public class Align {
private static final int CORRECT_BASE_QUALITY_PENALTY = 20; // TODO: should be a parameter to SRMA
private static final List<String> saveTags = Arrays.asList("RG", "LB", "PU", "PG", "CS", "CQ");
public static void align(
Graph graph,
SAMRecord rec,
Node recNode,
ReferenceSequence sequence,
SAMProgramRecord programRecord,
int offset,
AlleleCoverageCutoffs alleleCoverageCutoffs,
boolean correctBases,
boolean useSequenceQualities,
int MAXIMUM_TOTAL_COVERAGE,
int MAX_HEAP_SIZE)
throws Exception {
int i;
AlignHeapNode curAlignHeapNode = null;
AlignHeapNode nextAlignHeapNode = null;
AlignHeapNode bestAlignHeapNode = null;
AlignHeap heap = null;
String read = null; // could be cs
String readBases = null; // always nt
String qualities = null; // could be cq
SRMAUtil.Space space = SRMAUtil.Space.NTSPACE;
ListIterator<NodeRecord> iter = null;
AlignHeapNodeComparator comp = null;
int alignmentStart = -1;
int numStartNodesAdded = 0;
boolean strand = rec.getReadNegativeStrandFlag(); // false -> forward, true -> reverse
String softClipStartBases = null;
String softClipStartQualities = null;
String softClipEndBases = null;
String softClipEndQualities = null;
// Debugging stuff
String readName = rec.getReadName();
assert SRMAUtil.Space.COLORSPACE != space;
// Get space
read = (String) rec.getAttribute("CS");
if (null == read) {
// Use base space
space = SRMAUtil.Space.NTSPACE;
} else {
// assumes CS and CQ are always in sequencing order
space = SRMAUtil.Space.COLORSPACE;
}
// Get read and qualities
if (space == SRMAUtil.Space.NTSPACE) {
byte tmpRead[] = rec.getReadString().getBytes();
byte tmpQualities[] = rec.getBaseQualityString().getBytes();
// Reverse once
if (strand) { // reverse
SAMRecordUtil.reverseArray(tmpRead);
SAMRecordUtil.reverseArray(tmpQualities);
}
read = new String(tmpRead);
readBases = new String(tmpRead);
qualities = new String(tmpQualities);
// Reverse again
if (strand) { // reverse
SAMRecordUtil.reverseArray(tmpRead);
SAMRecordUtil.reverseArray(tmpQualities);
}
} else {
byte tmpRead[] = rec.getReadString().getBytes();
// Reverse once
if (strand) { // reverse
SAMRecordUtil.reverseArray(tmpRead);
}
readBases = new String(tmpRead);
// Reverse again
if (strand) { // reverse
SAMRecordUtil.reverseArray(tmpRead);
}
read = SRMAUtil.normalizeColorSpaceRead(read);
qualities = (String) rec.getAttribute("CQ");
// Some aligners include a quality value for the adapter. A quality value
// IMHO should not be given for an unobserved (assumed) peice of data. Trim
// the first quality in this case
if (qualities.length() == 1 + read.length()) { // trim the first quality
qualities = qualities.substring(1);
}
}
// Reverse back
if (readBases.length() <= 0) {
throw new Exception("Error. The current alignment has no bases.");
}
if (read.length() <= 0) {
throw new Exception("Error. The current alignment has no bases.");
}
if (qualities.length() <= 0) {
throw new Exception("Error. The current alignment has no qualities.");
}
if (readBases.length() != read.length()) {
if (space == SRMAUtil.Space.COLORSPACE) {
throw new Exception(
"Error. The current alignment's read bases length does not match the length of the colors in the CS tag ["
+ rec.getReadName()
+ "].");
} else {
throw new Exception("Error. Internal error: readBases.length() != read.length()");
}
}
// Deal with soft-clipping
// - save the soft clipped sequence for latter
{
List<CigarElement> cigarElements = null;
cigarElements = rec.getCigar().getCigarElements();
CigarElement e1 = cigarElements.get(0); // first
CigarElement e2 = cigarElements.get(cigarElements.size() - 1); // last
// Soft-clipped
if (CigarOperator.S == e1.getOperator()) {
if (space == SRMAUtil.Space.COLORSPACE) {
throw new Exception(
"Error. Soft clipping with color-space data not currently supported.");
}
int l = e1.getLength();
if (strand) { // reverse
softClipStartBases = readBases.substring(readBases.length() - l);
softClipStartQualities = qualities.substring(qualities.length() - l);
readBases = readBases.substring(0, readBases.length() - l);
read = read.substring(0, read.length() - l);
qualities = qualities.substring(0, qualities.length() - l);
} else {
softClipStartBases = readBases.substring(0, l - 1);
softClipStartQualities = qualities.substring(0, l - 1);
readBases = readBases.substring(l);
read = read.substring(l);
qualities = qualities.substring(l);
}
}
if (CigarOperator.S == e2.getOperator()) {
if (space == SRMAUtil.Space.COLORSPACE) {
throw new Exception(
"Error. Soft clipping with color-space data not currently supported.");
}
int l = e2.getLength();
if (strand) { // reverse
