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* Generates simulated reads for variants

*

* <p>Given a set of variants, this tool will generate simulated reads that support the input variants.</p>

*

* <h3>Caveats</h3>

* <p>For practical reasons, only bi-allelic variants that are not too close to the ends of contigs (< 1/2 read length) are supported; all others will simply be ignored.</p>

*

* <h3>Input</h3>

* <p>A VCF file containing variants.</p>

*

* <h3>Output</h3>

* <p>A BAM file containing simulated sequence reads that support the input variants, with the requested error rate and coverage depth.</p>

*

* <h3>Example</h3>

* <pre>

* java -Xmx2g -jar GenomeAnalysisTK.jar \

*   -T SimulateReadsForVariants \

*   -R reference.fasta \

*   -V input_variants.vcf \

*   -o simulated_reads.bam \

*   --readDepth 50 \

*   --errorRate 25

* </pre>

*

*/

    private static Logger logger = Logger.getLogger(SimulateReadsForVariants.class);

    @ArgumentCollection protected StandardVariantContextInputArgumentCollection variantCollection = new StandardVariantContextInputArgumentCollection();

    /**

     * The simulated reads will be written to a BAM file.

     */

    @Output(doc="Reads corresponding to variants", required=true)

    protected GATKSAMFileWriter readWriter;

    /**

     * Use this argument to set the desired target read depth. See the readSamplingMode argument for options that

     * determine whether coverage distribution will be exactly this value or an approximation.

     */

    @Argument(fullName="readDepth", shortName="DP", doc="Read depth to generate", required=false, minValue = 0, minRecommendedValue = 1, maxRecommendedValue = 1000, maxValue = Integer.MAX_VALUE)

    public int readDepth = 20;

    /**

     * Errors will be generated at this rate in the simulated reads. Base qualities are therefore also assigned this value.

     */

    @Argument(fullName="errorRate", shortName="ER", doc="Base error rate (Phred-scaled)", required=false, minValue = 0, maxValue = Integer.MAX_VALUE)

    public int phredErrorRate = 20;

    /**

     * All simulated reads will be exactly this length.

     */

    @Argument(fullName="readLength", shortName="RL", doc="Read lengths (bp)", required=false, minValue = 1, maxValue = Integer.MAX_VALUE)

    public int readLength = 101;

    /**

     * Use this argument to simulate events at a non-50/50 allele fraction represented in the VCF by AF (used for somatic event simulation)

     */

    @Hidden

    @Argument(fullName="useAFAsAlleleFraction", shortName="AF", doc="Use AF in VCF as event allele fraction ", required=false)

    public boolean useAFAsAlleleFraction = false;

    /**

     * The corresponding platform identifier will be specified in the simulated read group PL tag. This setting does not

     * affect the properties of the simulated reads.

     */

    @Advanced

    @Argument(fullName="rgPlatform", shortName="RGPL", doc="Sequencing platform", required=false)

    public NGSPlatform rgPlatform = NGSPlatform.ILLUMINA;

    /**

     * This determines how read sampling is achieved, and affects the coverage distribution of simulated reads.

     * CONSTANT sampling will produce uniform depth at all positions, while POISSON sampling will produce a

     * distribution of coverages around the requested value.

     */

    @Advanced

    @Argument(fullName="readSamplingMode", shortName="RSM", doc="Sampling mode", required=false)

    public ReadSamplingMode samplingMode = ReadSamplingMode.CONSTANT;

    public enum ReadSamplingMode { CONSTANT, POISSON };

    @Hidden

    @Argument(fullName = "no_pg_tag", shortName = "npt", doc ="Discard program tags, for integration tests", required=false)

    public boolean NO_PG_TAG = false;

    @Hidden

    @Argument(fullName="verbose", shortName="verbose", doc="Verbose", required=false)

    public boolean verbose = false;

    public static final String PROGRAM_RECORD_NAME = "GATK SimulateReadsForVariants";

    // variables used to store state

    private long readNameCounter = 1;

    private int halfReadLength;

    private double errorRate;

    private byte[] readQuals;

    private SAMFileHeader header = null;

    // randomness related variables

    private static final long RANDOM_SEED = 1252863495;

    private static final Random ran = GenomeAnalysisEngine.getRandomGenerator();

    private Poisson poissonRandom = null;

    // samples and read groups

    private final Map<String, SAMReadGroupRecord> sample2RG = new HashMap<String, SAMReadGroupRecord>();

    private SAMReadGroupRecord sampleRG(String name) { return sample2RG.get(name); }

    private SAMReadGroupRecord createRG(String name) {

        SAMReadGroupRecord rg = new SAMReadGroupRecord(name);

        rg.setPlatform(rgPlatform.getDefaultPlatform());

        rg.setSample(name);

        return rg;

    }

    // class to store the bases, offset, and representative CIGAR of a haplotype

        public final byte[] bases;

        public final int offset;

        public final String cigar;

        public ArtificialHaplotype(final byte[] bases, final int offset, final String cigar) {

            this.bases = bases;

            this.offset = offset;

            this.cigar = cigar;

