/**
* Returns the coverage distribution of a single read within the desired region.
*
* <p>Note: This function counts DELETIONS as coverage (since the main purpose is to downsample
* reads for variant regions, and deletions count as variants)
*
* @param read the read to get the coverage distribution of
* @param startLocation the first reference coordinate of the region (inclusive)
* @param stopLocation the last reference coordinate of the region (inclusive)
* @return an array with the coverage of each position from startLocation to stopLocation
*/
public static int[] getCoverageDistributionOfRead(
GATKSAMRecord read, int startLocation, int stopLocation) {
int[] coverage = new int[stopLocation - startLocation + 1];
int refLocation = read.getSoftStart();
for (CigarElement cigarElement : read.getCigar().getCigarElements()) {
switch (cigarElement.getOperator()) {
case S:
case M:
case EQ:
case N:
case X:
case D:
for (int i = 0; i < cigarElement.getLength(); i++) {
if (refLocation >= startLocation && refLocation <= stopLocation) {
int baseCount =
read.isReducedRead()
? read.getReducedCount(refLocation - read.getSoftStart())
: 1;
coverage[refLocation - startLocation] +=
baseCount; // this may be a reduced read, so add the proper number of bases
}
refLocation++;
}
break;
case P:
case I:
case H:
break;
}
if (refLocation > stopLocation) break;
}
return coverage;
}
/**
* Returns the read coordinate corresponding to the requested reference coordinate.
*
* <p>WARNING: if the requested reference coordinate happens to fall inside a deletion in the
* read, this function will return the last read base before the deletion. This function returns a
* Pair(int readCoord, boolean fallsInsideDeletion) so you can choose which readCoordinate to use
* when faced with a deletion.
*
* <p>SUGGESTION: Use getReadCoordinateForReferenceCoordinate(GATKSAMRecord, int, ClippingTail)
* instead to get a pre-processed result according to normal clipping needs. Or you can use this
* function and tailor the behavior to your needs.
*
* @param read
* @param refCoord
* @return the read coordinate corresponding to the requested reference coordinate. (see warning!)
*/
@Requires({"refCoord >= read.getSoftStart()", "refCoord <= read.getSoftEnd()"})
@Ensures({"result.getFirst() >= 0", "result.getFirst() < read.getReadLength()"})
public static Pair<Integer, Boolean> getReadCoordinateForReferenceCoordinate(
GATKSAMRecord read, int refCoord) {
return getReadCoordinateForReferenceCoordinate(
read.getSoftStart(), read.getCigar(), refCoord, false);
}
public static int getMeanRepresentativeReadCount(GATKSAMRecord read) {
if (!read.isReducedRead()) return 1;
// compute mean representative read counts
final byte[] counts = read.getReducedReadCounts();
return (int) Math.round((double) MathUtils.sum(counts) / counts.length);
}
/**
* is this base inside the adaptor of the read?
*
* <p>There are two cases to treat here:
*
* <p>1) Read is in the negative strand => Adaptor boundary is on the left tail 2) Read is in the
* positive strand => Adaptor boundary is on the right tail
*
* <p>Note: We return false to all reads that are UNMAPPED or have an weird big insert size
* (probably due to mismapping or bigger event)
*
* @param read the read to test
* @param basePos base position in REFERENCE coordinates (not read coordinates)
* @return whether or not the base is in the adaptor
*/
public static boolean isBaseInsideAdaptor(final GATKSAMRecord read, long basePos) {
Integer adaptorBoundary = getAdaptorBoundary(read);
if (adaptorBoundary == null || read.getInferredInsertSize() > DEFAULT_ADAPTOR_SIZE)
return false;
return read.getReadNegativeStrandFlag()
? basePos <= adaptorBoundary
: basePos >= adaptorBoundary;
}
/**
* Pre-processes the results of getReadCoordinateForReferenceCoordinate(GATKSAMRecord, int) to
* take care of two corner cases:
*
* <p>1. If clipping the right tail (end of the read) getReadCoordinateForReferenceCoordinate and
* fall inside a deletion return the base after the deletion. If clipping the left tail (beginning
* of the read) it doesn't matter because it already returns the previous base by default.
