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/**
* Find contigs that overlap and end due to a lack of coverage.
* Written by Shaun Jackman <sjackman@bcgsc.ca>.
*/
#include "config.h"
#include "Common/Options.h"
#include "ContigProperties.h"
#include "Estimate.h"
#include "FastaReader.h"
#include "IOUtil.h"
#include "Uncompress.h"
#include "Graph/ContigGraph.h"
#include "Graph/ContigGraphAlgorithms.h"
#include "Graph/DirectedGraph.h"
#include "Graph/GraphIO.h"
#include "Graph/GraphUtil.h"
#include <boost/lambda/bind.hpp>
#include <boost/lambda/lambda.hpp>
#include <boost/ref.hpp>
#include <algorithm>
#include <cassert>
#include <cctype>
#include <climits> // for UINT_MAX
#include <cstdlib>
#include <fstream>
#include <getopt.h>
#include <iostream>
#include <sstream>
#include <string>
#include <vector>
using namespace std;
using namespace boost::lambda;
#define PROGRAM "Overlap"
static const char VERSION_MESSAGE[] =
PROGRAM " (" PACKAGE_NAME ") " VERSION "\n"
"Written by Shaun Jackman.\n"
"\n"
"Copyright 2014 Canada's Michael Smith Genome Sciences Centre\n";
static const char USAGE_MESSAGE[] =
"Usage: " PROGRAM " -k<kmer> -o<out.fa> [OPTION]... CONTIGS ADJ DIST\n"
"Find overlaps between blunt contigs that have negative distance\n"
"estimates. Add edges to the overlap graph.\n"
"\n"
" Options:\n"
"\n"
" -k, --kmer=KMER_SIZE k-mer size\n"
" -m, --min=OVERLAP require a minimum of OVERLAP bases\n"
" default is 5 bases\n"
" --scaffold join contigs with Ns [default]\n"
" --no-scaffold do not scaffold\n"
" --mask-repeat join contigs at a simple repeat and mask\n"
" the repeat sequence [default]\n"
" --no-merge-repeat don't join contigs at a repeat\n"
" --SS expect contigs to be oriented correctly\n"
" --no-SS no assumption about contig orientation [default]\n"
" -g, --graph=FILE write the contig adjacency graph to FILE\n"
" --adj output the graph in ADJ format [default]\n"
" --asqg output the graph in ASQG format\n"
" --dot output the graph in GraphViz format\n"
" --gfa output the graph in GFA1 format\n"
" --gfa1 output the graph in GFA1 format\n"
" --gfa2 output the graph in GFA2 format\n"
" --gv output the graph in GraphViz format\n"
" --sam output the graph in SAM format\n"
" -o, --out=FILE write result to FILE\n"
" -v, --verbose display verbose output\n"
" --help display this help and exit\n"
" --version output version information and exit\n"
"\n"
"Report bugs to <" PACKAGE_BUGREPORT ">.\n";
namespace opt {
unsigned k; // used by ContigGraph
static unsigned minimum_overlap = 5;
static int mask = 1;
static int scaffold = 1;
/** Run a strand-specific RNA-Seq assembly. */
static int ss;
/** The acceptable error of a distance estimate. */
unsigned distanceError = 6;
/** Write the contig adjacency graph to this file. */
static string graphPath;
/** Write the new contigs to this file. */
static string out;
/** Output format */
int format = ADJ; // used by ContigProperties
}
static const char shortopts[] = "g:k:m:o:v";
enum { OPT_HELP = 1, OPT_VERSION };
static const struct option longopts[] = {
{ "kmer", required_argument, NULL, 'k' },
{ "min", required_argument, NULL, 'm' },
{ "scaffold", no_argument, &opt::scaffold, 1 },
{ "no-scaffold", no_argument, &opt::scaffold, 0 },
{ "mask-repeat", no_argument, &opt::mask, 1 },
{ "no-merge-repeat", no_argument, &opt::mask, 0 },
{ "SS", no_argument, &opt::ss, 1 },
{ "no-SS", no_argument, &opt::ss, 0 },
{ "graph", required_argument, NULL, 'g' },
{ "adj", no_argument, &opt::format, ADJ },
{ "asqg", no_argument, &opt::format, ASQG },
{ "dot", no_argument, &opt::format, DOT },
{ "gfa", no_argument, &opt::format, GFA1 },
{ "gfa1", no_argument, &opt::format, GFA1 },
{ "gfa2", no_argument, &opt::format, GFA2 },
