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package consensus;
import java.io.PrintStream;
import java.util.ArrayList;
import java.util.LinkedHashMap;
import java.util.Map.Entry;
import java.util.concurrent.atomic.AtomicIntegerArray;
import java.util.concurrent.atomic.AtomicLongArray;
import java.util.concurrent.locks.ReadWriteLock;
import java.util.concurrent.locks.ReentrantReadWriteLock;
import fileIO.ByteFile;
import fileIO.FileFormat;
import fileIO.ReadWrite;
import shared.Parse;
import shared.Parser;
import shared.PreParser;
import shared.Shared;
import shared.Timer;
import shared.Tools;
import stream.ConcurrentReadInputStream;
import stream.ConcurrentReadOutputStream;
import stream.FastaReadInputStream;
import stream.Read;
import stream.SamLine;
import stream.SamReadStreamer;
import stream.SamStreamer;
import structures.ByteBuilder;
import structures.ListNum;
import template.Accumulator;
import template.ThreadWaiter;
import tracker.ReadStats;
import var2.SamFilter;
/**
* Resizes scaffold gaps to represent the best estimate
* based on the insert size distribution of paired reads.
*
* @author Brian Bushnell
* @date September 11, 2019
*
*/
public class FixScaffoldGaps implements Accumulator<FixScaffoldGaps.ProcessThread> {
/*--------------------------------------------------------------*/
/*---------------- Initialization ----------------*/
/*--------------------------------------------------------------*/
/**
* Code entrance from the command line.
* @param args Command line arguments
*/
public static void main(String[] args){
//Start a timer immediately upon code entrance.
Timer t=new Timer();
//Create an instance of this class
FixScaffoldGaps x=new FixScaffoldGaps(args);
//Run the object
x.process(t);
//Close the print stream if it was redirected
Shared.closeStream(x.outstream);
}
/**
* Constructor.
* @param args Command line arguments
*/
public FixScaffoldGaps(String[] args){
{//Preparse block for help, config files, and outstream
PreParser pp=new PreParser(args, getClass(), false);
args=pp.args;
outstream=pp.outstream;
}
//Set shared static variables prior to parsing
ReadWrite.USE_PIGZ=ReadWrite.USE_UNPIGZ=true;
ReadWrite.setZipThreads(Shared.threads());
samFilter.includeUnmapped=false;
samFilter.includeSupplimentary=false;
// samFilter.includeDuplicate=false;
samFilter.includeNonPrimary=false;
samFilter.includeQfail=false;
// samFilter.minMapq=4;
{//Parse the arguments
final Parser parser=parse(args);
Parser.processQuality();
maxReads=parser.maxReads;
overwrite=ReadStats.overwrite=parser.overwrite;
append=ReadStats.append=parser.append;
in=parser.in1;
extin=parser.extin;
out=parser.out1;
extout=parser.extout;
}
{
// if("auto".equalsIgnoreCase(atomic)){Scaffold.setCA3A(Shared.threads()>8);}
// else{Scaffold.setCA3A(Parse.parseBoolean(atomic));}
samFilter.setSamtoolsFilter();
streamerThreads=Tools.max(1, Tools.min(streamerThreads, Shared.threads()));
assert(streamerThreads>0) : streamerThreads;
}
validateParams();
fixExtensions(); //Add or remove .gz or .bz2 as needed
checkFileExistence(); //Ensure files can be read and written
checkStatics(); //Adjust file-related static fields as needed for this program
//Create output FileFormat objects
ffout=FileFormat.testOutput(out, FileFormat.FASTA, extout, true, overwrite, append, ordered);
//Create input FileFormat objects
ffin=FileFormat.testInput(in, FileFormat.SAM, extin, true, true);
ffref=FileFormat.testInput(ref, FileFormat.FASTA, null, true, true);
}
/*--------------------------------------------------------------*/
/*---------------- Initialization Helpers ----------------*/
