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#include "Aligner.h"
#include "Common/Options.h"
#include "DataLayer/Options.h"
#include "KAligner/Options.h"
#include "FastaReader.h"
#include "Iterator.h"
#include "IOUtil.h"
#include "MemoryUtil.h"
#include "SAM.h"
#include "StringUtil.h" // for toSI
#include "Uncompress.h"
#include "Pipe.h"
#include "PipeMux.h"
#include <algorithm>
#include <cassert>
#include <cctype>
#include <cerrno>
#include <cstdlib>
#include <cstring>
#include <fstream>
#include <getopt.h>
#include <iostream>
#include <pthread.h>
#include <sstream>
#include <string>
#include <sys/stat.h>
#include <sys/time.h>
using namespace std;
#define PROGRAM "KAligner"
static const char VERSION_MESSAGE[] =
PROGRAM " (" PACKAGE_NAME ") " VERSION "\n"
"Written by Jared Simpson and Shaun Jackman.\n"
"\n"
"Copyright 2014 Canada's Michael Smith Genome Sciences Centre\n";
static const char USAGE_MESSAGE[] =
"Usage: " PROGRAM " -k<kmer> [OPTION]... QUERY... TARGET\n"
"Align the sequences of the files QUERY to those of TARGET.\n"
"All perfect matches of at least k bases will be found.\n"
"\n"
" Options:\n"
"\n"
" -k, -l, --kmer=N k-mer size and minimum alignment length\n"
" -s, --section=S/N split the target into N sections and align\n"
" reads to section S [1/1]\n"
" -i, --ignore-multimap ignore duplicate k-mer in the target\n"
" [default]\n"
" -m, --multimap allow duplicate k-mer in the target\n"
" --no-multimap disallow duplicate k-mer in the target\n"
" -j, --threads=N use N threads [2] up to one per query file\n"
" or if N is 0 use one thread per query file\n"
" -v, --verbose display verbose output\n"
" --no-sam output the results in KAligner format\n"
" --sam output the results in SAM format\n"
" --seq print the sequence with the alignments\n"
" --help display this help and exit\n"
" --version output version information and exit\n"
"\n"
"Report bugs to <" PACKAGE_BUGREPORT ">.\n";
/** Enumeration of output formats */
enum format { KALIGNER, SAM };
namespace opt {
static unsigned k;
static int threads = 2;
static int printSeq;
static unsigned section = 1;
static unsigned nsections = 1;
/** Output formats */
static int format;
}
static const char shortopts[] = "ij:k:l:mo:s:v";
enum { OPT_HELP = 1, OPT_VERSION, OPT_SYNC };
static const struct option longopts[] = {
{ "kmer", required_argument, NULL, 'k' },
{ "section", required_argument, NULL, 's' },
{ "no-multi", no_argument, &opt::multimap, opt::ERROR },
{ "multimap", no_argument, &opt::multimap, opt::MULTIMAP },
{ "ignore-multimap", no_argument, &opt::multimap, opt::IGNORE },
{ "threads", required_argument, NULL, 'j' },
{ "verbose", no_argument, NULL, 'v' },
{ "no-sam", no_argument, &opt::format, KALIGNER },
{ "sam", no_argument, &opt::format, SAM },
{ "no-seq", no_argument, &opt::printSeq, 0 },
{ "seq", no_argument, &opt::printSeq, 1 },
{ "help", no_argument, NULL, OPT_HELP },
{ "version", no_argument, NULL, OPT_VERSION },
{ NULL, 0, NULL, 0 }
};
/** Return the number of k-mer in the specified file. */
static size_t countKmer(const string& path)
{
struct stat st;
if (stat(path.c_str(), &st) == -1) {
perror(path.c_str());
exit(EXIT_FAILURE);
}
if (!S_ISREG(st.st_mode)) {
cerr << "Not calculating k-mer in `" << path
<< "', because it is not a regular file.\n";
return 500000000;
}
if (opt::verbose > 0)