        }

    }

    @Override

    public void initialize() {

        // initialize sample -> read group map

        final List<SAMReadGroupRecord> sampleRGs = new ArrayList<SAMReadGroupRecord>();

        for ( final String sample : SampleUtils.getUniqueSamplesFromRods(getToolkit(), Arrays.asList(variantCollection.variants.getName())) ) {

            final SAMReadGroupRecord rg = createRG(sample);

            sampleRGs.add(rg);

            sample2RG.put(sample, rg);

        }

        // initialize BAM headers

        header = new SAMFileHeader();

        header.setSequenceDictionary(getToolkit().getReferenceDataSource().getReference().getSequenceDictionary());

        header.setSortOrder(SAMFileHeader.SortOrder.coordinate);

        header.setReadGroups(sampleRGs);

        final SAMProgramRecord programRecord = new SAMProgramRecord(PROGRAM_RECORD_NAME);

        if ( !NO_PG_TAG ) {

            final ResourceBundle headerInfo = TextFormattingUtils.loadResourceBundle("GATKText");

            programRecord.setProgramVersion(headerInfo.getString("org.broadinstitute.gatk.tools.version"));

            programRecord.setCommandLine(getToolkit().createApproximateCommandLineArgumentString(getToolkit(), this));

        }

        header.setProgramRecords(Arrays.asList(programRecord));

        readWriter.setPresorted(false);

        readWriter.writeHeader(header);

        halfReadLength = readLength / 2;

        errorRate = QualityUtils.qualToErrorProb((byte)phredErrorRate);

        readQuals = new byte[readLength];

        Arrays.fill(readQuals, (byte)phredErrorRate);

        if ( samplingMode == ReadSamplingMode.POISSON )

           poissonRandom = new Poisson(readDepth, new MersenneTwister((int)RANDOM_SEED));

    }

    public Integer map(RefMetaDataTracker tracker, ReferenceContext ref, AlignmentContext context) {

        if ( tracker == null ) // RodWalkers can make funky map calls

            return 0;

        if ( ref.getLocus().getStart() < readLength || ! BaseUtils.isRegularBase(ref.getBase()) )

            return 0;

        final VariantContext vc = tracker.getFirstValue(variantCollection.variants, context.getLocation());

        if ( vc == null || !vc.isBiallelic() )

            return 0;

        if ( !generateReadsForVariant(vc, ref, useAFAsAlleleFraction) )

            return 0;

        if ( verbose ) logger.info(String.format("Generating reads for %s", vc));

        return 1;

    }

    /**

     * Constructs an artificial haplotype given an allele and original reference context

     *

     * @param allele     the allele to model (can be reference)

     * @param refLength  the length of the reference allele

     * @param ref        the original reference context

     * @return the artificial haplotype or null if the readLength parameter is too small to hold the allele and reference

     */

    private ArtificialHaplotype constructHaplotype(final Allele allele, final int refLength, final ReferenceContext ref) {

        final byte[] haplotype = new byte[readLength];

        final int alleleLength = allele.getBases().length;

        final int halfAlleleLength = (alleleLength + 1) / 2;

        final int refContextLength = ref.getBases().length;

        // this is how far back to move from the event to start copying bases

        final int offset = halfReadLength - halfAlleleLength;

        // number of bases copied to the haplotype

        int copiedCount = 0;

        // copy bases before the event

        final int locusPosOnRefContext = (int)(ref.getLocus().getStart() - ref.getWindow().getStart());

        int posOnRefContext = locusPosOnRefContext - offset;

        if ( offset >= 0 && posOnRefContext >= 0 && posOnRefContext + offset <= refContextLength )

        {

            System.arraycopy(ref.getBases(), posOnRefContext, haplotype, 0, offset);

            copiedCount = offset;

        }

        else

        {

            String msg = new String("Can not copy reference bases to haplotype: ");

            if ( offset < 0 )

                msg += "Read length(" + readLength + ") < Allele length(" +  alleleLength + ")";

            else

                msg += "Reference position(" + posOnRefContext + ") < 0";

            logger.info(msg);

            return null;

        }

        // copy the event bases

        if ( copiedCount + alleleLength <= readLength )

        {

            System.arraycopy(allele.getBases(), 0, haplotype, copiedCount, alleleLength);

            copiedCount += alleleLength;

        }

        else

        {

            String msg = new String("Can not copy allele bases to haplotype: ");

            msg += "Read length(" + readLength + ") < Allele length(" +  alleleLength + ") +  copied count(" + copiedCount + ")";

            logger.info(msg);

            return null;

        }

        // copy bases after the event

        posOnRefContext = locusPosOnRefContext + refLength;

        final int remainder = readLength - copiedCount;

        if ( remainder > 0 && posOnRefContext + remainder <= refContextLength )

        {

            System.arraycopy(ref.getBases(), posOnRefContext, haplotype, copiedCount, remainder);

            copiedCount += remainder;

        }

        else

        {

            String msg = new String("Can not copy remaining reference bases to haplotype: ");

            msg += "Read length(" + readLength + ") <= Copied count(" +  copiedCount + ")";

            logger.info(msg);

            return null;