softClipEndBases = readBases.substring(0, l - 1);
softClipEndQualities = qualities.substring(0, l - 1);
readBases = readBases.substring(l);
read = read.substring(l);
qualities = qualities.substring(l);
} else {
softClipEndBases = readBases.substring(readBases.length() - l);
softClipEndQualities = qualities.substring(qualities.length() - l);
readBases = readBases.substring(0, readBases.length() - l);
read = read.substring(0, read.length() - l);
qualities = qualities.substring(0, qualities.length() - l);
}
}
}
// Remove mate pair information
Align.removeMateInfo(rec);
comp =
new AlignHeapNodeComparator(
(strand) ? AlignHeap.HeapType.MAXHEAP : AlignHeap.HeapType.MINHEAP);
// Bound by original alignment if possible
bestAlignHeapNode =
Align.boundWithOriginalAlignment(
rec,
graph,
recNode,
comp,
strand,
read,
qualities,
readBases,
space,
sequence,
alleleCoverageCutoffs,
useSequenceQualities,
MAXIMUM_TOTAL_COVERAGE,
MAX_HEAP_SIZE);
/*
System.err.println("readName="+rec.getReadName());
if(null != bestAlignHeapNode) {
System.err.println("\nFOUND BEST:" + rec.toString());
}
else {
System.err.println("\nNOT FOUND (BEST): " + rec.toString());
}
Align.updateSAM(rec, programRecord, bestAlignHeapNode, space, read, qualities, softClipStartBases, softClipStartQualities, softClipEndBases, softClipEndQualities, strand, correctBases);
return;
*/
heap = new AlignHeap((strand) ? AlignHeap.HeapType.MAXHEAP : AlignHeap.HeapType.MINHEAP);
// Add start nodes
if (strand) { // reverse
alignmentStart = rec.getAlignmentEnd();
for (i = alignmentStart + offset; alignmentStart - offset <= i; i--) {
int position = graph.getPriorityQueueIndexAtPositionOrBefore(i);
PriorityQueue<Node> startNodeQueue = graph.getPriorityQueue(position);
if (0 != position && null != startNodeQueue) {
Iterator<Node> startNodeQueueIter = startNodeQueue.iterator();
while (startNodeQueueIter.hasNext()) {
Node startNode = startNodeQueueIter.next();
int f = passFilters(graph, startNode, alleleCoverageCutoffs, MAXIMUM_TOTAL_COVERAGE);
if (0 == f) {
heap.add(
new AlignHeapNode(
null,
startNode,
startNode.coverage,
read.charAt(0),
qualities.charAt(0),
useSequenceQualities,
space));
} else if (f < 0) {
return;
}
if (startNode.position < i) {
i = startNode.position;
}
numStartNodesAdded++;
}
}
}
} else {
alignmentStart = rec.getAlignmentStart();
for (i = alignmentStart - offset; i <= alignmentStart + offset; i++) {
int position = graph.getPriorityQueueIndexAtPositionOrGreater(i);
PriorityQueue<Node> startNodeQueue = graph.getPriorityQueue(position);
if (0 != position && null != startNodeQueue) {
Iterator<Node> startNodeQueueIter = startNodeQueue.iterator();
while (startNodeQueueIter.hasNext()) {
Node startNode = startNodeQueueIter.next();
int f = passFilters(graph, startNode, alleleCoverageCutoffs, MAXIMUM_TOTAL_COVERAGE);
if (0 == f) {
heap.add(
new AlignHeapNode(
null,
startNode,
startNode.coverage,
read.charAt(0),
qualities.charAt(0),
useSequenceQualities,
space));
} else if (f < 0) {
return;
}
if (i < startNode.position) {
i = startNode.position;
}
numStartNodesAdded++;
}
}
}
}
if (numStartNodesAdded == 0) {
throw new Exception("Did not add any start nodes!");
}
// Get first node off the heap
curAlignHeapNode = heap.poll();
while (null != curAlignHeapNode) {
if (MAX_HEAP_SIZE <= heap.size()) {
// too many to consider
return;
}
// System.err.println("strand:" + strand + "\tsize:" + heap.size() + "\talignmentStart:" +
// alignmentStart + "\toffset:" + offset + "\treadOffset:" + curAlignHeapNode.readOffset);
// System.err.print("size:" + heap.size() + ":" + curAlignHeapNode.readOffset + ":" +
// curAlignHeapNode.score + ":" + curAlignHeapNode.alleleCoverageSum + ":" +
// curAlignHeapNode.startPosition + "\t");
// curAlignHeapNode.node.print(System.err);
// System.err.print("\rposition:" + curAlignHeapNode.node.position + "\treadOffset:" +
// curAlignHeapNode.readOffset);
// Remove all non-insertions with the same contig/pos/read-offset/type/base and lower score
nextAlignHeapNode = heap.peek();
while (Node.INSERTION != curAlignHeapNode.node.type
&& null != nextAlignHeapNode
&& 0 == comp.compare(curAlignHeapNode, nextAlignHeapNode)) {
if (curAlignHeapNode.score < nextAlignHeapNode.score
|| (curAlignHeapNode.score == nextAlignHeapNode.score
&& curAlignHeapNode.alleleCoverageSum < nextAlignHeapNode.alleleCoverageSum)) {
// Update current node
curAlignHeapNode = heap.poll();
} else {
// Ignore next node
heap.poll();
}
nextAlignHeapNode = heap.peek();
}
nextAlignHeapNode = null;
// Check if the alignment is complete
if (curAlignHeapNode.readOffset == read.length() - 1) {
// All read bases examined, store if has the best alignment.