{ "gv", no_argument, &opt::format, DOT },
{ "sam", no_argument, &opt::format, SAM },
{ "out", required_argument, NULL, 'o' },
{ "verbose", no_argument, NULL, 'v' },
{ "help", no_argument, NULL, OPT_HELP },
{ "version", no_argument, NULL, OPT_VERSION },
{ NULL, 0, NULL, 0 }
};
/** Contig sequences. */
static vector<string> g_contigs;
/** Contig adjacency graph. */
typedef ContigGraph<DirectedGraph<ContigProperties, Distance> > Graph;
static struct {
unsigned overlap;
unsigned scaffold;
unsigned none;
unsigned tooshort;
unsigned homopolymer;
unsigned motif;
unsigned ambiguous;
} stats;
/** Return the sequence of the specified contig. */
static string sequence(const ContigNode& id)
{
const string& seq = g_contigs[id.id()];
return id.sense() ? reverseComplement(seq) : seq;
}
static unsigned findOverlap(const Graph& g,
const ContigNode& t_id,
const ContigNode& h_id,
bool& mask)
{
mask = false;
string t = sequence(t_id);
string h = sequence(h_id);
unsigned len = min(t.length(), h.length());
vector<unsigned> overlaps;
overlaps.reserve(len);
for (unsigned overlap = len; overlap >= 1; overlap--) {
if (t.substr(t.length()-overlap, overlap)
== h.substr(0, overlap))
overlaps.push_back(overlap);
}
if (opt::verbose > 0) {
cout << get(vertex_name, g, t_id)
<< '\t' << get(vertex_name, g, h_id);
for (vector<unsigned>::const_iterator i = overlaps.begin();
i != overlaps.end(); ++i)
cout << '\t' << *i;
cout << '\n';
}
if (overlaps.empty()) {
stats.none++;
return 0;
}
if (overlaps[0] < opt::minimum_overlap) {
stats.tooshort++;
return 0;
}
if (overlaps.size() >= 3
&& overlaps[0]-overlaps[1] == overlaps[1]-overlaps[2]) {
// Homopolymer run or motif.
if (overlaps[0]-overlaps[1] == 1)
stats.homopolymer++;
else
stats.motif++;
mask = true;
}
return overlaps[0];
}
static FastaRecord newContig(const Graph& g,
const ContigNode& t, const ContigNode& v,
int dist, const string& seq)
{
ostringstream comment;
comment << seq.length() << " 0 "
<< get(vertex_name, g, t) << ' '
<< get(vertex_name, g, v) << ' ' << dist;
return FastaRecord(createContigName(), comment.str(), seq);
}
/** An overlap of two sequences. */
struct Overlap : public DistanceEst {
unsigned overlap;
bool mask;
Overlap() : overlap(UINT_MAX), mask(false) { }
Overlap(int) { assert(false); }
Overlap(const DistanceEst& est, unsigned overlap, bool mask)
: DistanceEst(est), overlap(overlap), mask(mask) { }
bool operator==(const Overlap& o) const
{
return overlap == o.overlap;
}
operator Distance() const
{
assert(overlap > 0);
return -overlap;
}
friend ostream& operator<<(ostream& out, const Overlap& o)
{
return out << "d="
<< (o.overlap > 0 ? -(int)o.overlap : o.distance);
}
};
/** Create a contig representing the gap between contigs u and v. */
static FastaRecord createGapContig(const Graph& g,
const ContigNode& u, const ContigNode& v,
const Overlap& o)
{
assert(opt::scaffold);
assert(o.overlap == 0);
stats.scaffold++;
int distance = o.distance;
if (opt::verbose > 0)
cout << get(vertex_name, g, u)
<< '\t' << get(vertex_name, g, v)
<< "\t(" << distance << ")\n";
assert(distance < 100000);
string gap = distance <= 0 ? string("n")
: string(distance, 'N');
const string& useq = sequence(u);
const string& vseq = sequence(v);
unsigned overlap = opt::k - 1; // by convention
return newContig(g, u, v, distance,
useq.substr(useq.length() - overlap) + gap
+ vseq.substr(0, overlap));
}
/** The scaffold graph. Edges join two blunt contigs that are joined
* by a distance estimate. */
typedef ContigGraph<DirectedGraph<NoProperty, Overlap> > OverlapGraph;
/**
* Check for an overlap between the specified pair of contigs.
* Add the size of the overlap to the edge properties. Add the
* complementary edge if it does not exist in the graph.
