package org.broadinstitute.sting.playground.fourbasecaller; import edu.mit.broad.picard.illumina.BustardFileParser; import edu.mit.broad.picard.illumina.BustardReadData; import edu.mit.broad.picard.illumina.AbstractBustardFileParser; import edu.mit.broad.picard.illumina.BustardFileParser_1_1; import net.sf.samtools.SAMFileHeader; import net.sf.samtools.SAMFileWriter; import net.sf.samtools.SAMFileWriterFactory; import net.sf.samtools.SAMRecord; import org.broadinstitute.sting.playground.illumina.FirecrestFileParser; import org.broadinstitute.sting.playground.illumina.FirecrestReadData; import org.broadinstitute.sting.utils.QualityUtils; import org.broadinstitute.sting.utils.cmdLine.CommandLineProgram; import java.io.File; import java.io.IOException; import java.io.PrintWriter; public class FourBaseRecaller extends CommandLineProgram { public static FourBaseRecaller Instance = null; public File DIR; public int LANE; public String RUN_BARCODE; public File OUT; public int END = 0; public int TRAINING_LIMIT = 1000000000; public int CALLING_LIMIT = 1000000000; public Boolean RAW = false; public Boolean OLD = false; public static void main(String[] argv) { Instance = new FourBaseRecaller(); start(Instance, argv); } protected void setupArgs() { m_parser.addRequiredArg("dir", "D", "Illumina Bustard directory", "DIR"); m_parser.addRequiredArg("lane", "L", "Illumina flowcell lane", "LANE"); m_parser.addOptionalArg("end", "E", "End of read to process (0 = whole read, i.e. unpaired; 1 = first end; 2 = second end)", "END"); m_parser.addRequiredArg("run_barcode", "B", "Illumina Run Barcode (e.g. 305PJAAXX080716)", "RUN_BARCODE"); m_parser.addRequiredArg("out", "O", "Output path for sam file", "OUT"); m_parser.addOptionalArg("tlim", "T", "Number of reads to use for parameter initialization", "TRAINING_LIMIT"); m_parser.addOptionalArg("clim", "C", "Number of reads to basecall", "CALLING_LIMIT"); m_parser.addOptionalFlag("raw", "R", "Use raw intensities?", "RAW"); m_parser.addOptionalFlag("old", "1", "Old Bustard 1.1 mode?", "OLD"); } protected int execute() { boolean isPaired = (END > 0); // Set up debugging paths File debugdir = new File(OUT.getPath() + ".debug/"); debugdir.mkdir(); PrintWriter debugout = null; try { debugout = new PrintWriter(debugdir.getPath() + "/debug.out"); } catch (IOException e) { } AbstractBustardFileParser bfp; BustardReadData bread; FirecrestFileParser ffp; FirecrestReadData fread; bfp = OLD ? new BustardFileParser_1_1(DIR, LANE, isPaired, RUN_BARCODE) : new BustardFileParser(DIR, LANE, isPaired, RUN_BARCODE); bread = bfp.next(); ffp = new FirecrestFileParser(DIR.getParentFile(), LANE); fread = ffp.next(); int cycle_offset = (END <= 1) ? 0 : bread.getIntensities().length/2; BasecallingReadModel model = new BasecallingReadModel(bread.getFirstReadSequence().length()); int queryid; // learn mean parameters System.err.println("Computing mean parameters..."); queryid = 0; do { assert(bread.getXCoordinate() == fread.getXCoordinate() && bread.getYCoordinate() == fread.getYCoordinate()); String bases = (END <= 1) ? bread.getFirstReadSequence() : bread.getSecondReadSequence(); byte[] quals = (END <= 1) ? bread.getFirstReadPhredBinaryQualities() : bread.getSecondReadPhredBinaryQualities(); double[][] intensities = bread.getIntensities(); double[][] rawintensities = fread.getIntensities(); for (int cycle = 0; cycle < bases.length(); cycle++) { char baseCur = bases.charAt(cycle); byte qualCur = quals[cycle]; double[] fourintensity = (RAW || OLD) ? rawintensities[cycle + cycle_offset] : intensities[cycle + cycle_offset]; model.addMeanPoint(cycle, baseCur, qualCur, fourintensity); } queryid++; } while (queryid < TRAINING_LIMIT && bfp.hasNext() && (bread = bfp.next()) != null && (fread = ffp.next()) != null); // learn covariance parameters System.err.println("Computing covariance parameters..."); bfp = OLD ? new BustardFileParser_1_1(DIR, LANE, isPaired, RUN_BARCODE) : new