// System.err.print(curAlignHeapNode.alleleCoverageSum + ":" + curAlignHeapNode.score +
// ":");
// System.err.print(curAlignHeapNode.startPosition + ":");
// curAlignHeapNode.node.print(System.err);
if (null == bestAlignHeapNode
|| bestAlignHeapNode.score < curAlignHeapNode.score
|| (bestAlignHeapNode.score == curAlignHeapNode.score
&& bestAlignHeapNode.alleleCoverageSum < curAlignHeapNode.alleleCoverageSum)) {
bestAlignHeapNode = curAlignHeapNode;
}
} else if (null != bestAlignHeapNode && curAlignHeapNode.score < bestAlignHeapNode.score) {
// ignore, under the assumption that scores can only become more negative.
} else {
if (strand) { // reverse
// Go to all the "prev" nodes
iter = curAlignHeapNode.node.prev.listIterator();
} else { // forward
// Go to all "next" nodes
iter = curAlignHeapNode.node.next.listIterator();
}
while (iter.hasNext()) {
NodeRecord next = iter.next();
int f =
passFilters(
graph, next.node, next.coverage, alleleCoverageCutoffs, MAXIMUM_TOTAL_COVERAGE);
if (0 == f) {
heap.add(
new AlignHeapNode(
curAlignHeapNode,
next.node,
next.coverage,
read.charAt(curAlignHeapNode.readOffset + 1),
qualities.charAt(curAlignHeapNode.readOffset + 1),
useSequenceQualities,
space));
} else if (f < 0) {
return;
}
}
iter = null;
}
// Get next node
curAlignHeapNode = heap.poll();
}
// Recover alignment
Align.updateSAM(
rec,
sequence,
programRecord,
bestAlignHeapNode,
space,
read,
qualities,
softClipStartBases,
softClipStartQualities,
softClipEndBases,
softClipEndQualities,
strand,
correctBases);
}
private static void removeMateInfo(SAMRecord rec) {
if (rec.getReadPairedFlag()) {
// Remove all information of its mate
// flag
rec.setProperPairFlag(false); // not paired any more
rec.setMateUnmappedFlag(false);
rec.setMateNegativeStrandFlag(false);
// entries
rec.setMateReferenceIndex(SAMRecord.NO_ALIGNMENT_REFERENCE_INDEX);
rec.setMateAlignmentStart(0);
rec.setInferredInsertSize(0);
// TODO: remove tags and values that are mate pair inclined.
}
}
private static AlignHeapNode boundWithOriginalAlignment(
SAMRecord rec,
Graph graph,
Node recNode,
AlignHeapNodeComparator comp,
boolean strand,
String read, // could be cs
String qualities, // could be cq
String readBases, // always nt
SRMAUtil.Space space,
ReferenceSequence sequence,
AlleleCoverageCutoffs alleleCoverageCutoffs,
boolean useSequenceQualities,
int MAXIMUM_TOTAL_COVERAGE,
int MAX_HEAP_SIZE)
throws Exception {
AlignHeapNode curAlignHeapNode = null;
AlignHeapNode nextAlignHeapNode = null;
AlignHeapNode bestAlignHeapNode = null;
ListIterator<NodeRecord> iter = null;
AlignHeap heap = null;
// Cannot bound
if (0 != passFilters(graph, recNode, alleleCoverageCutoffs, MAXIMUM_TOTAL_COVERAGE)) {
return null;
}
// Initialize heap
if (strand) { // reverse
heap = new AlignHeap(AlignHeap.HeapType.MAXHEAP);
} else { // forward
heap = new AlignHeap(AlignHeap.HeapType.MINHEAP);
}
// Add start nodes
heap.add(
new AlignHeapNode(
null,
recNode,
recNode.coverage,
read.charAt(0),
qualities.charAt(0),
useSequenceQualities,
space));
curAlignHeapNode = heap.poll();
while (null != curAlignHeapNode) {
if (MAX_HEAP_SIZE <= heap.size()) {
// too many to consider
return null;
}
// Remove all non-insertions with the same contig/pos/read-offset/type/base and lower score
nextAlignHeapNode = heap.peek();
while (Node.INSERTION != curAlignHeapNode.node.type
&& null != nextAlignHeapNode
&& 0 == comp.compare(curAlignHeapNode, nextAlignHeapNode)) {
if (curAlignHeapNode.score < nextAlignHeapNode.score
|| (curAlignHeapNode.score == nextAlignHeapNode.score
&& curAlignHeapNode.alleleCoverageSum < nextAlignHeapNode.alleleCoverageSum)) {
// Update current node
curAlignHeapNode = heap.poll();
} else {
// Ignore next node
heap.poll();
}
nextAlignHeapNode = heap.peek();
}
nextAlignHeapNode = null;
if (curAlignHeapNode.readOffset == readBases.length() - 1) { // found, keep beset
if (null == bestAlignHeapNode
|| bestAlignHeapNode.score < curAlignHeapNode.score
|| (bestAlignHeapNode.score == curAlignHeapNode.score