/*--------------------------------------------------------------*/
/** Parse arguments from the command line */
private Parser parse(String[] args){
//Create a parser object
Parser parser=new Parser();
//Set any necessary Parser defaults here
//parser.foo=bar;
//Parse each argument
for(int i=0; i<args.length; i++){
String arg=args[i];
//Break arguments into their constituent parts, in the form of "a=b"
String[] split=arg.split("=");
String a=split[0].toLowerCase();
String b=split.length>1 ? split[1] : null;
if(b!=null && b.equalsIgnoreCase("null")){b=null;}
if(a.equals("verbose")){
verbose=Parse.parseBoolean(b);
}else if(a.equals("ref") || a.equals("scaffolds")){
ref=b;
}else if(a.equals("insertlist")){
insertList=b;
}else if(a.equals("ordered")){
ordered=Parse.parseBoolean(b);
}else if(a.equalsIgnoreCase("ns") || a.equalsIgnoreCase("n") || a.equalsIgnoreCase("scaffoldbreak") || a.equalsIgnoreCase("gap")){
scaffoldBreakNs=Integer.parseInt(b);
assert(scaffoldBreakNs>0);
}else if(a.equalsIgnoreCase("mindepth")){
minDepth=Integer.parseInt(b);
assert(minDepth>0);
}else if(a.equalsIgnoreCase("trim") || a.equalsIgnoreCase("trimFraction") || a.equalsIgnoreCase("border")){
trimFraction=Float.parseFloat(b);
assert(trimFraction>=0 && trimFraction<=1) : "trimFraction should be between 0 and 1";
}else if(a.equals("clearfilters") || a.equals("clearfilter")){
if(Parse.parseBoolean(b)){
samFilter.clear();
}
}else if(a.equals("parse_flag_goes_here")){
long fake_variable=Parse.parseKMG(b);
//Set a variable here
}else if(samFilter.parse(arg, a, b)){
//do nothing
}else if(parser.parse(arg, a, b)){//Parse standard flags in the parser
//do nothing
}else{
outstream.println("Unknown parameter "+args[i]);
assert(false) : "Unknown parameter "+args[i];
}
}
return parser;
}
/** Add or remove .gz or .bz2 as needed */
private void fixExtensions(){
in=Tools.fixExtension(in);
ref=Tools.fixExtension(ref);
}
/** Ensure files can be read and written */
private void checkFileExistence(){
//Ensure there is an input file
if(in==null){throw new RuntimeException("Error - an input file is required.");}
//Ensure there is an input file
if(ref==null){throw new RuntimeException("Error - a reference file is required.");}
//Ensure output files can be written
if(!Tools.testOutputFiles(overwrite, append, false, out)){
outstream.println((out==null)+", "+out);
throw new RuntimeException("\n\noverwrite="+overwrite+"; Can't write to output file "+out+"\n");
}
//Ensure input files can be read
if(!Tools.testInputFiles(false, true, in, ref)){
throw new RuntimeException("\nCan't read some input files.\n");
}
//Ensure that no file was specified multiple times
if(!Tools.testForDuplicateFiles(true, in, ref, out)){
throw new RuntimeException("\nSome file names were specified multiple times.\n");
}
}
/** Adjust file-related static fields as needed for this program */
private static void checkStatics(){
//Adjust the number of threads for input file reading
if(!ByteFile.FORCE_MODE_BF1 && !ByteFile.FORCE_MODE_BF2 && Shared.threads()>2){
ByteFile.FORCE_MODE_BF2=true;
}
assert(FastaReadInputStream.settingsOK());
}
/** Ensure parameter ranges are within bounds and required parameters are set */
private boolean validateParams(){
// assert(minfoo>0 && minfoo<=maxfoo) : minfoo+", "+maxfoo;
return true;
}
/*--------------------------------------------------------------*/
/*---------------- Outer Methods ----------------*/
/*--------------------------------------------------------------*/
/** Create read streams and process all data */
void process(Timer t){
//Turn off read validation in the input threads to increase speed