*
* <p>2. If clipping the left tail (beginning of the read) getReadCoordinateForReferenceCoordinate
* and the read starts with an insertion, and you're requesting the first read based coordinate,
* it will skip the leading insertion (because it has the same reference coordinate as the
* following base).
*
* @param read
* @param refCoord
* @param tail
* @return the read coordinate corresponding to the requested reference coordinate for clipping.
*/
@Requires({
"refCoord >= read.getUnclippedStart()",
"refCoord <= read.getUnclippedEnd() || (read.getUnclippedEnd() < read.getUnclippedStart())"
})
@Ensures({"result >= 0", "result < read.getReadLength()"})
public static int getReadCoordinateForReferenceCoordinate(
GATKSAMRecord read, int refCoord, ClippingTail tail) {
return getReadCoordinateForReferenceCoordinate(
read.getSoftStart(), read.getCigar(), refCoord, tail, false);
}
private GATKSAMRecord makeRead(final int fragmentSize, final int mateStart) {
final byte[] bases = {'A', 'C', 'G', 'T', 'A', 'C', 'G', 'T'};
final byte[] quals = {30, 30, 30, 30, 30, 30, 30, 30};
final String cigar = "8M";
GATKSAMRecord read = ArtificialSAMUtils.createArtificialRead(bases, quals, cigar);
read.setProperPairFlag(true);
read.setReadPairedFlag(true);
read.setMateAlignmentStart(mateStart);
read.setInferredInsertSize(fragmentSize);
return read;
}
@Test(enabled = true)
public void testGetBasesReverseComplement() {
int iterations = 1000;
Random random = GenomeAnalysisEngine.getRandomGenerator();
while (iterations-- > 0) {
final int l = random.nextInt(1000);
GATKSAMRecord read = GATKSAMRecord.createRandomRead(l);
byte[] original = read.getReadBases();
byte[] reconverted = new byte[l];
String revComp = ReadUtils.getBasesReverseComplement(read);
for (int i = 0; i < l; i++) {
reconverted[l - 1 - i] = BaseUtils.getComplement((byte) revComp.charAt(i));
}
Assert.assertEquals(reconverted, original);
}
}
/**
* Creates a map with each event in the read (cigar operator) and the read coordinate where it
* happened.
*
* <p>Example: D -> 2, 34, 75 I -> 55 S -> 0, 101 H -> 101
*
* @param read the read
* @return a map with the properties described above. See example
*/
public static Map<CigarOperator, ArrayList<Integer>> getCigarOperatorForAllBases(
GATKSAMRecord read) {
Map<CigarOperator, ArrayList<Integer>> events =
new HashMap<CigarOperator, ArrayList<Integer>>();
int position = 0;
for (CigarElement cigarElement : read.getCigar().getCigarElements()) {
CigarOperator op = cigarElement.getOperator();
if (op.consumesReadBases()) {
ArrayList<Integer> list = events.get(op);
if (list == null) {
list = new ArrayList<Integer>();
events.put(op, list);
}
for (int i = position; i < cigarElement.getLength(); i++) list.add(position++);
} else {
ArrayList<Integer> list = events.get(op);
if (list == null) {
list = new ArrayList<Integer>();
events.put(op, list);
}
list.add(position);
}
}
return events;
}
@Test(enabled = true)
public void testReadWithNsRefAfterDeletion() throws FileNotFoundException {
final IndexedFastaSequenceFile seq =
new CachingIndexedFastaSequenceFile(new File(b37KGReference));
final SAMFileHeader header =
ArtificialSAMUtils.createArtificialSamHeader(seq.getSequenceDictionary());
final int readLength = 76;
final GATKSAMRecord read =
ArtificialSAMUtils.createArtificialRead(header, "myRead", 0, 8975, readLength);
read.setReadBases(Utils.dupBytes((byte) 'A', readLength));
read.setBaseQualities(Utils.dupBytes((byte) 30, readLength));
read.setCigarString("3M414N1D73M");
final int result =
ReadUtils.getReadCoordinateForReferenceCoordinateUpToEndOfRead(
read, 9393, ReadUtils.ClippingTail.LEFT_TAIL);
Assert.assertEquals(result, 3);
}
/**
* Calculates the reference coordinate for a read coordinate
*
* @param read the read
* @param offset the base in the read (coordinate in the read)
* @return the reference coordinate correspondent to this base
*/
public static long getReferenceCoordinateForReadCoordinate(GATKSAMRecord read, int offset) {
if (offset > read.getReadLength())
throw new ReviewedStingException(
String.format(OFFSET_OUT_OF_BOUNDS_EXCEPTION, offset, read.getReadLength()));
long location = read.getAlignmentStart();
Iterator<CigarElement> cigarElementIterator = read.getCigar().getCigarElements().iterator();
while (offset > 0 && cigarElementIterator.hasNext()) {
CigarElement cigarElement = cigarElementIterator.next();
long move = 0;
if (cigarElement.getOperator().consumesReferenceBases())
move = (long) Math.min(cigarElement.getLength(), offset);
location += move;
offset -= move;
}
if (offset > 0 && !cigarElementIterator.hasNext())
throw new ReviewedStingException(OFFSET_NOT_ZERO_EXCEPTION);
return location;
}
/**
* Determines what is the position of the read in relation to the interval. Note: This function
* uses the UNCLIPPED ENDS of the reads for the comparison.