&& bestAlignHeapNode.alleleCoverageSum < curAlignHeapNode.alleleCoverageSum)) {
bestAlignHeapNode = curAlignHeapNode;
}
} else {
if (strand) { // reverse
// Go to all the "prev" nodes
iter = curAlignHeapNode.node.prev.listIterator();
} else { // forward
// Go to all "next" nodes
iter = curAlignHeapNode.node.next.listIterator();
}
// Get the expected next position in the alignment
while (iter.hasNext()) {
NodeRecord next = iter.next();
// Base should match alignment
if (next.node.base == readBases.charAt(curAlignHeapNode.readOffset + 1)) {
int f =
passFilters(
graph, next.node, next.coverage, alleleCoverageCutoffs, MAXIMUM_TOTAL_COVERAGE);
if (0 == f) {
heap.add(
new AlignHeapNode(
curAlignHeapNode,
next.node,
next.coverage,
read.charAt(curAlignHeapNode.readOffset + 1),
qualities.charAt(curAlignHeapNode.readOffset + 1),
useSequenceQualities,
space));
} else if (f < 0) {
return null;
}
}
}
iter = null;
}
// Get next
curAlignHeapNode = heap.poll();
}
return bestAlignHeapNode;
}
private static byte getColorQuality(byte e1, byte e2, byte q1, byte q2) {
int val;
if (e1 == (byte) Alignment.GAP && e2 == (byte) Alignment.GAP) {
val = q1 + q2 + 10;
} else if (e1 == (byte) Alignment.GAP) {
val = q1 - q2;
} else if (e2 == (byte) Alignment.GAP) {
val = q2 - q1;
} else {
val = 1;
}
if (val <= 0) {
val = 1;
} else if (63 < val) {
val = 63;
}
return (byte) val;
}
private static void updateSAM(
SAMRecord rec,
ReferenceSequence sequence,
SAMProgramRecord programRecord,
AlignHeapNode bestAlignHeapNode,
SRMAUtil.Space space,
String read,
String qualities,
String softClipStartBases,
String softClipStartQualities,
String softClipEndBases,
String softClipEndQualities,
boolean strand,
boolean correctBases)
throws Exception {
AlignHeapNode curAlignHeapNode = null;
AlignHeapNode prevAlignHeapNode = null;
int alignmentStart = 0;
int readIndex = -1;
byte readBases[] = null;
byte baseQualities[] = null;
byte colorErrors[] = null;
int i;
int numEdits = 0;
List<String> optFieldTags = new LinkedList<String>();
List<Object> optFieldValues = new LinkedList<Object>();
Object attr;
// Debugging stuff
String readName = rec.getReadName();
if (null == bestAlignHeapNode) {
// Do not modify the alignment
return;
}
// To generate a new CIGAR
List<CigarElement> cigarElements = null;
CigarOperator prevCigarOperator = null, curCigarOperator = null;
int prevCigarOperatorLength = 0;
// TODO
// setInferredInsertSize (invalidates paired end reads)
// setMappingQuality (?)
// setFlag
// update base qualities for color space reads
// clear attributes, but save some
Align.clearAttributes(rec, optFieldTags, optFieldValues);
readBases = new byte[read.length()];
baseQualities = new byte[qualities.length()];
for (i = 0; i < qualities.length(); i++) {
// Must subtract 33 for PHRED scaling
baseQualities[i] = (byte) (qualities.charAt(i) - 33);
}
if (strand) {
readIndex = 0;
} else {
readIndex = read.length() - 1;
}
cigarElements = new LinkedList<CigarElement>();
if (strand) { // reverse strand is the current position
alignmentStart = bestAlignHeapNode.node.position;
} else {
alignmentStart = bestAlignHeapNode.startPosition;
}
assert null != bestAlignHeapNode;
curAlignHeapNode = bestAlignHeapNode;
while (null != curAlignHeapNode) {
// Get the current cigar operator
if (null != prevAlignHeapNode
&& CigarOperator.DELETION != prevCigarOperator
&& 1 < Math.abs(curAlignHeapNode.node.position - prevAlignHeapNode.node.position)) {
curCigarOperator = CigarOperator.DELETION;
} else {
switch (curAlignHeapNode.node.type) {
case Node.MISMATCH: // Fall through
case Node.MATCH:
curCigarOperator = CigarOperator.MATCH_OR_MISMATCH;
break;
case Node.INSERTION:
// System.out.println("INS");
curCigarOperator = CigarOperator.INSERTION;
break;
default:
throw new Exception("Unknown node type");
}
if (space == SRMAUtil.Space.COLORSPACE || correctBases) {
readBases[readIndex] = (byte) curAlignHeapNode.node.base;
if (strand) {
readIndex++;
} else {
readIndex--;
}
// count the number of mismatches