final boolean vic=Read.VALIDATE_IN_CONSTRUCTOR;
Read.VALIDATE_IN_CONSTRUCTOR=Shared.threads()<4;
//Create a read input stream
final SamStreamer ss=makeStreamer(ffin);
//Load reference
loadReferenceCustom();
//Reset counters
readsProcessed=readsOut=0;
basesProcessed=basesOut=0;
//Process the reads in separate threads
spawnThreads(ss);
//Optionally create a read output stream
final ConcurrentReadOutputStream ros=makeCros();
if(verbose){outstream.println("Fixing reference.");}
fixScaffolds(ros);
if(verbose){outstream.println("Finished; closing streams.");}
//Write anything that was accumulated by ReadStats
errorState|=ReadStats.writeAll();
//Close the read streams
errorState|=ReadWrite.closeStream(ros);
//Reset read validation
Read.VALIDATE_IN_CONSTRUCTOR=vic;
//Report timing and results
t.stop();
outstream.println(Tools.timeReadsBasesProcessed(t, readsProcessed, basesProcessed, 8));
outstream.println(Tools.readsBasesOut(readsProcessed, basesProcessed, scaffoldsOut, scaffoldLengthOut, 8, false));
outstream.println();
outstream.println(Tools.number("Average Insert", totalAverageInsert, 2, 8));
outstream.println(Tools.number("Gaps Unchanged", gapsUnchanged, 8));
outstream.println(Tools.number("Gaps Widened ", gapsWidened, 8));
outstream.println(Tools.number("Gaps Narrowed ", gapsNarrowed, 8));
outstream.println(Tools.number("Ns Total ", nsTotal, 8));
outstream.println(Tools.number("Ns Added ", nsAdded, 8));
outstream.println(Tools.number("Ns Removed ", nsRemoved, 8));
//Throw an exception of there was an error in a thread
if(errorState){
throw new RuntimeException(getClass().getName()+" terminated in an error state; the output may be corrupt.");
}
}
private synchronized void loadReferenceCustom(){
assert(!loadedRef);
ConcurrentReadInputStream cris=makeRefCris();
for(ListNum<Read> ln=cris.nextList(); ln!=null && ln.size()>0; ln=cris.nextList()) {
for(Read r : ln){
String name=r.id;
String name2=Tools.trimToWhitespace(r.id);
Scaffold scaf=new Scaffold(name, r.bases, r.numericID);
refMap.put(name, scaf);
refMap2.put(name2, scaf);
}
}
loadedRef=true;
}
private ConcurrentReadInputStream makeRefCris(){
ConcurrentReadInputStream cris=ConcurrentReadInputStream.getReadInputStream(maxReads, true, ffref, null);
cris.start(); //Start the stream
if(verbose){outstream.println("Started cris");}
boolean paired=cris.paired();
assert(!paired) : "References should not be paired.";
return cris;
}
private SamStreamer makeStreamer(FileFormat ff){
if(ff==null){return null;}
SamStreamer ss=new SamReadStreamer(ff, streamerThreads, true, maxReads);
ss.start(); //Start the stream
if(verbose){outstream.println("Started Streamer");}
return ss;
}
private ConcurrentReadOutputStream makeCros(){
if(ffout==null){return null;}
//Select output buffer size based on whether it needs to be ordered
final int buff=(ordered ? Tools.mid(16, 128, (Shared.threads()*2)/3) : 8);
final ConcurrentReadOutputStream ros=ConcurrentReadOutputStream.getStream(ffout, null, buff, null, false);
ros.start(); //Start the stream
return ros;
}
/*--------------------------------------------------------------*/
/*---------------- Thread Management ----------------*/
/*--------------------------------------------------------------*/
/** Spawn process threads */
private void spawnThreads(final SamStreamer ss){
//Do anything necessary prior to processing
//Determine how many threads may be used
final int threads=Shared.threads();
//Fill a list with ProcessThreads
ArrayList<ProcessThread> alpt=new ArrayList<ProcessThread>(threads);
for(int i=0; i<threads; i++){