cerr << "Reading target `" << path << "'..." << endl;
ifstream in(path.c_str());
assert(in.is_open());
size_t scaffolds = 0, contigs = 0, bases = 0;
enum { ID, SEQUENCE, GAP } state = SEQUENCE;
for (char c; in.get(c);) {
c = toupper(c);
switch (state) {
case ID:
if (c == '\n')
state = SEQUENCE;
break;
case SEQUENCE:
case GAP:
switch (c) {
case '>':
scaffolds++;
contigs++;
state = ID;
break;
case 'N':
case 'B': case 'D': case 'H': case 'K': case 'M':
case 'R': case 'S': case 'V': case 'W': case 'Y':
if (state != GAP)
contigs++;
state = GAP;
break;
case 'A': case 'C': case 'G': case 'T':
case '0': case '1': case '2': case '3':
bases++;
state = SEQUENCE;
break;
case '\n':
break;
default:
cerr << "error: unexpected character: "
"`" << c << "'\n";
exit(EXIT_FAILURE);
}
break;
}
}
size_t overlaps = contigs * (opt::k-1);
size_t kmer = bases - overlaps;
if (opt::verbose > 0) {
cerr << "Read " << bases << " bases, "
<< contigs << " contigs, " << scaffolds << " scaffolds"
" from `" << path << "'. "
"Expecting " << kmer << " k-mer.\n";
cerr << "Index will use at least "
<< toSI(kmer * sizeof(pair<Kmer, Position>))
<< "B.\n";
}
assert(bases > overlaps);
return kmer;
}
template <class SeqPosHashMap>
static void readContigsIntoDB(string refFastaFile,
Aligner<SeqPosHashMap>& aligner);
static void *alignReadsToDB(void *arg);
static void *readFile(void *arg);
/** Unique aligner using map */
static Aligner<SeqPosHashUniqueMap> *g_aligner_u;
/** Multimap aligner using multimap */
static Aligner<SeqPosHashMultiMap> *g_aligner_m;
/** Number of reads. */
static unsigned g_readCount;
/** Number of reads that aligned. */
static unsigned g_alignedCount;
/** Guard cerr. */
static pthread_mutex_t g_mutexCerr = PTHREAD_MUTEX_INITIALIZER;
/** Stores the output string and the read index number for an
* alignment. */
struct OutData
{
string s;
size_t index;
OutData(string s = string(), size_t index = 0)
: s(s), index(index) { }
/** Operator needed for sorting priority queue. */
bool operator<(const OutData& a) const
{
// Smaller index number has higher priority.
return index > a.index;
}
};
/** Shares data between workers and the output thread. */
static Pipe<OutData> g_pipeOut(1<<7);
/** Shares data between producer and worker threads. */
static PipeMux<FastaRecord> g_pipeMux(1);
/** Notification of the current size of the g_pqueue. */
static size_t g_pqSize;
static const size_t MAX_PQ_SIZE = 1000;
/** Conditional variable used to block workers until the g_pqueue has
* become small enough. */
static pthread_cond_t g_pqSize_cv = PTHREAD_COND_INITIALIZER;
static pthread_mutex_t g_mutexPqSize = PTHREAD_MUTEX_INITIALIZER;
static void* printAlignments(void*)
{
priority_queue<OutData> pqueue;
size_t index = 1;
for (pair<OutData, size_t> p = g_pipeOut.pop();
p.second > 0; p = g_pipeOut.pop()) {
pqueue.push(p.first);
while (!pqueue.empty()) {
const OutData& rec = pqueue.top();
if (index == rec.index) {
// Print the record at the current index.
index++;
assert(rec.index > 0);
cout << rec.s;
assert_good(cout, "stdout");
pqueue.pop();
} else if (g_pipeMux.invalidEntry(index)) {
// Skip this index since it is invalid.
index++;
} else {
// The record for this index has not been added, get
// another record from the pipe.
break;
}
}
// Let waiting workers continue if the pqueue is small enough.
