/// <summary> /// Runs a two-pass STAR alignment for a given set of RNA-Seq fastq files, /// or it performs a Bowtie2 alignment for WGS or exome sequencing files. /// </summary> public void PerformAlignment() { int starThreads = Math.Min(18, Parameters.Threads); // 18 max, otherwise it throws a segmentation fault in sorting the BAM files if (Parameters.ExperimentType == ExperimentType.RNASequencing) { // Alignment preparation WrapperUtility.GenerateAndRunScript(WrapperUtility.GetAnalysisScriptPath(Parameters.AnalysisDirectory, "GenomeGenerate.bash"), STARWrapper.GenerateGenomeIndex( Parameters.SpritzDirectory, Parameters.Threads, Parameters.GenomeStarIndexDirectory, new string[] { Parameters.ReorderedFastaPath }, Parameters.GeneModelGtfOrGffPath, Parameters.Fastqs)) .WaitForExit(); // there's trouble with the number of open files for sorting and stuff, which increases with the number of threads // 18 is the max that works with the default max number of open files TwoPassAlignment(starThreads, Parameters.OverwriteStarAlignment); } else { foreach (string[] fastq in Parameters.Fastqs) { SkewerWrapper.Trim(Parameters.SpritzDirectory, Parameters.AnalysisDirectory, Parameters.Threads, 19, fastq, false, out string[] trimmedFastqs, out string skewerLog); FastqsForAlignment.Add(trimmedFastqs); } TopHatWrapper.GenerateBowtieIndex(Parameters.SpritzDirectory, Parameters.AnalysisDirectory, Parameters.ReorderedFastaPath, out string bowtieIndexPrefix); List <string> alignmentCommands = new List <string> { "echo \"Aligning reads with bowtie2.\"" }; foreach (string[] fastq in FastqsForAlignment) { // alignment alignmentCommands.AddRange(TopHatWrapper.Bowtie2Align(Parameters.SpritzDirectory, Parameters.AnalysisDirectory, bowtieIndexPrefix, Parameters.Threads, fastq, Parameters.StrandSpecific, out string sortedBamPath)); alignmentCommands.Add(SamtoolsWrapper.IndexBamCommand(sortedBamPath)); // mark duplicates GATKWrapper gatk = new GATKWrapper(1); alignmentCommands.AddRange(gatk.PrepareBamAndFasta(Parameters.SpritzDirectory, Parameters.AnalysisDirectory, Parameters.Threads, sortedBamPath, Parameters.ReorderedFastaPath, Parameters.Reference)); alignmentCommands.Add(SamtoolsWrapper.IndexBamCommand(gatk.PreparedBamPath)); SortedBamFiles.Add(sortedBamPath); DedupedBamFiles.Add(gatk.PreparedBamPath); } WrapperUtility.GenerateAndRunScript(WrapperUtility.GetAnalysisScriptPath(Parameters.AnalysisDirectory, "BowtieAlignment.bash"), alignmentCommands).WaitForExit(); } }
/// <summary> /// Performs the bulk of two-pass alignments /// </summary> private void TwoPassAlignment(int threads, bool overWriteStarAlignment) { // Trimming and strand specificity Genome genome = new Genome(Parameters.ReorderedFastaPath); foreach (string[] fq in Parameters.Fastqs) { // Infer strand specificity before trimming because trimming can change read pairings string[] fqForAlignment = fq; bool localStrandSpecific = Parameters.StrandSpecific; if (Parameters.InferStrandSpecificity || Parameters.UseReadSubset) { STARWrapper.SubsetFastqs(Parameters.SpritzDirectory, Parameters.AnalysisDirectory, fqForAlignment, Parameters.ReadSubset, Parameters.AnalysisDirectory, out string[] subsetFastqs); if (Parameters.UseReadSubset) { fqForAlignment = subsetFastqs; } if (Parameters.InferStrandSpecificity) { string subsetOutPrefix = Path.Combine(Path.GetDirectoryName(subsetFastqs[0]), Path.GetFileNameWithoutExtension(subsetFastqs[0])); WrapperUtility.GenerateAndRunScript(WrapperUtility.GetAnalysisScriptPath(Parameters.AnalysisDirectory, "AlignSubset.bash"), STARWrapper.BasicAlignReadCommands(Parameters.SpritzDirectory, threads, Parameters.GenomeStarIndexDirectory, subsetFastqs, subsetOutPrefix, false, STARGenomeLoadOption.LoadAndKeep)) .WaitForExit(); BAMProperties bamProperties = new BAMProperties(subsetOutPrefix + STARWrapper.BamFileSuffix, Parameters.GeneModelGtfOrGffPath, new Genome(Parameters.ReorderedFastaPath), 0.8); localStrandSpecific = bamProperties.Strandedness != Strandedness.None; } } SkewerWrapper.Trim(Parameters.SpritzDirectory, Parameters.AnalysisDirectory, threads, 19, fqForAlignment, false, out string[] trimmedFastqs, out string skewerLog); fqForAlignment = trimmedFastqs; StrandSpecificities.Add(localStrandSpecific); FastqsForAlignment.Add(fqForAlignment); } // Alignment List <string> alignmentCommands = new List <string>(); foreach (string[] fq in FastqsForAlignment) { string outPrefix = Path.Combine(Path.GetDirectoryName(fq[0]), Path.GetFileNameWithoutExtension(fq[0])); if (!File.Exists(outPrefix + STARWrapper.SpliceJunctionFileSuffix) || overWriteStarAlignment) { alignmentCommands.AddRange(STARWrapper.FirstPassAlignmentCommands(Parameters.SpritzDirectory, threads, Parameters.GenomeStarIndexDirectory, fq, outPrefix, StrandSpecificities[FastqsForAlignment.IndexOf(fq)], STARGenomeLoadOption.LoadAndKeep)); } FirstPassSpliceJunctions.Add(outPrefix + STARWrapper.SpliceJunctionFileSuffix); } int uniqueSuffix = 1; foreach (string f in FastqsForAlignment.SelectMany(f => f)) { uniqueSuffix = uniqueSuffix ^ f.GetHashCode(); } alignmentCommands.AddRange(STARWrapper.RemoveGenome(Parameters.SpritzDirectory, Parameters.GenomeStarIndexDirectory)); alignmentCommands.AddRange(STARWrapper.ProcessFirstPassSpliceCommands(FirstPassSpliceJunctions, uniqueSuffix, out string spliceJunctionStartDatabase)); SecondPassGenomeDirectory = Parameters.GenomeStarIndexDirectory + "SecondPass" + uniqueSuffix.ToString(); alignmentCommands.AddRange(STARWrapper.GenerateGenomeIndex(Parameters.SpritzDirectory, threads, SecondPassGenomeDirectory, new string[] { Parameters.ReorderedFastaPath }, Parameters.GeneModelGtfOrGffPath, Parameters.Fastqs, spliceJunctionStartDatabase)); foreach (string[] fq in FastqsForAlignment) { string outPrefix = Path.Combine(Path.GetDirectoryName(fq[0]), Path.GetFileNameWithoutExtension(fq[0])); OutputPrefixes.Add(outPrefix); alignmentCommands.AddRange(STARWrapper.AlignRNASeqReadsForVariantCalling(Parameters.SpritzDirectory, threads, SecondPassGenomeDirectory, fq, outPrefix, overWriteStarAlignment, StrandSpecificities[FastqsForAlignment.IndexOf(fq)], STARGenomeLoadOption.LoadAndKeep)); SortedBamFiles.Add(outPrefix + STARWrapper.SortedBamFileSuffix); DedupedBamFiles.Add(outPrefix + STARWrapper.DedupedBamFileSuffix); ChimericSamFiles.Add(outPrefix + STARWrapper.ChimericSamFileSuffix); ChimericJunctionFiles.Add(outPrefix + STARWrapper.ChimericJunctionsFileSuffix); } alignmentCommands.AddRange(STARWrapper.RemoveGenome(Parameters.SpritzDirectory, SecondPassGenomeDirectory)); WrapperUtility.GenerateAndRunScript(WrapperUtility.GetAnalysisScriptPath(Parameters.AnalysisDirectory, "AlignReads.bash"), alignmentCommands).WaitForExit(); }