alpt.add(new ProcessThread(ss, i));
}
//Start the threads
for(ProcessThread pt : alpt){
pt.start();
}
//Wait for threads to finish
boolean success=ThreadWaiter.waitForThreadsToFinish(alpt, this);
errorState&=!success;
//Do anything necessary after processing
totalAverageInsert=totalInsertSum/(double)totalInsertCount;
insertByPercentile=Tools.makeHistogram(insertCounts, buckets);
}
@Override
public final void accumulate(ProcessThread pt){
readsProcessed+=pt.readsProcessedT;
basesProcessed+=pt.basesProcessedT;
readsOut+=pt.readsOutT;
basesOut+=pt.basesOutT;
totalInsertSum+=pt.totalInsertSumT;
totalInsertCount+=pt.totalInsertCountT;
errorState|=(!pt.success);
}
@Override
public final boolean success(){return !errorState;}
/*--------------------------------------------------------------*/
/*---------------- Inner Methods ----------------*/
/*--------------------------------------------------------------*/
private void fixScaffolds(ConcurrentReadOutputStream ros){
ByteBuilder bb=new ByteBuilder(1000000);
ArrayList<Read> list=new ArrayList<Read>(200);
long num=0;
long lengthSum=0;
for(Entry<String, Scaffold> e : refMap.entrySet()){
Scaffold scaf=e.getValue();
Read r=scaf.fixScaffold(bb);
lengthSum+=r.length();
list.add(r);
scaffoldsOut++;
scaffoldLengthOut+=r.length();
if(list.size()>=200 || lengthSum>=100000){
if(ros!=null){ros.add(list, num);}
list=new ArrayList<Read>(200);
num++;
lengthSum=0;
}
}
if(list.size()>0){
if(ros!=null){ros.add(list, num);}
}
}
private static int calcInsertSize(SamLine sl) {
assert(sl.mapped() && sl.pairedOnSameChrom());
assert(sl.primary());
assert(!sl.supplementary());
assert(sl.leftmost());
assert(sl.tlen>0) : sl.tlen+"\n\n"+sl;
return sl.tlen>0 ? sl.tlen : -sl.tlen;
// final int insertSize;
// String insertTag=null;
// if(sl.optional!=null){
// for(String s : sl.optional){
// if(s.startsWith("X8:Z:")){
// insertTag=s;
// break;
// }
// }
// }
// if(insertTag!=null){
// insertSize=Integer.parseInt(insertTag.substring(5));
// }else{
// insertSize=sl.tlen;//This is unsafe due to indels.
// assert(false) : "Reads need insert size tags.";
// }
// assert(insertSize>0) : sl;
//
// return insertSize;
}
/*--------------------------------------------------------------*/
/*---------------- Inner Classes ----------------*/
/*--------------------------------------------------------------*/
/** This class is static to prevent accidental writing to shared variables.
* It is safe to remove the static modifier. */
class ProcessThread extends Thread {
//Constructor
ProcessThread(final SamStreamer ss_, final int tid_){
ss=ss_;
tid=tid_;
}
//Called by start()
@Override
public void run(){
//Do anything necessary prior to processing
//Process the reads
processInner();
//Do anything necessary after processing
//Indicate successful exit status
success=true;
}
/** Iterate through the reads */
void processInner(){
//Grab and process all lists
for(ListNum<Read> ln=ss.nextReads(); ln!=null; ln=ss.nextReads()){
// if(verbose){outstream.println("Got list of size "+list.size());} //Disabled due to non-static access
processList(ln);
}
}
void processList(ListNum<Read> ln){
//Grab the actual read list from the ListNum
final ArrayList<Read> reads=ln.list;
//Loop through each read in the list
for(int idx=0; idx<reads.size(); idx++){
final Read r=reads.get(idx);
//Validate reads in worker threads
if(!r.validated()){r.validate(true);}
//Track the initial length for statistics
final int initialLength=r.length();
//Increment counters
readsProcessedT+=r.pairCount();
basesProcessedT+=initialLength;
processRead(r);
}
}
/**
* Process a read or a read pair.
