/// <summary>
/// lncRNA discovery from fastq files
/// </summary>
public void LncRNADiscoveryFromFastqs()
{
// Setup and Alignments
EnsemblDownloadsWrapper ensemblDownloads = new EnsemblDownloadsWrapper();
ensemblDownloads.PrepareEnsemblGenomeFasta(Parameters.AnalysisDirectory, Parameters.GenomeFasta);
AlignmentFlow alignment = new AlignmentFlow();
alignment.Parameters = new AlignmentParameters();
alignment.Parameters.SpritzDirectory = Parameters.SpritzDirectory;
alignment.Parameters.AnalysisDirectory = Parameters.AnalysisDirectory;
alignment.Parameters.Reference = Parameters.Reference;
alignment.Parameters.Threads = Parameters.Threads;
alignment.Parameters.Fastqs = Parameters.Fastqs;
alignment.Parameters.ExperimentType = ExperimentType.RNASequencing;
alignment.Parameters.StrandSpecific = Parameters.StrandSpecific;
alignment.Parameters.InferStrandSpecificity = Parameters.InferStrandSpecificity;
alignment.Parameters.OverwriteStarAlignment = Parameters.OverwriteStarAlignment;
alignment.Parameters.GenomeStarIndexDirectory = Parameters.GenomeStarIndexDirectory;
alignment.Parameters.ReorderedFastaPath = ensemblDownloads.ReorderedFastaPath;
alignment.Parameters.GeneModelGtfOrGffPath = Parameters.GeneModelGtfOrGff;
alignment.Parameters.UseReadSubset = Parameters.UseReadSubset;
alignment.Parameters.ReadSubset = Parameters.ReadSubset;
alignment.PerformAlignment();
ensemblDownloads.GetImportantProteinAccessions(Parameters.SpritzDirectory, Parameters.ProteinFasta);
EnsemblDownloadsWrapper.FilterGeneModel(Parameters.AnalysisDirectory, Parameters.GeneModelGtfOrGff, ensemblDownloads.EnsemblGenome, out string filteredGeneModelForScalpel);
string sortedBed12Path = BEDOPSWrapper.GffOrGtf2Bed12(Parameters.SpritzDirectory, Parameters.AnalysisDirectory, filteredGeneModelForScalpel);
// Transcript Reconstruction
StringtieWrapper stringtie = new StringtieWrapper();
stringtie.TranscriptReconstruction(Parameters.SpritzDirectory, Parameters.AnalysisDirectory, Parameters.Threads, Parameters.GeneModelGtfOrGff, ensemblDownloads.EnsemblGenome,
Parameters.StrandSpecific, Parameters.InferStrandSpecificity, alignment.SortedBamFiles, true);
ReconstructedTranscriptModels = stringtie.FilteredTranscriptGtfPaths;
MergedTranscriptModel = stringtie.FilteredMergedGtfPath;
// Annotate lncRNAs
foreach (string gtf in ReconstructedTranscriptModels)
{
string slnckyScriptName = WrapperUtility.GetAnalysisScriptPath(Parameters.AnalysisDirectory, "SlnckyAnnotation.bash");
SlnckyOutPrefix = Path.Combine(Path.GetDirectoryName(gtf), Path.GetFileNameWithoutExtension(gtf) + ".slnckyOut", "annotated");
WrapperUtility.GenerateAndRunScript(slnckyScriptName,
SlnckyWrapper.Annotate(Parameters.SpritzDirectory, Parameters.AnalysisDirectory, Parameters.Threads,
gtf, Parameters.Reference, SlnckyOutPrefix)).WaitForExit();
}
// Write quantification tables for differential expression analysis (using stringtie TPM values)
}
/// <summary>
/// Generate sample specific protein database starting with fastq files
/// </summary>
public void GenerateSampleSpecificProteinDatabases()
{
// Download references and align reads
Downloads.PrepareEnsemblGenomeFasta(Parameters.AnalysisDirectory, Parameters.GenomeFasta);
if (Parameters.Fastqs != null)