*
* @param read the read
* @param interval the interval
* @return the overlap type as described by ReadAndIntervalOverlap enum (see above)
*/
public static ReadAndIntervalOverlap getReadAndIntervalOverlapType(
GATKSAMRecord read, GenomeLoc interval) {
int sStart = read.getSoftStart();
int sStop = read.getSoftEnd();
int uStart = read.getUnclippedStart();
int uStop = read.getUnclippedEnd();
if (!read.getReferenceName().equals(interval.getContig()))
return ReadAndIntervalOverlap.NO_OVERLAP_CONTIG;
else if (uStop < interval.getStart()) return ReadAndIntervalOverlap.NO_OVERLAP_LEFT;
else if (uStart > interval.getStop()) return ReadAndIntervalOverlap.NO_OVERLAP_RIGHT;
else if (sStop < interval.getStart()) return ReadAndIntervalOverlap.NO_OVERLAP_HARDCLIPPED_LEFT;
else if (sStart > interval.getStop())
return ReadAndIntervalOverlap.NO_OVERLAP_HARDCLIPPED_RIGHT;
else if ((sStart >= interval.getStart()) && (sStop <= interval.getStop()))
return ReadAndIntervalOverlap.OVERLAP_CONTAINED;
else if ((sStart < interval.getStart()) && (sStop > interval.getStop()))
return ReadAndIntervalOverlap.OVERLAP_LEFT_AND_RIGHT;
else if ((sStart < interval.getStart())) return ReadAndIntervalOverlap.OVERLAP_LEFT;
else return ReadAndIntervalOverlap.OVERLAP_RIGHT;
}
// copied from LocusViewTemplate
protected GATKSAMRecord buildSAMRecord(
final String readName, final String contig, final int alignmentStart) {
GATKSAMRecord record = new GATKSAMRecord(header);
record.setReadName(readName);
record.setReferenceIndex(dictionary.getSequenceIndex(contig));
record.setAlignmentStart(alignmentStart);
record.setCigarString("1M");
record.setReadString("A");
record.setBaseQualityString("A");
record.setReadGroup(readGroup);
return record;
}
@DataProvider(name = "HasWellDefinedFragmentSizeData")
public Object[][] makeHasWellDefinedFragmentSizeData() throws Exception {
final List<Object[]> tests = new LinkedList<Object[]>();
// setup a basic read that will work
final SAMFileHeader header = ArtificialSAMUtils.createArtificialSamHeader();
final GATKSAMRecord read = ArtificialSAMUtils.createArtificialRead(header, "read1", 0, 10, 10);
read.setReadPairedFlag(true);
read.setProperPairFlag(true);
read.setReadUnmappedFlag(false);
read.setMateUnmappedFlag(false);
read.setAlignmentStart(100);
read.setCigarString("50M");
read.setMateAlignmentStart(130);
read.setInferredInsertSize(80);
read.setFirstOfPairFlag(true);
read.setReadNegativeStrandFlag(false);
read.setMateNegativeStrandFlag(true);
tests.add(new Object[] {"basic case", read.clone(), true});
{
final GATKSAMRecord bad1 = (GATKSAMRecord) read.clone();
bad1.setReadPairedFlag(false);
tests.add(new Object[] {"not paired", bad1, false});
}
{
final GATKSAMRecord bad = (GATKSAMRecord) read.clone();
bad.setProperPairFlag(false);
// we currently don't require the proper pair flag to be set
tests.add(new Object[] {"not proper pair", bad, true});
// tests.add( new Object[]{ "not proper pair", bad, false });
}
{
final GATKSAMRecord bad = (GATKSAMRecord) read.clone();
bad.setReadUnmappedFlag(true);
tests.add(new Object[] {"read is unmapped", bad, false});
}
{