* @param r Read 1
* @param r2 Read 2 (may be null)
* @return True if the reads should be kept, false if they should be discarded.
*/
void processRead(final Read r){
final SamLine sl=r.samline;
assert(sl!=null) : sl;
if(samFilter!=null && !samFilter.passesFilter(sl)){return;}
//sl.nextMapped();
if(sl.mapped() && sl.pairedOnSameChrom() && sl.properPair() && sl.primary() && !sl.supplementary() && sl.leftmost()){
final String rname=sl.rnameS();
Scaffold scaf=refMap.get(rname);
if(scaf==null){scaf=refMap2.get(Tools.trimToWhitespace(rname));}
assert(scaf!=null) : "Can't find graph for "+rname;
if(scaf!=null){
final int insertSize=calcInsertSize(sl);
insertCounts.incrementAndGet(Tools.mid(0, insertSize, insertCounts.length()));
scaf.add(sl, insertSize);
readsOutT++;
basesOutT+=r.length();
totalInsertSumT+=insertSize;
totalInsertCountT++;
}
}
}
/** Number of reads processed by this thread */
protected long readsProcessedT=0;
/** Number of bases processed by this thread */
protected long basesProcessedT=0;
/** Number of reads retained by this thread */
protected long readsOutT=0;
/** Number of bases retained by this thread */
protected long basesOutT=0;
protected long totalInsertSumT=0;
protected long totalInsertCountT=0;
long insertSum=0;
/** True only if this thread has completed successfully */
boolean success=false;
/** Shared input stream */
private final SamStreamer ss;
/** Thread ID */
final int tid;
}
/*--------------------------------------------------------------*/
private class Scaffold {
Scaffold(String name_, byte[] bases_, long numericID_){
name=name_;
bases=bases_;
numericID=(int)numericID_;
depthArray=new AtomicIntegerArray(bases.length);
insertArray=new AtomicLongArray(bases.length);
}
void add(SamLine sl, int insertSize){
assert(sl.mapped() && sl.pairedOnSameChrom());
assert(sl.primary());
assert(!sl.supplementary());
assert(sl.leftmost());
// final int insertSize=calcInsertSize(sl);
int start=sl.pos-1;
int stop=start+sl.tlen;
int trim=(int)(sl.length()*trimFraction);
start+=trim;
stop-=trim;
for(int i=start; i<stop; i++){
if(i>=0 && i<bases.length){
depthArray.incrementAndGet(i);
insertArray.addAndGet(i, insertSize);
}
}
}
Read fixScaffold(ByteBuilder bb){
int streak=0;
bb.clear();
if(insertList!=null){
for(int i=0; i<bases.length; i++) {
bb.append(i).tab().append(depthArray.get(i)).tab().append(insertArray.get(i)/(Tools.max(1, depthArray.get(i)))).nl();
}
ReadWrite.writeString(bb, insertList, false);
bb.clear();
}
for(int i=0; i<bases.length; i++){
byte b=bases[i];
if(b=='N'){
streak++;
}else{
if(streak>=scaffoldBreakNs && i-streak>300 && i<bases.length-300){
int pivot=i-streak/2-1;
long depthSum=depthArray.get(pivot);
long insertSum=insertArray.get(pivot);
double avgInsert=(insertSum/(double)depthSum);
int avgDepth=((depthArray.get(i-200-streak)+depthArray.get(i+200))/2);
int percentile=(int)(buckets*Tools.max(0.5, 1.0-depthSum/(double)(avgDepth+depthSum)));
int insertProxy=insertByPercentile[percentile];
// assert(false) : Arrays.toString(insertByPercentile);
int dif=(int)Math.round(insertProxy-avgInsert);
int toAdd=Tools.max(scaffoldBreakNs, streak+dif);
// System.err.println("totalAverageInsert="+(int)totalAverageInsert+", avg="+(int)avgInsert+", dif="+dif);
// System.err.println("proxy="+insertProxy+", percentile="+percentile+", avgDepth="+(int)avgDepth+", depth="+depthSum);
// System.err.println("pivot="+pivot+", depthSum="+depthSum+", avg="+(int)avgInsert+", streak="+streak+", dif="+dif+", toAdd="+toAdd);
if(dif>0){
//TODO: This is tricky because long-insert reads will be self-selected.