switch (curAlignHeapNode.node.type) {
case Node.MISMATCH:
case Node.INSERTION:
numEdits++;
break;
default:
break;
}
} else {
// count the number of mismatches
switch (curAlignHeapNode.node.type) {
case Node.MATCH:
if (read.charAt(curAlignHeapNode.readOffset) != curAlignHeapNode.node.base) {
numEdits++;
}
break;
case Node.MISMATCH: // Fall through
if (read.charAt(curAlignHeapNode.readOffset)
!= sequence.getBases()[curAlignHeapNode.node.position - 1]) {
numEdits++;
}
break;
case Node.INSERTION:
numEdits++;
break;
default:
break;
}
}
}
if (prevCigarOperator != curCigarOperator) {
// different cigar operator
// add the previous cigar operator
if (null != prevCigarOperator) {
if (strand) { // reverse
// append
cigarElements.add(new CigarElement(prevCigarOperatorLength, prevCigarOperator));
} else {
// prepend
cigarElements.add(0, new CigarElement(prevCigarOperatorLength, prevCigarOperator));
}
}
// update prevCigarOperator
prevCigarOperator = curCigarOperator;
if (curCigarOperator == CigarOperator.DELETION) {
// length of deletion
prevCigarOperatorLength =
Math.abs(curAlignHeapNode.node.position - prevAlignHeapNode.node.position) - 1;
numEdits += prevCigarOperatorLength; // deletions
} else {
prevCigarOperatorLength = 1;
}
} else {
// same cigar operator
prevCigarOperatorLength++;
}
// Update
if (CigarOperator.DELETION != curCigarOperator) {
prevAlignHeapNode = curAlignHeapNode;
curAlignHeapNode = curAlignHeapNode.prev;
}
}
if (0 < prevCigarOperatorLength) {
if (null == prevCigarOperator || CigarOperator.DELETION == prevCigarOperator) {
throw new Exception("Ended with a null cigar operator or a deletion cigar operator");
}
if (strand) { // reverse
// append
cigarElements.add(new CigarElement(prevCigarOperatorLength, prevCigarOperator));
} else {
// prepend
cigarElements.add(0, new CigarElement(prevCigarOperatorLength, prevCigarOperator));
}
}
if (space == SRMAUtil.Space.COLORSPACE) { // color space, read bases already inferred
// Get color error string
colorErrors = new byte[read.length()];
char prevBase = SRMAUtil.COLORSPACE_ADAPTOR;
if (strand) { // reverse
for (i = 0; i < read.length(); i++) {
char nextBase = SRMAUtil.colorSpaceNextBase(prevBase, read.charAt(i));
if (nextBase == SRMAUtil.getCompliment((char) readBases[read.length() - i - 1])) {
colorErrors[i] = (byte) Alignment.GAP;
} else {
colorErrors[i] = (byte) read.charAt(i);
}
if (0 < i) {
// qualities are assumed to be always in the same direction as the color errors
baseQualities[read.length() - i] =
getColorQuality(
colorErrors[i - 1],
colorErrors[i],
(byte) (qualities.charAt(i - 1) - 33),
(byte) (qualities.charAt(i) - 33));
}
prevBase = SRMAUtil.getCompliment((char) readBases[read.length() - i - 1]);
}
// last color
baseQualities[0] = (byte) (qualities.charAt(read.length() - 1) - 33);
} else {
for (i = 0; i < read.length(); i++) {
char nextBase = SRMAUtil.colorSpaceNextBase(prevBase, read.charAt(i));
if (nextBase == readBases[i]) {
colorErrors[i] = (byte) Alignment.GAP;
} else {
colorErrors[i] = (byte) read.charAt(i);
}
if (0 < i) {
baseQualities[i - 1] =
getColorQuality(
colorErrors[i - 1],
colorErrors[i],
(byte) (qualities.charAt(i - 1) - 33),
(byte) (qualities.charAt(i) - 33));
}
prevBase = (char) readBases[i];
}
// last color
baseQualities[read.length() - 1] = (byte) (qualities.charAt(read.length() - 1) - 33);
}
} else if (correctBases) { // bases were corrected
if (strand) {
for (i = 0; i < read.length(); i++) {
if (readBases[i] == (byte) read.charAt(read.length() - i - 1)) {
baseQualities[i] = (byte) (qualities.charAt(read.length() - i - 1) - 33);
} else {
// TODO: how much to down-weight ?
baseQualities[i] =
(byte)
(SRMAUtil.QUAL2CHAR(
SRMAUtil.CHAR2QUAL(qualities.charAt(read.length() - i - 1))
- CORRECT_BASE_QUALITY_PENALTY)
- 33);
if (baseQualities[i] <= 0) {
baseQualities[i] = 1;
}
}
}
} else {
for (i = 0; i < read.length(); i++) {
if (readBases[i] == (byte) read.charAt(i)) {
baseQualities[i] = (byte) (qualities.charAt(i) - 33);
} else {
// TODO: how much to down-weight ?