final GATKSAMRecord bad = (GATKSAMRecord) read.clone();
bad.setMateUnmappedFlag(true);
tests.add(new Object[] {"mate is unmapped", bad, false});
}
{
final GATKSAMRecord bad = (GATKSAMRecord) read.clone();
bad.setMateNegativeStrandFlag(false);
tests.add(new Object[] {"read and mate both on positive strand", bad, false});
}
{
final GATKSAMRecord bad = (GATKSAMRecord) read.clone();
bad.setReadNegativeStrandFlag(true);
tests.add(new Object[] {"read and mate both on negative strand", bad, false});
}
{
final GATKSAMRecord bad = (GATKSAMRecord) read.clone();
bad.setInferredInsertSize(0);
tests.add(new Object[] {"insert size is 0", bad, false});
}
{
final GATKSAMRecord bad = (GATKSAMRecord) read.clone();
bad.setAlignmentStart(1000);
tests.add(new Object[] {"positve read starts after mate end", bad, false});
}
{
final GATKSAMRecord bad = (GATKSAMRecord) read.clone();
bad.setReadNegativeStrandFlag(true);
bad.setMateNegativeStrandFlag(false);
bad.setMateAlignmentStart(1000);
tests.add(new Object[] {"negative strand read ends before mate starts", bad, false});
}
return tests.toArray(new Object[][] {});
}
/**
* Is this read all insertion?
*
* @param read
* @return whether or not the only element in the cigar string is an Insertion
*/
public static boolean readIsEntirelyInsertion(GATKSAMRecord read) {
for (CigarElement cigarElement : read.getCigar().getCigarElements()) {
if (cigarElement.getOperator() != CigarOperator.INSERTION) return false;
}
return true;
}
/**
* Is a base inside a read?
*
* @param read the read to evaluate
* @param referenceCoordinate the reference coordinate of the base to test
* @return true if it is inside the read, false otherwise.
*/
public static boolean isInsideRead(final GATKSAMRecord read, final int referenceCoordinate) {
return referenceCoordinate >= read.getAlignmentStart()
&& referenceCoordinate <= read.getAlignmentEnd();
}
@Test(dataProvider = "AdaptorGetter")
public void testGetAdaptorBoundary(final GetAdaptorFunc get) {
final int fragmentSize = 10;
final int mateStart = 1000;
final int BEFORE = mateStart - 2;
final int AFTER = mateStart + 2;
int myStart, boundary;
GATKSAMRecord read;
// Test case 1: positive strand, first read
read = makeRead(fragmentSize, mateStart);
myStart = BEFORE;
read.setAlignmentStart(myStart);
read.setReadNegativeStrandFlag(false);
read.setMateNegativeStrandFlag(true);
boundary = get.getAdaptor(read);
Assert.assertEquals(boundary, myStart + fragmentSize + 1);
// Test case 2: positive strand, second read
read = makeRead(fragmentSize, mateStart);
myStart = AFTER;
read.setAlignmentStart(myStart);
read.setReadNegativeStrandFlag(false);
read.setMateNegativeStrandFlag(true);
boundary = get.getAdaptor(read);
Assert.assertEquals(boundary, myStart + fragmentSize + 1);
// Test case 3: negative strand, second read
read = makeRead(fragmentSize, mateStart);
myStart = AFTER;
read.setAlignmentStart(myStart);
read.setReadNegativeStrandFlag(true);
read.setMateNegativeStrandFlag(false);
boundary = get.getAdaptor(read);
Assert.assertEquals(boundary, mateStart - 1);
// Test case 4: negative strand, first read
read = makeRead(fragmentSize, mateStart);