* @param goverlap the contig overlap graph
* @param g the scaffold graph
* @return true if the contigs overlap
*/
static bool checkEdgeForOverlap(const Graph& goverlap,
OverlapGraph& g,
graph_traits<OverlapGraph>::edge_descriptor e)
{
typedef graph_traits<Graph>::vertex_descriptor V;
typedef graph_traits<Graph>::edge_descriptor E;
typedef edge_bundle_type<OverlapGraph>::type EP;
V u = source(e, g), v = target(e, g);
V uc = get(vertex_complement, g, u);
V vc = get(vertex_complement, g, v);
assert(u != v);
assert(u != vc);
EP& ep = g[e];
if (ep.overlap != UINT_MAX) {
// Found the complementary overlap.
return ep.overlap > 0 || opt::scaffold;
}
if (ep.distance >= 0 && !opt::scaffold) {
// Positive distance estimate and not scaffolding.
return false;
}
if (out_degree(u, goverlap) > 0 || in_degree(v, goverlap) > 0) {
// Not blunt.
return false;
}
bool mask = false;
unsigned overlap
= ep.distance - (int)allowedError(ep.stdDev) <= 0
? findOverlap(goverlap, u, v, mask) : 0;
if (mask && !opt::mask) {
// Ambiguous overlap.
return false;
}
if (overlap == 0 && !opt::scaffold) {
// No overlap and not scaffolding.
return false;
}
ep.overlap = overlap;
ep.mask = mask;
pair<E, bool> ecomplement = edge(vc, uc, g);
if (ecomplement.second) {
// Modify the complementary edge.
g[ecomplement.first] = ep;
} else {
// Add the complementary edge.
assert(vc != u);
add_edge(vc, uc, ep,
static_cast<OverlapGraph::base_type&>(g));
}
return true;
}
static void findOverlap(const Graph& g,
ContigID refID, bool rc,
const ContigNode& pair,
const DistanceEst& est,
OverlapGraph& out)
{
if (refID == pair.id()
|| (est.distance >= 0 && !opt::scaffold))
return;
ContigNode ref(refID, false);
const ContigNode& t = rc ? pair : ref;
const ContigNode& h = rc ? ref : pair;
if (out_degree(t, g) > 0 || in_degree(h, g) > 0
|| edge(t, h, out).second)
return;
bool mask = false;
unsigned overlap
= est.distance - (int)allowedError(est.stdDev) <= 0
? findOverlap(g, t, h, mask) : 0;
if (mask && !opt::mask)
return;
if (overlap > 0 || opt::scaffold)
add_edge(t, h, Overlap(est, overlap, mask), out);
}
static void readContigs(const char *contigPath)
{
FastaReader in(contigPath, FastaReader::FOLD_CASE);
for (FastaRecord rec; in >> rec;)
g_contigs.push_back(rec.seq);
assert(in.eof());
assert(!g_contigs.empty());
opt::colourSpace = isdigit(g_contigs[0][0]);
}
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();
}
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 'g': arg >> opt::graphPath; break;
case 'k': arg >> opt::k; break;
case 'm': arg >> opt::minimum_overlap; break;
case 'o': arg >> opt::out; break;
case 'v': opt::verbose++; 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::k <= 0) {
cerr << PROGRAM ": " << "missing -k,--kmer option\n";
die = true;
}
if (opt::out.empty()) {
cerr << PROGRAM ": " << "missing -o,--out option\n";
die = true;
}
if (argc - optind < 3) {
cerr << PROGRAM ": missing arguments\n";
die = true;
}
if (argc - optind > 3) {
cerr << PROGRAM ": too many arguments\n";
die = true;
}
if (die) {
cerr << "Try `" << PROGRAM
<< " --help' for more information.\n";
exit(EXIT_FAILURE);
}
const char* contigPath(argv[optind++]);
string adjPath(argv[optind++]);
string estPath(argv[optind++]);
readContigs(contigPath);
// Read the contig adjacency graph.
ifstream fin(adjPath.c_str());
assert_good(fin, adjPath);
Graph graph;
fin >> graph;
assert(fin.eof());
g_contigNames.lock();
// Open the output file.
ofstream out(opt::out.c_str());
assert_good(out, opt::out);
// Read the scaffold graph.
ifstream in(estPath.c_str());
assert_good(in, estPath);
// Find overlapping contigs.