baseQualities[i] =
(byte)
(SRMAUtil.QUAL2CHAR(
SRMAUtil.CHAR2QUAL(qualities.charAt(i)) - CORRECT_BASE_QUALITY_PENALTY)
- 33);
if (baseQualities[i] <= 0) {
baseQualities[i] = 1;
}
}
}
}
rec.setAttribute("XO", read);
rec.setAttribute("XQ", qualities);
} else { // bases not corrected
readBases = new byte[read.length()];
baseQualities = new byte[qualities.length()]; // qualities.length() == read.length()
if (strand) { // reverse
for (i = 0; i < read.length(); i++) {
readBases[i] = (byte) read.charAt(read.length() - i - 1);
baseQualities[i] = (byte) (qualities.charAt(read.length() - i - 1) - 33);
}
} else {
for (i = 0; i < read.length(); i++) {
readBases[i] = (byte) read.charAt(i);
baseQualities[i] = (byte) (qualities.charAt(i) - 33);
}
}
}
// Add in soft-clipping
if (null != softClipStartBases) { // prepend
cigarElements.add(0, new CigarElement(softClipStartBases.length(), CigarOperator.S));
byte tmpBases[] = new byte[readBases.length + softClipStartBases.length()];
System.arraycopy(readBases, 0, tmpBases, softClipStartBases.length(), readBases.length);
readBases = tmpBases;
for (i = 0; i < softClipStartBases.length(); i++) {
readBases[i] = (byte) softClipStartBases.charAt(i);
}
byte tmpQualities[] = new byte[baseQualities.length + softClipStartQualities.length()];
System.arraycopy(
baseQualities, 0, tmpQualities, softClipStartQualities.length(), baseQualities.length);
baseQualities = tmpQualities;
for (i = 0; i < softClipStartQualities.length(); i++) {
baseQualities[i] = (byte) softClipStartQualities.charAt(i);
}
}
if (null != softClipEndBases) { // append
cigarElements.add(new CigarElement(softClipEndBases.length(), CigarOperator.S));
byte tmpBases[] = new byte[readBases.length + softClipEndBases.length()];
System.arraycopy(readBases, 0, tmpBases, 0, readBases.length);
for (i = 0; i < softClipEndBases.length(); i++) {
tmpBases[i + readBases.length] = (byte) softClipEndBases.charAt(i);
}
readBases = tmpBases;
byte tmpQualities[] = new byte[baseQualities.length + softClipEndQualities.length()];
System.arraycopy(baseQualities, 0, tmpQualities, 0, baseQualities.length);
for (i = 0; i < softClipEndQualities.length(); i++) {
tmpQualities[i + baseQualities.length] = (byte) softClipEndQualities.charAt(i);
}
baseQualities = tmpQualities;
}
// Update SAM record
rec.setCigar(new Cigar(cigarElements));
rec.setAlignmentStart(alignmentStart);
rec.setReadBases(readBases);
rec.setBaseQualities(baseQualities);
// Reset saved attributes
Align.resetAttributes(rec, optFieldTags, optFieldValues);
// Set new attributes
if (space == SRMAUtil.Space.COLORSPACE) {
// set the XE attribute for colorError string
rec.setAttribute("XE", new String(colorErrors));
}
rec.setAttribute("AS", bestAlignHeapNode.score);
rec.setAttribute("XC", bestAlignHeapNode.alleleCoverageSum);
rec.setAttribute("PG", programRecord.getId());
rec.setAttribute("NM", numEdits);
}
/*
* -1 if the alignment process should be aborted
* 0 if the alignment should continue
* 1 if the alignment should not be considered any further
* */
private static int passFilters(
Graph graph,
Node node,
int toNodeCoverage,
AlleleCoverageCutoffs alleleCoverageCutoffs,
int MAXIMUM_TOTAL_COVERAGE) {
int totalCoverage = graph.getCoverage(node.position);
if (MAXIMUM_TOTAL_COVERAGE < totalCoverage) {
return -1;
} else if (alleleCoverageCutoffs.getQ(totalCoverage) <= toNodeCoverage) {
return 0;
} else {
return 1;
}
}
private static int passFilters(
Graph graph,
Node node,
AlleleCoverageCutoffs alleleCoverageCutoffs,
int MAXIMUM_TOTAL_COVERAGE) {
return passFilters(graph, node, node.coverage, alleleCoverageCutoffs, MAXIMUM_TOTAL_COVERAGE);
}
private static void clearAttributes(
SAMRecord rec, List<String> optFieldTags, List<Object> optFieldValues) {
ListIterator<String> iter = saveTags.listIterator();
while (iter.hasNext()) {
String tag = iter.next();
Object attr = rec.getAttribute(tag);
if (null != attr) {
optFieldTags.add(tag);
optFieldValues.add(attr);
}
}
rec.clearAttributes();
}
private static void resetAttributes(
SAMRecord rec, List<String> optFieldTags, List<Object> optFieldValues) {
ListIterator<String> iterTags = optFieldTags.listIterator();
ListIterator<Object> iterValues = optFieldValues.listIterator();
while (iterTags.hasNext()) {
rec.setAttribute(iterTags.next(), iterValues.next());
}
}
}
public class ReadMetricsUnitTest extends BaseTest {
@Test
public void testReadsSeenDoNotOverflowInt() {
final ReadMetrics metrics = new ReadMetrics();
final long moreThanMaxInt = ((long) Integer.MAX_VALUE) + 1L;
for (long i = 0L; i < moreThanMaxInt; i++) {
metrics.incrementNumReadsSeen();
}
Assert.assertEquals(metrics.getNumReadsSeen(), moreThanMaxInt);
Assert.assertTrue(metrics.getNumReadsSeen() > (long) Integer.MAX_VALUE);
logger.warn(String.format("%d %d %d", Integer.MAX_VALUE, moreThanMaxInt, Long.MAX_VALUE));