//Should be compared to average coverage, or nearby coverage, and then consult a size distribution histogram.
gapsWidened++;
nsAdded+=dif;
}else if(dif<0){
gapsNarrowed++;
nsRemoved-=dif;
}else{
gapsUnchanged++;
}
nsTotal+=toAdd;
for(int j=0; j<toAdd; j++){
bb.append('N');
}
}
streak=0;
bb.append(b);
}
}
return new Read(bb.toBytes(), null, name, numericID);
}
final int numericID;
final String name;
final byte[] bases;
final AtomicIntegerArray depthArray;
final AtomicLongArray insertArray;
}
/*--------------------------------------------------------------*/
/*---------------- Fields ----------------*/
/*--------------------------------------------------------------*/
/** Primary input file path */
private String in=null;
/** Secondary input file path */
private String ref=null;
/** Primary output file path */
private String out=null;
/** Override input file extension */
private String extin=null;
/** Override output file extension */
private String extout=null;
private String insertList=null;
/*--------------------------------------------------------------*/
/** Number of reads processed */
protected long readsProcessed=0;
/** Number of bases processed */
protected long basesProcessed=0;
/** Number of reads retained */
protected long readsOut=0;
/** Number of bases retained */
protected long basesOut=0;
protected long scaffoldsOut=0;
protected long scaffoldLengthOut=0;
protected long gapsUnchanged=0;
protected long gapsWidened=0;
protected long gapsNarrowed=0;
protected long nsAdded=0;
protected long nsRemoved=0;
protected long nsTotal=0;
protected long totalInsertSum=0;
protected long totalInsertCount=0;
protected double totalAverageInsert;
protected AtomicLongArray insertCounts=new AtomicLongArray(20000);
protected int[] insertByPercentile;
/** Quit after processing this many input reads; -1 means no limit */
private long maxReads=-1;
/*--------------------------------------------------------------*/
/** Threads dedicated to reading the sam file */
private int streamerThreads=SamStreamer.DEFAULT_THREADS;
private boolean loadedRef=false;
private int scaffoldBreakNs=10;
int buckets=1000;
private int minDepth=10;
private float trimFraction=0.4f;
public final SamFilter samFilter=new SamFilter();
/** Uses full ref names */
public LinkedHashMap<String, Scaffold> refMap=new LinkedHashMap<String, Scaffold>();
/** Uses truncated ref names */
public LinkedHashMap<String, Scaffold> refMap2=new LinkedHashMap<String, Scaffold>();;
/*--------------------------------------------------------------*/
/*---------------- Final Fields ----------------*/
/*--------------------------------------------------------------*/
/** Primary input file */
private final FileFormat ffin;
/** Secondary input file */
private final FileFormat ffref;
/** Primary output file */
private final FileFormat ffout;
@Override
public final ReadWriteLock rwlock() {return rwlock;}
private final ReadWriteLock rwlock=new ReentrantReadWriteLock();
/*--------------------------------------------------------------*/
/*---------------- Common Fields ----------------*/
/*--------------------------------------------------------------*/
/** Print status messages to this output stream */
private PrintStream outstream=System.err;
/** Print verbose messages */
public static boolean verbose=false;
/** True if an error was encountered */
public boolean errorState=false;
/** Overwrite existing output files */
private boolean overwrite=true;
/** Append to existing output files */
private boolean append=false;
/** Reads are output in input order */
private boolean ordered=false;
}
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