OverlapGraph scaffoldGraph(graph.num_vertices() / 2);
if (in.peek() == 'd') {
// dot graph format
in >> scaffoldGraph;
assert(in.eof());
if (opt::verbose > 0)
printGraphStats(cout, scaffoldGraph);
remove_edge_if(
!boost::lambda::bind(checkEdgeForOverlap,
boost::cref(graph), boost::ref(scaffoldGraph),
_1),
static_cast<OverlapGraph::base_type&>(scaffoldGraph));
} else {
// dist graph format
for (EstimateRecord er; in >> er;) {
for (int sense = false; sense <= true; ++sense) {
typedef vector<
pair<ContigNode, DistanceEst> > Estimates;
const Estimates& ests = er.estimates[sense];
for (Estimates::const_iterator it = ests.begin();
it != ests.end(); ++it)
findOverlap(graph, er.refID, sense,
it->first, it->second,
scaffoldGraph);
}
}
assert(in.eof());
}
in.close();
if (opt::verbose > 1)
cout << dot_writer(scaffoldGraph);
typedef graph_traits<OverlapGraph>::vertex_descriptor
vertex_descriptor;
typedef graph_traits<OverlapGraph>::vertex_iterator
vertex_iterator;
typedef graph_traits<OverlapGraph>::edge_descriptor
edge_descriptor;
typedef graph_traits<OverlapGraph>::out_edge_iterator
out_edge_iterator;
/** The overlapping edges (d<0) of scaffoldGraph. */
OverlapGraph overlapGraph(num_vertices(graph) / 2);
/** The canonical edges of scaffoldGraph. */
unsigned numOverlaps = num_edges(scaffoldGraph) / 2;
typedef vector<edge_descriptor> Edges;
Edges edges;
edges.reserve(numOverlaps);
// Create the set of canonical edges and the overlap subgraph.
std::pair<vertex_iterator, vertex_iterator>
uit = vertices(scaffoldGraph);
for (vertex_iterator u = uit.first; u != uit.second; ++u) {
std::pair<out_edge_iterator, out_edge_iterator>
vit = out_edges(*u, scaffoldGraph);
for (out_edge_iterator e = vit.first; e != vit.second; ++e) {
vertex_descriptor v = target(*e, scaffoldGraph);
assert(*u != v);
if (v < *u)
continue;
edges.push_back(*e);
const Overlap& ep = get(edge_bundle, scaffoldGraph, e);
if (ep.overlap > 0)
add_edge(*u, v, ep, overlapGraph);
}
}
assert(edges.size() == numOverlaps);
// First, give priority to overlapping edges (not scaffolded).
for (Edges::const_iterator it = edges.begin();
it != edges.end(); ++it) {
const ContigNode& t = source(*it, overlapGraph),
h = target(*it, overlapGraph);
if (!edge(t, h, overlapGraph).second) {
// This edge is scaffolded.
continue;
}
const Overlap& overlap = get(edge_bundle, overlapGraph, *it);
assert(overlap.overlap > 0);
if (contiguous_out(overlapGraph, t)) {
stats.overlap++;
assert(*adjacent_vertices(t, overlapGraph).first == h);
add_edge(t, h, overlap, graph);
// Clear the out-edges of t and the in-edges of h.
clear_out_edges(t, scaffoldGraph);
clear_in_edges(h, scaffoldGraph);
} else
stats.ambiguous++;
}
overlapGraph.clear();
// Second, handle scaffolded edges.
g_contigNames.unlock();
for (Edges::const_iterator it = edges.begin();
it != edges.end(); ++it) {
const ContigNode& t = source(*it, scaffoldGraph),
h = target(*it, scaffoldGraph);
if (!edge(t, h, scaffoldGraph).second) {
// This edge involved a vertex that has already been used
// and removed.
continue;
}
const Overlap& overlap = get(edge_bundle,
scaffoldGraph, *it);
if (overlap.overlap > 0) {
// This edge is not scaffolded.
} else if (contiguous_out(scaffoldGraph, t)) {
assert(*adjacent_vertices(t, scaffoldGraph).first == h);
ContigNode t1 = t, h1 = h;
if (opt::ss && t.sense() && h.sense()) {
t1 = h ^ true;
h1 = t ^ true;
}
FastaRecord contig = createGapContig(graph,
t1, h1, overlap);
out << contig;
assert(out.good());
// Add the new contig to the adjacency graph.
vertex_descriptor v = add_vertex(
ContigProperties(contig.seq.length(), 0), graph);
put(vertex_name, graph, v, contig.id);
add_edge(t1, v, graph);
add_edge(v, h1, graph);
} else
stats.ambiguous++;
}
g_contigNames.lock();
out.close();
if (!opt::graphPath.empty()) {
// Output the updated adjacency graph.
ofstream fout(opt::graphPath.c_str());
assert_good(fout, opt::graphPath);
write_graph(fout, graph, PROGRAM, commandLine);
assert_good(fout, opt::graphPath);
}
cout << "Overlap: " << stats.overlap << "\n"
"Scaffold: " << stats.scaffold << "\n"
"No overlap: " << stats.none << "\n"
"Insignificant (<" << opt::minimum_overlap << "bp): "
<< stats.tooshort << "\n"
"Homopolymer: " << stats.homopolymer << "\n"
"Motif: " << stats.motif << "\n"
"Ambiguous: " << stats.ambiguous << "\n";
return 0;
}
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