}
// Test the accuracy of the read metrics
private IndexedFastaSequenceFile reference;
private SAMSequenceDictionary dictionary;
private SAMFileHeader header;
private GATKSAMReadGroupRecord readGroup;
private GenomeLocParser genomeLocParser;
private File testBAM;
private static final int numReadsPerContig = 250000;
private static final List<String> contigs = Arrays.asList("1", "2", "3");
@BeforeClass
private void init() throws IOException {
reference = new CachingIndexedFastaSequenceFile(new File(b37KGReference));
dictionary = reference.getSequenceDictionary();
genomeLocParser = new GenomeLocParser(dictionary);
header = ArtificialSAMUtils.createDefaultReadGroup(new SAMFileHeader(), "test", "test");
header.setSequenceDictionary(dictionary);
header.setSortOrder(SAMFileHeader.SortOrder.coordinate);
readGroup = new GATKSAMReadGroupRecord(header.getReadGroup("test"));
final List<GATKSAMRecord> reads = new ArrayList<>();
for (final String contig : contigs) {
for (int i = 1; i <= numReadsPerContig; i++) {
reads.add(buildSAMRecord("read" + contig + "_" + i, contig, i));
}
}
createBAM(reads);
}
private void createBAM(final List<GATKSAMRecord> reads) throws IOException {
testBAM = File.createTempFile("TraverseActiveRegionsUnitTest", ".bam");
testBAM.deleteOnExit();
SAMFileWriter out =
new SAMFileWriterFactory()
.setCreateIndex(true)
.makeBAMWriter(reads.get(0).getHeader(), true, testBAM);
for (GATKSAMRecord read : reads) {
out.addAlignment(read);
}
out.close();
new File(testBAM.getAbsolutePath().replace(".bam", ".bai")).deleteOnExit();
new File(testBAM.getAbsolutePath() + ".bai").deleteOnExit();
}
// 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;
}
@Test
public void testCountsFromReadTraversal() {
final GenomeAnalysisEngine engine = new GenomeAnalysisEngine();
engine.setGenomeLocParser(genomeLocParser);
final Collection<SAMReaderID> samFiles = new ArrayList<>();
final SAMReaderID readerID = new SAMReaderID(testBAM, new Tags());
samFiles.add(readerID);
final SAMDataSource dataSource =
new SAMDataSource(
samFiles,
new ThreadAllocation(),
null,
genomeLocParser,
false,
SAMFileReader.ValidationStringency.STRICT,
null,
null,
new ValidationExclusion(),
new ArrayList<ReadFilter>(),
new ArrayList<ReadTransformer>(),
false,
(byte) 30,
false,
true);
engine.setReadsDataSource(dataSource);
final TraverseReadsNano traverseReadsNano = new TraverseReadsNano(1);
final DummyReadWalker walker = new DummyReadWalker();
traverseReadsNano.initialize(engine, walker, null);
for (final Shard shard : dataSource.createShardIteratorOverAllReads(new ReadShardBalancer())) {
final ReadShardDataProvider dataProvider =
new ReadShardDataProvider(
shard,
engine.getGenomeLocParser(),
dataSource.seek(shard),
reference,
new ArrayList<ReferenceOrderedDataSource>());
traverseReadsNano.traverse(walker, dataProvider, 0);
dataProvider.close();
}
Assert.assertEquals(
engine.getCumulativeMetrics().getNumReadsSeen(), contigs.size() * numReadsPerContig);
Assert.assertEquals(
engine.getCumulativeMetrics().getNumIterations(), contigs.size() * numReadsPerContig);
}
@Test
public void testCountsFromLocusTraversal() {
final GenomeAnalysisEngine engine = new GenomeAnalysisEngine();
engine.setGenomeLocParser(genomeLocParser);
final Collection<SAMReaderID> samFiles = new ArrayList<>();
final SAMReaderID readerID = new SAMReaderID(testBAM, new Tags());
samFiles.add(readerID);
final SAMDataSource dataSource =
new SAMDataSource(
samFiles,
new ThreadAllocation(),
null,
genomeLocParser,
false,
SAMFileReader.ValidationStringency.STRICT,
null,
null,
new ValidationExclusion(),
new ArrayList<ReadFilter>(),
new ArrayList<ReadTransformer>(),
false,
(byte) 30,
false,
true);
engine.setReadsDataSource(dataSource);
final Set<String> samples = SampleUtils.getSAMFileSamples(dataSource.getHeader());
final TraverseLociNano traverseLociNano = new TraverseLociNano(1);
final DummyLocusWalker walker = new DummyLocusWalker();
traverseLociNano.initialize(engine, walker, null);
for (final Shard shard : dataSource.createShardIteratorOverAllReads(new LocusShardBalancer())) {
final WindowMaker windowMaker =
new WindowMaker(
shard, genomeLocParser, dataSource.seek(shard), shard.getGenomeLocs(), samples);
for (WindowMaker.WindowMakerIterator window : windowMaker) {
final LocusShardDataProvider dataProvider =
new LocusShardDataProvider(
shard,
shard.getReadProperties(),
genomeLocParser,
window.getLocus(),
window,
reference,
new ArrayList<ReferenceOrderedDataSource>());
traverseLociNano.traverse(walker, dataProvider, 0);
dataProvider.close();
}
windowMaker.close();
}
// dataSource.close();
Assert.assertEquals(
engine.getCumulativeMetrics().getNumReadsSeen(), contigs.size() * numReadsPerContig);
Assert.assertEquals(
engine.getCumulativeMetrics().getNumIterations(), contigs.size() * numReadsPerContig);