{
Alignment.Parameters = new AlignmentParameters();
Alignment.Parameters.SpritzDirectory = Parameters.SpritzDirectory;
Alignment.Parameters.AnalysisDirectory = Parameters.AnalysisDirectory;
Alignment.Parameters.Reference = Parameters.Reference;
Alignment.Parameters.Threads = Parameters.Threads;
Alignment.Parameters.Fastqs = Parameters.Fastqs;
Alignment.Parameters.ExperimentType = Parameters.ExperimentType;
Alignment.Parameters.StrandSpecific = Parameters.StrandSpecific;
Alignment.Parameters.InferStrandSpecificity = Parameters.InferStrandSpecificity;
Alignment.Parameters.OverwriteStarAlignment = Parameters.OverwriteStarAlignment;
Alignment.Parameters.GenomeStarIndexDirectory = Parameters.GenomeStarIndexDirectory;
Alignment.Parameters.ReorderedFastaPath = Downloads.ReorderedFastaPath;
Alignment.Parameters.GeneModelGtfOrGffPath = Parameters.ReferenceGeneModelGtfOrGff;
Alignment.Parameters.UseReadSubset = Parameters.UseReadSubset;
Alignment.Parameters.ReadSubset = Parameters.ReadSubset;
Alignment.PerformAlignment();
Downloads.GetImportantProteinAccessions(Parameters.SpritzDirectory, Parameters.ProteinFastaPath);
}
EnsemblDownloadsWrapper.FilterGeneModel(Parameters.AnalysisDirectory, Parameters.ReferenceGeneModelGtfOrGff, Downloads.EnsemblGenome, out string filteredGeneModelForScalpel);
string sortedBed12Path = BEDOPSWrapper.GffOrGtf2Bed12(Parameters.SpritzDirectory, Parameters.AnalysisDirectory, filteredGeneModelForScalpel);
GeneModel referenceGeneModel = new GeneModel(Downloads.EnsemblGenome, Parameters.ReferenceGeneModelGtfOrGff);
string referenceGeneModelProteinXml = Path.Combine(Path.GetDirectoryName(Parameters.ReferenceGeneModelGtfOrGff), Path.GetFileNameWithoutExtension(Parameters.ReferenceGeneModelGtfOrGff) + ".protein.xml"); // used if no fastqs are provided
// Merge reference gene model and a new gene model (either specified or stringtie-generated)
string newGeneModelPath = Parameters.NewGeneModelGtfOrGff;
string mergedGeneModelWithCdsPath = null;
string mergedGeneModelProteinXml = null;
string reference = Parameters.Reference;
if (Parameters.DoTranscriptIsoformAnalysis)
{
StringtieWrapper stringtie = new StringtieWrapper();
if (newGeneModelPath == null)
{
stringtie.TranscriptReconstruction(Parameters.SpritzDirectory, Parameters.AnalysisDirectory, Parameters.Threads, Parameters.ReferenceGeneModelGtfOrGff, Downloads.EnsemblGenome, Parameters.StrandSpecific, Parameters.InferStrandSpecificity, Alignment.SortedBamFiles, true);
newGeneModelPath = stringtie.FilteredMergedGtfPath;
}
else
{
newGeneModelPath = EnsemblDownloadsWrapper.ConvertFirstColumnUCSC2Ensembl(Parameters.SpritzDirectory, Parameters.Reference, Parameters.NewGeneModelGtfOrGff);
string mergedGeneModelPath = Path.Combine(Path.GetDirectoryName(newGeneModelPath), Path.GetFileNameWithoutExtension(newGeneModelPath) + ".merged.gtf");
WrapperUtility.GenerateAndRunScript(WrapperUtility.GetAnalysisScriptPath(Parameters.AnalysisDirectory, "MergeTranscriptModels.bash"),
StringtieWrapper.MergeTranscriptPredictions(Parameters.SpritzDirectory, Parameters.ReferenceGeneModelGtfOrGff, new List <string> {
newGeneModelPath
}, mergedGeneModelPath)).WaitForExit();
newGeneModelPath = mergedGeneModelPath;
}