pthread_mutex_lock(&g_mutexPqSize);
g_pqSize = pqueue.size();
if (g_pqSize < MAX_PQ_SIZE)
pthread_cond_broadcast(&g_pqSize_cv);
pthread_mutex_unlock(&g_mutexPqSize);
}
return NULL;
}
/** Store a FastaReader and Pipe. */
struct WorkerArg {
FastaReader& in;
Pipe<FastaRecord>& out;
WorkerArg(FastaReader& in, Pipe<FastaRecord>& out)
: in(in), out(out) { }
~WorkerArg() { delete ∈ }
};
static pthread_t getReadFiles(const char *readsFile)
{
if (opt::verbose > 0) {
pthread_mutex_lock(&g_mutexCerr);
cerr << "Reading `" << readsFile << "'...\n";
pthread_mutex_unlock(&g_mutexCerr);
}
FastaReader* in = new FastaReader(
readsFile, FastaReader::FOLD_CASE);
WorkerArg* arg = new WorkerArg(*in, *g_pipeMux.addPipe());
pthread_t thread;
pthread_create(&thread, NULL, readFile, static_cast<void*>(arg));
return thread;
}
int main(int argc, char** argv)
{
string commandLine;
{
ostringstream ss;
char** last = argv + argc - 1;
copy(argv, last, ostream_iterator<const char *>(ss, " "));
ss << *last;
commandLine = ss.str();
}
char delim = '/';
bool die = false;
for (int c; (c = getopt_long(argc, argv,
shortopts, longopts, NULL)) != -1;) {
istringstream arg(optarg != NULL ? optarg : "");
switch (c) {
case '?': die = true; break;
case 'k': case 'l':
arg >> opt::k;
break;
case 'm': opt::multimap = opt::MULTIMAP; break;
case 'i': opt::multimap = opt::IGNORE; break;
case 'j': arg >> opt::threads; break;
case 'v': opt::verbose++; break;
case 's': arg >> opt::section >> delim >>
opt::nsections; break;
case OPT_HELP:
cout << USAGE_MESSAGE;
exit(EXIT_SUCCESS);
case OPT_VERSION:
cout << VERSION_MESSAGE;
exit(EXIT_SUCCESS);
}
if (optarg != NULL && !arg.eof()) {
cerr << PROGRAM ": invalid option: `-"
<< (char)c << optarg << "'\n";
exit(EXIT_FAILURE);
}
}
if (opt::section == 0 || opt::section > opt::nsections
|| delim != '/') {
cerr << PROGRAM ": -s, --section option is incorrectly set\n";
die = true;
}
if (opt::k <= 0) {
cerr << PROGRAM ": missing -k,--kmer option\n";
die = true;
}
Kmer::setLength(opt::k);
if (argc - optind < 2) {
cerr << PROGRAM ": missing arguments\n";
die = true;
}
if (die) {
cerr << "Try `" << PROGRAM
<< " --help' for more information.\n";
exit(EXIT_FAILURE);
}
string refFastaFile(argv[--argc]);
int numQuery = argc - optind;
if (opt::threads <= 0)
opt::threads = numQuery;
// SAM headers.
cout << "@HD\tVN:1.0\n"
"@PG\tID:" PROGRAM "\tVN:" VERSION "\t"
"CL:" << commandLine << '\n';
size_t numKmer = countKmer(refFastaFile);
if (opt::multimap == opt::MULTIMAP) {
g_aligner_m = new Aligner<SeqPosHashMultiMap>(opt::k,
numKmer);
readContigsIntoDB(refFastaFile, *g_aligner_m);
} else {
#if HAVE_GOOGLE_SPARSE_HASH_MAP
g_aligner_u = new Aligner<SeqPosHashUniqueMap>(opt::k,
numKmer, 0.3);
#else
g_aligner_u = new Aligner<SeqPosHashUniqueMap>(opt::k,
numKmer);
#endif
readContigsIntoDB(refFastaFile, *g_aligner_u);
}
g_readCount = 0;
vector<pthread_t> producer_threads;
transform(argv + optind, argv + argc,
back_inserter(producer_threads), getReadFiles);
vector<pthread_t> threads;
for (int i = 0; i < opt::threads; i++) {
pthread_t thread;
pthread_create(&thread, NULL, alignReadsToDB, NULL);
threads.push_back(thread);
}
pthread_t out_thread;
pthread_create(&out_thread, NULL, printAlignments, NULL);
void *status;
// Wait for all threads to finish.
for (size_t i = 0; i < producer_threads.size(); i++)
pthread_join(producer_threads[i], &status);
for (size_t i = 0; i < threads.size(); i++)
pthread_join(threads[i], &status);
g_pipeOut.close();
pthread_join(out_thread, &status);
if (opt::verbose > 0)
cerr << "Aligned " << g_alignedCount
<< " of " << g_readCount << " reads ("
<< (float)100 * g_alignedCount / g_readCount << "%)\n";
if (opt::multimap == opt::MULTIMAP)
delete g_aligner_m;
else
delete g_aligner_u;
return 0;
}
template <class SeqPosHashMap>
static void printProgress(const Aligner<SeqPosHashMap>& align,
unsigned count)
{
size_t size = align.size();
size_t buckets = align.bucket_count();
cerr << "Read " << count << " contigs. "
"Hash load: " << size << " / " << buckets
<< " = " << (float)size / buckets
<< " using " << toSI(getMemoryUsage()) << "B." << endl;
}
template <class SeqPosHashMap>
static void readContigsIntoDB(string refFastaFile,
Aligner<SeqPosHashMap>& aligner)
{
if (opt::verbose > 0)
cerr << "Reading target `" << refFastaFile << "'..." << endl;
unsigned count = 0;
FastaReader in(refFastaFile.c_str(), FastaReader::FOLD_CASE);
if (opt::nsections > 1)
in.split(opt::section, opt::nsections);
for (FastaRecord rec; in >> rec;) {
if (count == 0) {
// Detect colour-space contigs.
