMassDrift = (from x in goodPsms
select x.MassDrift)
.ToArray().Percentile(50);
qcData.IdentificationRate = IdRate;
qcData.MissedCleavageRate = missedCleavageRate;
qcData.DigestionEfficiency = digestionEfficiency;
qcData.ChargeRatio3to2 = chargeRatio3to2;
qcData.ChargeRatio4to2 = chargeRatio4to2;
}
public static QcDataContainer ProcessQcData(this QcDataCollection Data, RawDataCollection rawData, IRawDataPlus rawFile, string qcDirectory, string fastaDB = null)
{
DateTime dateAcquired = rawFile.CreationDate;
//RawDataCollection rawData = new RawDataCollection(rawFile);
MetricsData metricsData = new MetricsData();
metricsData.GetMetricsData(metaData: rawData.metaData, rawData: rawData, rawFile: rawFile);
QcDataContainer qcData = new QcDataContainer(rawData.rawFileName, dateAcquired);
qcData.Instrument = rawData.instrument;
qcData.ExperimentMsOrder = rawData.methodData.AnalysisOrder;
qcData.Ms1Analyzer = rawData.methodData.MassAnalyzers[MSOrderType.Ms].ToString();
qcData.Ms2Analyzer = rawData.methodData.MassAnalyzers[MSOrderType.Ms2].ToString();
if (qcData.ExperimentMsOrder == MSOrderType.Ms3)
{
qcData.Ms3Analyzer = rawData.methodData.MassAnalyzers[MSOrderType.Ms3].ToString();
}
else
{
qcData.Ms3Analyzer = "None";
}
qcData.TotalScans = metricsData.TotalScans;
qcData.NumMs1Scans = metricsData.MS1Scans;
qcData.NumMs2Scans = metricsData.MS2Scans;
qcData.NumMs3Scans = metricsData.MS3Scans;
qcData.Ms1ScanRate = metricsData.MS1ScanRate;
qcData.Ms2ScanRate = metricsData.MS2ScanRate;
qcData.MeanDutyCycle = metricsData.MeanDutyCycle;
qcData.MeanTopN = metricsData.MeanTopN;
qcData.MedianPrecursorIntensity = metricsData.MedianPrecursorIntensity;
qcData.MedianSummedMs2Intensity = metricsData.MedianSummedMS2Intensity;
qcData.MedianMs1IsolationInterference = metricsData.MedianMs1IsolationInterference;
qcData.MedianMs2FractionConsumingTop80PercentTotalIntensity = metricsData.MedianMs2FractionConsumingTop80PercentTotalIntensity;
qcData.NumEsiStabilityFlags = NumberOfEsiFlags(rawData);
qcData.QuantMeta = metricsData.QuantMeta;
qcData.GradientTime = metricsData.Gradient;
qcData.ColumnPeakCapacity = metricsData.PeakCapacity;
qcData.ChromIntMetrics(rawData, metricsData);
if (!rawData.isBoxCar)
{
qcData.PeakShape.Asymmetry.P10 = rawData.peakData.PeakShapeMedians.Asymmetry.P10;
qcData.PeakShape.Asymmetry.P50 = rawData.peakData.PeakShapeMedians.Asymmetry.P50;
qcData.PeakShape.Width.P10 = rawData.peakData.PeakShapeMedians.Width.P10;
qcData.PeakShape.Width.P50 = rawData.peakData.PeakShapeMedians.Width.P50;
}
// add the signal-to-noise distribution to the QC data. These are presented as "median of the ith percentile", so for example we take all the 10th percentile values of
// the S2N and put them in a list, then report the median of that list
qcData.MedianSummedMs1Intensity = (from x in rawData.scanIndex.ScanEnumerators[MSOrderType.Ms] select rawData.centroidStreams[x].Intensities.Sum()).ToArray().Percentile(50);
// add the fill-time distribution to the QC data. This is more straightforward. Just put all the fill times in an array and use it to instantiate a new distribution.