// Determine CDS from start codons of reference gene model
// In the future, we could also try ORF finding to expand this (e.g. https://github.com/TransDecoder/TransDecoder/wiki)
GeneModel newGeneModel = new GeneModel(Downloads.EnsemblGenome, newGeneModelPath);
newGeneModel.CreateCDSFromAnnotatedStartCodons(referenceGeneModel);
mergedGeneModelWithCdsPath = Path.Combine(Path.GetDirectoryName(newGeneModelPath), Path.GetFileNameWithoutExtension(newGeneModelPath) + ".withcds.gtf");
newGeneModel.PrintToGTF(mergedGeneModelWithCdsPath);
}
// SnpEff databases or outputing protein XMLs from gene models
if (Parameters.DoTranscriptIsoformAnalysis) // isoform analysis, so generate a new snpeff database
{
reference = SnpEffWrapper.GenerateDatabase(Parameters.SpritzDirectory, Parameters.AnalysisDirectory, Downloads.ReorderedFastaPath, Parameters.ProteinFastaPath, mergedGeneModelWithCdsPath);
if (Parameters.Fastqs == null || Parameters.SkipVariantAnalysis) // isoform analysis without variant analysis, so generate a protein database directly from merged gtf
{
mergedGeneModelProteinXml = SnpEffWrapper.GenerateXmlDatabaseFromReference(Parameters.SpritzDirectory, Parameters.AnalysisDirectory, reference, mergedGeneModelWithCdsPath);
}
}
else // no isoform analysis
{
new SnpEffWrapper(1).DownloadSnpEffDatabase(Parameters.SpritzDirectory, Parameters.AnalysisDirectory, Parameters.Reference);
if (Parameters.Fastqs == null) // no isoform analysis and no fastqs
{
referenceGeneModelProteinXml = SnpEffWrapper.GenerateXmlDatabaseFromReference(Parameters.SpritzDirectory, Parameters.AnalysisDirectory, Parameters.Reference, Parameters.ReferenceGeneModelGtfOrGff);
}
}
// Gene Fusion Discovery
List <Protein> fusionProteins = new List <Protein>();
if (Parameters.DoFusionAnalysis)
{
Fusion.Parameters.SpritzDirectory = Parameters.SpritzDirectory;
Fusion.Parameters.AnalysisDirectory = Parameters.AnalysisDirectory;
Fusion.Parameters.Reference = Parameters.Reference;
Fusion.Parameters.Threads = Parameters.Threads;
Fusion.Parameters.Fastqs = Parameters.Fastqs;
Fusion.DiscoverGeneFusions();
fusionProteins = Fusion.FusionProteins;
}
// Variant Calling
if (Parameters.Fastqs != null && !Parameters.SkipVariantAnalysis)
{
VariantCalling.CallVariants(
Parameters.SpritzDirectory,
Parameters.AnalysisDirectory,
Parameters.ExperimentType,
reference,
Parameters.Threads,
sortedBed12Path,
Parameters.EnsemblKnownSitesPath,
Alignment.DedupedBamFiles,
Downloads.ReorderedFastaPath,
Downloads.EnsemblGenome,
Parameters.QuickSnpEffWithoutStats,
Parameters.IndelFinder,
Parameters.VariantCallingWorkers);
}
// Transfer features from UniProt
List <string> xmlsToUse = null;
if (VariantCalling.CombinedAnnotatedProteinXmlPaths.Count > 0)
{
xmlsToUse = VariantCalling.CombinedAnnotatedProteinXmlPaths;
}
// keep, since it might be useful for making a final database: .Concat(new[] { VariantCalling.CombinedAnnotatedProteinXmlPath }).ToList()
else
{
xmlsToUse = new List <string> {
Parameters.DoTranscriptIsoformAnalysis?mergedGeneModelProteinXml : referenceGeneModelProteinXml
}
};
VariantAnnotatedProteinXmlDatabases = new TransferModificationsFlow().TransferModifications(Parameters.SpritzDirectory, Parameters.UniProtXmlPath, xmlsToUse, fusionProteins);
}
