/// <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>
/// Infers the strandedness of reads based on aligning a subset.
/// </summary>
/// <param name="spritzDirectory"></param>
/// <param name="analysisDirectory"></param>
/// <param name="threads"></param>
/// <param name="fastqPaths"></param>
/// <param name="genomeStarIndexDirectory"></param>
/// <param name="reorderedFasta"></param>
/// <param name="geneModelGtfOrGff"></param>
/// <returns></returns>
public static BAMProperties InferStrandedness(string spritzDirectory, string analysisDirectory, int threads, string[] fastqPaths, string genomeStarIndexDirectory,
string reorderedFasta, string geneModelGtfOrGff)
{
// Alignment preparation
WrapperUtility.GenerateAndRunScript(WrapperUtility.GetAnalysisScriptPath(analysisDirectory, "GenomeGenerate.bash"),
STARWrapper.GenerateGenomeIndex(spritzDirectory, threads, genomeStarIndexDirectory, new string[] { reorderedFasta }, geneModelGtfOrGff, new List <string[]> {
fastqPaths
}))
.WaitForExit();
STARWrapper.SubsetFastqs(spritzDirectory, analysisDirectory, fastqPaths, 30000, analysisDirectory, out string[] subsetFastqs);
string subsetOutPrefix = Path.Combine(Path.GetDirectoryName(subsetFastqs[0]), Path.GetFileNameWithoutExtension(subsetFastqs[0]));
WrapperUtility.GenerateAndRunScript(WrapperUtility.GetAnalysisScriptPath(analysisDirectory, "AlignSubset.bash"),
STARWrapper.BasicAlignReadCommands(spritzDirectory, threads, genomeStarIndexDirectory, subsetFastqs, subsetOutPrefix, false, STARGenomeLoadOption.LoadAndKeep))
.WaitForExit();
BAMProperties bamProperties = new BAMProperties(subsetOutPrefix + STARWrapper.BamFileSuffix, geneModelGtfOrGff, new Genome(reorderedFasta), 0.8);
return(bamProperties);
}
/// <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();
}