qcData.Ms1FillTime = new Distribution((from x in rawData.scanIndex.ScanEnumerators[MSOrderType.Ms] select rawData.trailerExtras[x].InjectionTime).ToArray());
qcData.Ms2FillTime = new Distribution((from x in rawData.scanIndex.ScanEnumerators[MSOrderType.Ms2] select rawData.trailerExtras[x].InjectionTime).ToArray());
qcData.Ms3FillTime = new Distribution((from x in rawData.scanIndex.ScanEnumerators[MSOrderType.Ms3] select rawData.trailerExtras[x].InjectionTime).ToArray());
//Data.QcData.Add(dateAcquired, newData);
//Data.ProcessedRawFiles.Add(Path.GetFileName(rawData.rawFileName));
return(qcData);
}
public static void DoQc(QcParameters qcParameters)
{
QcDataCollection qcDataCollection;
string dataDirectory = qcParameters.RawFileDirectory;
string qcDirectory = qcParameters.QcDirectory;
string qcSearchDataDirecotry = qcParameters.QcSearchDataDirectory;
SearchParameters searchParameters = qcParameters.searchParameters;
// our qc file
string qcFile = Path.Combine(qcDirectory, "QC.xml");
// see if the file exists
if (File.Exists(qcFile))
{
// if so, open it
try
{
qcDataCollection = XmlSerialization.ReadFromXmlFile <QcDataCollection>(qcFile);
Log.Information("QC data file loaded successfully");
}
catch (Exception e)
{
Log.Error(e, "Failed while loading QC data");
throw e;
}
}
else
{
// if not, check if the directory exists
if (!Directory.Exists(qcDirectory))
{
Directory.CreateDirectory(qcDirectory);
}
qcDataCollection = new QcDataCollection(dataDirectory, qcDirectory);
Log.Information("Appears to be a new QC directory. New QC data collection created.");
}
// get our list of new raw files. it is every raw file in the directory that is not listed in the qc data
var fileList = Directory.GetFiles(dataDirectory, "*.*", SearchOption.TopDirectoryOnly)
.Where(s => s.EndsWith(".raw", StringComparison.OrdinalIgnoreCase)).ToList();
if (fileList.Count() == 0)
{
Log.Error("No raw files found in {Directory}", dataDirectory);
Console.WriteLine("{0} contains no raw files!", dataDirectory);
Environment.Exit(1);
}
fileList.RemoveAll(s => qcDataCollection.ProcessedRawFiles.Contains(Path.GetFileName(s)));
Log.Information("Raw files in QC queue: {Files}", fileList);
if (fileList.Count() == 0)
{
Log.Information("No new files to QC");
Console.WriteLine("No new files in the directory to QC!");
Environment.Exit(0);
}
Console.WriteLine("{0} file(s) to process", fileList.Count());
foreach (string fileName in fileList)
{
Console.WriteLine("Processing {0}", fileName);
IFileHeader rawHeader;
// try to open the raw file header
try
{
rawHeader = FileHeaderReaderFactory.ReadFile(fileName);;
}
catch (Exception)
{
Log.Information("{File} is not a valid raw file", fileName);
Console.WriteLine("{0} is not a valid raw file, continuing to next file.", fileName);
continue;
}
// is it a real raw file?
if (rawHeader.FileType == FileType.RawFile)
{
Log.Information("{File} is a valid raw file", fileName);
Log.Information("Creation date: {Date}", rawHeader.CreationDate);
Log.Information("File description: {Description}", rawHeader.FileDescription);
}
else
{
Log.Information("{File} is not a valid raw file", fileName);
Console.WriteLine("{0} is not a valid raw file, continuing to next file.", fileName);
continue;
}
// okay, it is probably a real raw file, let's do the QC
// check if the raw file already exists in the QC data with a different name
if (qcDataCollection.QcData.Keys.Contains(rawHeader.CreationDate))
{
Log.Information("A file with the same creation date and time as {File} already exists in the QC data", fileName);
Console.WriteLine("{0} appears to already exist in the QC data with the name {1}. Skipping to next file.",
fileName, qcDataCollection.QcData[rawHeader.CreationDate].RawFile);
continue;
}
using (IRawDataPlus rawFile = RawFileReaderFactory.ReadFile(fileName))
{
rawFile.SelectInstrument(Device.MS, 1);
RawDataCollection rawData = new RawDataCollection(rawFile);
rawData.ExtractAll(rawFile);
/*
* if (idpyPars?.QuantMods != null)
* {
* rawData.quantData.Quantify(rawData, rawFile, )
* }
*/
QcDataContainer newQcData = ProcessQcData(Data: qcDataCollection, rawData: rawData, rawFile: rawFile, qcDirectory: qcDirectory);
if (searchParameters != null)
{
Search.WriteSearchMGF(qcParameters, rawData, rawFile, searchParameters.FixedScans);
Search.RunSearch(qcParameters, rawData, rawFile);
newQcData.ParseSearchResults(rawData, rawFile, qcParameters);
/*
* if (searchParameters.SearchAlgorithm == SearchAlgorithm.XTandem)
* {
* SearchQC.ParseXTandem(newQcData, qcParameters);
* newQcData.IdentipyParameters = String.Format("\"Algorithm: X!Tandem; fmods: {0}; nmod: {1}; kmod: {2}; xmod: {3}; fastaDB: {4}; xtandemDirectory: {5}\"",
* searchParameters.FixedMods, searchParameters.NMod, searchParameters.KMod, searchParameters.XMod, searchParameters.FastaDatabase, searchParameters.XTandemDirectory);
* }
* else
* {
* SearchQC.ParseIdentipy(newQcData, rawData, rawFile, qcParameters);
* newQcData.IdentipyParameters = String.Format("\"Algorithm: IdentiPy; fmods: {0}; nmod: {1}; kmod: {2}; xmod: {3}; fastaDB: {4}; pythonExecutable: {5}; identipyScript: {6}\"",
* searchParameters.FixedMods, searchParameters.NMod, searchParameters.KMod, searchParameters.XMod, searchParameters.FastaDatabase, searchParameters.PythonExecutable, searchParameters.IdentipyScript);
* }
*/
}
qcDataCollection.QcData.Add(rawFile.CreationDate, newQcData);
qcDataCollection.ProcessedRawFiles.Add(Path.GetFileName(rawData.rawFileName));
qcDataCollection.WriteQcToTable();
}
Log.Information("QC finished: {File}", fileName);
}
Log.Information("QC of all files completed");
Console.WriteLine("QC of all files completed!");
try
{
XmlSerialization.WriteToXmlFile <QcDataCollection>(qcFile, qcDataCollection);
Log.Information("QC file saved successfully");
Console.WriteLine("QC file saved successfully");
}
catch (Exception e)
{
Log.Error(e, "Failed during serialization of QC data");
throw e;
}
}