        }

        final String cigar;

        if ( refLength == alleleLength )

            cigar = readLength + "M";

        else

            cigar = (offset + 1) + "M" + Math.abs(refLength - alleleLength) + (refLength > alleleLength ? "D" : "I") + remainder + "M";

        return new ArtificialHaplotype(haplotype, offset, cigar);

    }

    /**

     * Generates the artificial reads for a given variant

     *

     * @param vc         the (bi-allelic) variant context for which to generate artificial reads

     * @param ref        the original reference context

     * @param useAFAsAlleleFraction use AF tag to indicate allele fraction

     * @return true if successful generation of artificial reads for the variant, false otherwise

     */

    private boolean generateReadsForVariant(final VariantContext vc, final ReferenceContext ref, final boolean useAFAsAlleleFraction) {

        final int refLength = vc.getReference().getBases().length;

      final ArtificialHaplotype refHap = constructHaplotype(vc.getReference(), refLength, ref);

      if ( refHap == null )

            return false;

      final ArtificialHaplotype altHap = constructHaplotype(vc.getAlternateAllele(0), refLength, ref);

        if ( altHap == null )

            return false;

        final double refAlleleFraction = (useAFAsAlleleFraction)?1-vc.getAttributeAsDouble(VCFConstants.ALLELE_FREQUENCY_KEY, 0.5):0.5;

        if (refAlleleFraction < 0.0 || refAlleleFraction > 1.0 || Double.isNaN(refAlleleFraction) || Double.isInfinite(refAlleleFraction) ) {

            throw new UserException.MalformedVCF("Error in AF, must be between 0 and 1 but was " + refAlleleFraction);

        }

        int gi = 0;

        for ( final Genotype g : vc.getGenotypes() ) {

            final int myDepth = sampleDepth();

            for ( int d = 0; d < myDepth; d++ ) {

                final ArtificialHaplotype haplotype = chooseRefHaplotype(g, refAlleleFraction) ? refHap : altHap;

                final byte[] readBases = Arrays.copyOf(haplotype.bases, readLength);

                addMachineErrors(readBases, errorRate);

                writeRead(readBases, vc.getChr(), vc.getStart() - haplotype.offset, haplotype.cigar, g.getSampleName(), gi++ % 2 == 0);

            }

        }

        return true;

    }

    /**

     * Decides whether or not to choose the reference haplotype, depending on the given genotype

     *

     * @param g  the genotype of the given sample

     * @param pReferenceGivenHet probability of choosing reference for hets

     *

     * @return true if one should use the reference haplotype, false otherwise

     */

    private boolean chooseRefHaplotype(final Genotype g, final double pReferenceGivenHet) {

        final double refP;

        if ( g.isHomRef() )     refP = 1;

        else if ( g.isHet() )   refP = pReferenceGivenHet;

        else                    refP = 0.0;

        return ran.nextDouble() < refP;

    }

    /**

     * Generates the artificial read depth

     *

     * @return a non-negative int

     */

    private int sampleDepth() {

        switch ( samplingMode ) {

            case CONSTANT: return readDepth;

            case POISSON: return poissonRandom.nextInt();

            default:

                throw new IllegalStateException("Unexpected DepthSamplingType " + samplingMode);

        }

    }

    /**

     * Creates and writes an artificial read given the appropriate data

     *

     * @param readBases   the bases

     * @param contig      the contig

     * @param start       the read start

     * @param cigar       the cigar string

     * @param sample      the sample name (used to get the right read group)

     * @param isNegStrand should this read be on the negative strand?

     */

    private void writeRead(final byte[] readBases, final String contig, final int start,

         final String cigar, final String sample, final boolean isNegStrand) {

        final GATKSAMRecord read = new GATKSAMRecord(header);

        read.setBaseQualities(readQuals);

        read.setReadBases(readBases);

        read.setReadName("" + readNameCounter++);

        read.setCigarString(cigar);

        read.setReadPairedFlag(false);

        read.setAlignmentStart(start);

        read.setMappingQuality(60);

        read.setReferenceName(contig);

        read.setReadNegativeStrandFlag(isNegStrand);

        read.setAttribute("RG", sampleRG(sample).getReadGroupId());

        readWriter.addAlignment(read);

    }

    /**

     * Adds machine errors at the appropriate rate to the provided read bases

     *

     * @param readBases   the read bases

     * @param errorRate   the rate at which to produce errors

     */

    private void addMachineErrors(final byte[] readBases, final double errorRate) {

        for ( int i = 0; i < readBases.length; i++ ) {

            final double r = ran.nextDouble();

            if ( r < errorRate ) {

                byte errorBase = BaseUtils.baseIndexToSimpleBase(BaseUtils.getRandomBaseIndex(BaseUtils.simpleBaseToBaseIndex(readBases[i])));

                if ( errorBase == readBases[i] ) throw new IllegalStateException("Read and error bases are the same");

                readBases[i] = errorBase;

            }

        }

    }

    @Override

    public Integer reduceInit() {

        return 0;

    }

    @Override

    public Integer reduce(Integer counter, Integer sum) {

        return counter + sum;

    }

}