myStart = BEFORE;
read.setAlignmentStart(myStart);
read.setReadNegativeStrandFlag(true);
read.setMateNegativeStrandFlag(false);
boundary = get.getAdaptor(read);
Assert.assertEquals(boundary, mateStart - 1);
// Test case 5: mate is mapped to another chromosome (test both strands)
read = makeRead(fragmentSize, mateStart);
read.setInferredInsertSize(0);
read.setReadNegativeStrandFlag(true);
read.setMateNegativeStrandFlag(false);
boundary = get.getAdaptor(read);
Assert.assertEquals(boundary, ReadUtils.CANNOT_COMPUTE_ADAPTOR_BOUNDARY);
read.setReadNegativeStrandFlag(false);
read.setMateNegativeStrandFlag(true);
boundary = get.getAdaptor(read);
Assert.assertEquals(boundary, ReadUtils.CANNOT_COMPUTE_ADAPTOR_BOUNDARY);
read.setInferredInsertSize(10);
// Test case 6: read is unmapped
read = makeRead(fragmentSize, mateStart);
read.setReadUnmappedFlag(true);
boundary = get.getAdaptor(read);
Assert.assertEquals(boundary, ReadUtils.CANNOT_COMPUTE_ADAPTOR_BOUNDARY);
read.setReadUnmappedFlag(false);
// Test case 7: reads don't overlap and look like this:
// <--------|
// |------>
// first read:
read = makeRead(fragmentSize, mateStart);
myStart = 980;
read.setAlignmentStart(myStart);
read.setInferredInsertSize(20);
read.setReadNegativeStrandFlag(true);
boundary = get.getAdaptor(read);
Assert.assertEquals(boundary, ReadUtils.CANNOT_COMPUTE_ADAPTOR_BOUNDARY);
// second read:
read = makeRead(fragmentSize, mateStart);
myStart = 1000;
read.setAlignmentStart(myStart);
read.setInferredInsertSize(20);
read.setMateAlignmentStart(980);
read.setReadNegativeStrandFlag(false);
boundary = get.getAdaptor(read);
Assert.assertEquals(boundary, ReadUtils.CANNOT_COMPUTE_ADAPTOR_BOUNDARY);
// Test case 8: read doesn't have proper pair flag set
read = makeRead(fragmentSize, mateStart);
read.setReadPairedFlag(true);
read.setProperPairFlag(false);
Assert.assertEquals(get.getAdaptor(read), ReadUtils.CANNOT_COMPUTE_ADAPTOR_BOUNDARY);
// Test case 9: read and mate have same negative flag setting
for (final boolean negFlag : Arrays.asList(true, false)) {
read = makeRead(fragmentSize, mateStart);
read.setAlignmentStart(BEFORE);
read.setReadPairedFlag(true);
read.setProperPairFlag(true);
read.setReadNegativeStrandFlag(negFlag);
read.setMateNegativeStrandFlag(!negFlag);
Assert.assertTrue(
get.getAdaptor(read) != ReadUtils.CANNOT_COMPUTE_ADAPTOR_BOUNDARY,
"Get adaptor should have succeeded");
read = makeRead(fragmentSize, mateStart);
read.setAlignmentStart(BEFORE);
read.setReadPairedFlag(true);
read.setProperPairFlag(true);
read.setReadNegativeStrandFlag(negFlag);
read.setMateNegativeStrandFlag(negFlag);
Assert.assertEquals(
get.getAdaptor(read),
ReadUtils.CANNOT_COMPUTE_ADAPTOR_BOUNDARY,
"Get adaptor should have failed for reads with bad alignment orientation");
}
}
/**
* Checks if a read starts with an insertion. It looks beyond Hard and Soft clips if there are
* any.
*
* @param read
* @return A pair with the answer (true/false) and the element or null if it doesn't exist
*/
public static Pair<Boolean, CigarElement> readStartsWithInsertion(GATKSAMRecord read) {
return readStartsWithInsertion(read.getCigar());
}