BustardFileParser(DIR, LANE, isPaired, RUN_BARCODE); bread = bfp.next(); ffp = new FirecrestFileParser(DIR.getParentFile(), LANE); fread = ffp.next(); queryid = 0; do { assert(bread.getXCoordinate() == fread.getXCoordinate() && bread.getYCoordinate() == fread.getYCoordinate()); String bases = (END <= 1) ? bread.getFirstReadSequence() : bread.getSecondReadSequence(); byte[] quals = (END <= 1) ? bread.getFirstReadPhredBinaryQualities() : bread.getSecondReadPhredBinaryQualities(); double[][] intensities = bread.getIntensities(); double[][] rawintensities = fread.getIntensities(); for (int cycle = 0; cycle < bases.length(); cycle++) { char baseCur = bases.charAt(cycle); byte qualCur = quals[cycle]; double[] fourintensity = (RAW || OLD) ? rawintensities[cycle + cycle_offset] : intensities[cycle + cycle_offset]; model.addCovariancePoint(cycle, baseCur, qualCur, fourintensity); } queryid++; } while (queryid < TRAINING_LIMIT && bfp.hasNext() && (bread = bfp.next()) != null && (fread = ffp.next()) != null); // write model parameters model.write(debugdir); // call bases System.err.println("Calling bases..."); SAMFileHeader sfh = new SAMFileHeader(); SAMFileWriter sfw = new SAMFileWriterFactory().makeSAMOrBAMWriter(sfh, false, OUT); bfp = OLD ? new BustardFileParser_1_1(DIR, LANE, isPaired, RUN_BARCODE) : new BustardFileParser(DIR, LANE, isPaired, RUN_BARCODE); bread = bfp.next(); ffp = new FirecrestFileParser(DIR.getParentFile(), LANE); fread = ffp.next(); if (debugout != null) { debugout.println("cycle int_a int_c int_g int_t bustard_base kiran_base bustard_prob kiran_prob"); } queryid = 0; do { assert(bread.getXCoordinate() == fread.getXCoordinate() && bread.getYCoordinate() == fread.getYCoordinate()); String bases = (END <= 1) ? bread.getFirstReadSequence() : bread.getSecondReadSequence(); byte[] quals = (END <= 1) ? bread.getFirstReadPhredBinaryQualities() : bread.getSecondReadPhredBinaryQualities(); double[][] intensities = bread.getIntensities(); double[][] rawintensities = fread.getIntensities(); byte[] asciiseq = new byte[bases.length()]; byte[] bestqual = new byte[bases.length()]; byte[] nextbestqual = new byte[bases.length()]; for (int cycle = 0; cycle < bases.length(); cycle++) { double[] fourintensity = (RAW || OLD) ? rawintensities[cycle + cycle_offset] : intensities[cycle + cycle_offset]; FourProb fp = model.computeProbabilities(cycle, fourintensity); asciiseq[cycle] = (byte) fp.baseAtRank(0); bestqual[cycle] = fp.qualAtRank(0); nextbestqual[cycle] = QualityUtils.baseAndProbToCompressedQuality(fp.indexAtRank(1), fp.probAtRank(1)); if (debugout != null && queryid < 5000 && bases.charAt(cycle) != '.') { debugout.println(cycle + " " + fourintensity[0] + " " + fourintensity[1] + " " + fourintensity[2] + " " + fourintensity[3] + " " + (bases.charAt(cycle)) + " " + ((char) asciiseq[cycle]) + " " + bestqual[cycle] + " " + quals[cycle]); //System.out.println(cycle + " " + fourintensity[0] + " " + fourintensity[1] + " " + fourintensity[2] + " " + fourintensity[3] + " " + (bases.charAt(cycle)) + " " + ((char) asciiseq[cycle]) + " " + bestqual[cycle] + " " + quals[cycle]); } } sfw.addAlignment(constructSAMRecord("KIR_", new String(asciiseq), bestqual, nextbestqual, bread, sfh)); sfw.addAlignment(constructSAMRecord("BUS_", bases, quals, null, bread, sfh)); queryid++; } while (queryid < CALLING_LIMIT && bfp.hasNext() && (bread = bfp.next()) != null && (fread = ffp.next()) != null); if (debugout != null) { debugout.close(); } sfw.close(); return 0; } private SAMRecord constructSAMRecord(String readNamePrefix, String bases, byte[] bestqual, byte[] nextbestqual, BustardReadData bread, SAMFileHeader sfh) { SAMRecord sr = new SAMRecord(sfh); sr.setReadName(readNamePrefix + bread.getReadName()); sr.setReadUmappedFlag(true); sr.setReadString(bases); sr.setBaseQualities(bestqual); if (nextbestqual != null) { sr.setAttribute("SQ", nextbestqual); } sr.setReadFailsVendorQualityCheckFlag(!bread.isPf()); return sr; } }