}
@Test
public void testCountsFromActiveRegionTraversal() {
final GenomeAnalysisEngine engine = new GenomeAnalysisEngine();
engine.setGenomeLocParser(genomeLocParser);
final Collection<SAMReaderID> samFiles = new ArrayList<>();
final SAMReaderID readerID = new SAMReaderID(testBAM, new Tags());
samFiles.add(readerID);
final SAMDataSource dataSource =
new SAMDataSource(
samFiles,
new ThreadAllocation(),
null,
genomeLocParser,
false,
SAMFileReader.ValidationStringency.STRICT,
null,
null,
new ValidationExclusion(),
new ArrayList<ReadFilter>(),
new ArrayList<ReadTransformer>(),
false,
(byte) 30,
false,
true);
engine.setReadsDataSource(dataSource);
final Set<String> samples = SampleUtils.getSAMFileSamples(dataSource.getHeader());
final List<GenomeLoc> intervals = new ArrayList<>(contigs.size());
for (final String contig : contigs)
intervals.add(genomeLocParser.createGenomeLoc(contig, 1, numReadsPerContig));
final TraverseActiveRegions traverseActiveRegions = new TraverseActiveRegions();
final DummyActiveRegionWalker walker = new DummyActiveRegionWalker();
traverseActiveRegions.initialize(engine, walker, null);
for (final Shard shard :
dataSource.createShardIteratorOverIntervals(
new GenomeLocSortedSet(genomeLocParser, intervals), new ActiveRegionShardBalancer())) {
final WindowMaker windowMaker =
new WindowMaker(
shard, genomeLocParser, dataSource.seek(shard), shard.getGenomeLocs(), samples);
for (WindowMaker.WindowMakerIterator window : windowMaker) {
final LocusShardDataProvider dataProvider =
new LocusShardDataProvider(
shard,
shard.getReadProperties(),
genomeLocParser,
window.getLocus(),
window,
reference,
new ArrayList<ReferenceOrderedDataSource>());
traverseActiveRegions.traverse(walker, dataProvider, 0);
dataProvider.close();
}
windowMaker.close();
}
Assert.assertEquals(
engine.getCumulativeMetrics().getNumReadsSeen(), contigs.size() * numReadsPerContig);
Assert.assertEquals(
engine.getCumulativeMetrics().getNumIterations(), contigs.size() * numReadsPerContig);
}
@Test
public void testFilteredCounts() {
final GenomeAnalysisEngine engine = new GenomeAnalysisEngine();
engine.setGenomeLocParser(genomeLocParser);
final Collection<SAMReaderID> samFiles = new ArrayList<>();
final SAMReaderID readerID = new SAMReaderID(testBAM, new Tags());
samFiles.add(readerID);
final List<ReadFilter> filters = new ArrayList<>();
filters.add(new EveryTenthReadFilter());
final SAMDataSource dataSource =
new SAMDataSource(
samFiles,
new ThreadAllocation(),
null,
genomeLocParser,
false,
SAMFileReader.ValidationStringency.STRICT,
null,
null,
new ValidationExclusion(),
filters,
new ArrayList<ReadTransformer>(),
false,
(byte) 30,
false,
true);
engine.setReadsDataSource(dataSource);
final TraverseReadsNano traverseReadsNano = new TraverseReadsNano(1);
final DummyReadWalker walker = new DummyReadWalker();
traverseReadsNano.initialize(engine, walker, null);
for (final Shard shard : dataSource.createShardIteratorOverAllReads(new ReadShardBalancer())) {
final ReadShardDataProvider dataProvider =
new ReadShardDataProvider(
shard,
engine.getGenomeLocParser(),
dataSource.seek(shard),
reference,
new ArrayList<ReferenceOrderedDataSource>());
traverseReadsNano.traverse(walker, dataProvider, 0);
dataProvider.close();
}
Assert.assertEquals(
(long)
engine
.getCumulativeMetrics()
.getCountsByFilter()
.get(EveryTenthReadFilter.class.getSimpleName()),
contigs.size() * numReadsPerContig / 10);
}
class DummyLocusWalker extends LocusWalker<Integer, Integer> {
@Override
public Integer map(RefMetaDataTracker tracker, ReferenceContext ref, AlignmentContext context) {
return 0;
}
@Override
public Integer reduceInit() {
return 0;
}
@Override
public Integer reduce(Integer value, Integer sum) {
return 0;
}
}
class DummyReadWalker extends ReadWalker<Integer, Integer> {
@Override
public Integer map(
ReferenceContext ref, GATKSAMRecord read, RefMetaDataTracker metaDataTracker) {
return 0;
}
@Override
public Integer reduceInit() {
return 0;
}
@Override
public Integer reduce(Integer value, Integer sum) {
return 0;
}
}
class DummyActiveRegionWalker extends ActiveRegionWalker<Integer, Integer> {
@Override
public ActivityProfileState isActive(
RefMetaDataTracker tracker, ReferenceContext ref, AlignmentContext context) {
return new ActivityProfileState(ref.getLocus(), 0.0);
}
@Override
public Integer map(ActiveRegion activeRegion, RefMetaDataTracker metaDataTracker) {
return 0;
}
@Override
public Integer reduceInit() {
return 0;
}
@Override
public Integer reduce(Integer value, Integer sum) {
return 0;
}
}
private final class EveryTenthReadFilter extends ReadFilter {
private int myCounter = 0;
@Override
public boolean filterOut(final SAMRecord record) {
if (++myCounter == 10) {
myCounter = 0;
return true;
}
return false;
}
}
}