opt::colourSpace = isdigit(rec.seq[0]);
} else {
if (opt::colourSpace)
assert(isdigit(rec.seq[0]));
else
assert(isalpha(rec.seq[0]));
}
cout << "@SQ\tSN:" << rec.id
<< "\tLN:" << rec.seq.length() << '\n';
aligner.addReferenceSequence(rec.id, rec.seq);
count++;
if (opt::verbose > 0 && count % 100000 == 0)
printProgress(aligner, count);
}
assert(in.eof());
if (opt::verbose > 0)
printProgress(aligner, count);
if (opt::multimap == opt::IGNORE) {
// Count the number of duplicate k-mer in the target.
size_t duplicates = aligner.countDuplicates();
if (duplicates > 0)
cerr << "Found " << duplicates
<< " (" << (float)100 * duplicates / aligner.size()
<< "%) duplicate k-mer.\n";
}
}
/** Read each fasta record from 'in', and add it to 'pipe'. */
static void readFile(FastaReader& in, Pipe<FastaRecord>& pipe)
{
for (FastaRecord rec; in >> rec;)
pipe.push(rec);
assert(in.eof());
pipe.close();
}
/** Producer thread. */
static void* readFile(void* arg)
{
WorkerArg* p = static_cast<WorkerArg*>(arg);
readFile(p->in, p->out);
delete p;
return NULL;
}
/** @Returns the time in seconds between [start, end]. */
static double timeDiff(const timeval& start, const timeval& end)
{
double result = (double)end.tv_sec +
(double)end.tv_usec/1000000.0;
result -= (double)start.tv_sec +
(double)start.tv_usec/1000000.0;
return result;
}
static void* alignReadsToDB(void*)
{
opt::chastityFilter = false;
opt::trimMasked = false;
static timeval start, end;
pthread_mutex_lock(&g_mutexCerr);
gettimeofday(&start, NULL);
pthread_mutex_unlock(&g_mutexCerr);
for (pair<FastaRecord, size_t> recPair = g_pipeMux.nextValue();
recPair.second > 0; recPair = g_pipeMux.nextValue()) {
const FastaRecord& rec = recPair.first;
const Sequence& seq = rec.seq;
ostringstream output;
if (seq.find_first_not_of("ACGT0123") == string::npos) {
if (opt::colourSpace)
assert(isdigit(seq[0]));
else
assert(isalpha(seq[0]));
}
switch (opt::format) {
case KALIGNER:
if (opt::multimap == opt::MULTIMAP)
g_aligner_m->alignRead(rec.id, seq,
affix_ostream_iterator<Alignment>(
output, "\t"));
else
g_aligner_u->alignRead(rec.id, seq,
affix_ostream_iterator<Alignment>(
output, "\t"));
break;
case SAM:
if (opt::multimap == opt::MULTIMAP)
g_aligner_m->alignRead(rec.id, seq,
ostream_iterator<SAMRecord>(output, "\n"));
else
g_aligner_u->alignRead(rec.id, seq,
ostream_iterator<SAMRecord>(output, "\n"));
break;
}
ostringstream out;
string s = output.str();
switch (opt::format) {
case KALIGNER:
out << rec.id;
if (opt::printSeq) {
out << ' ';
if (opt::colourSpace)
out << rec.anchor;
out << seq;
}
out << s << '\n';
break;
case SAM:
out << s;
break;
}
g_pipeOut.push(OutData(out.str(), recPair.second));
// Prevent the priority_queue from growing too large by
// waiting for threads going far too slow.
pthread_mutex_lock(&g_mutexPqSize);
if (g_pqSize >= MAX_PQ_SIZE)
pthread_cond_wait(&g_pqSize_cv, &g_mutexPqSize);
pthread_mutex_unlock(&g_mutexPqSize);
if (opt::verbose > 0) {
pthread_mutex_lock(&g_mutexCerr);
if (!s.empty())
g_alignedCount++;
if (++g_readCount % 1000000 == 0) {
gettimeofday(&end, NULL);
double result = timeDiff(start, end);
cerr << "Aligned " << g_readCount << " reads at "
<< (int)(1000000 / result) << " reads/sec.\n";
start = end;
}
pthread_mutex_unlock(&g_mutexCerr);
}
}
return NULL;
}
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