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/*****************************************************************************
* GATB : Genome Assembly Tool Box
* Copyright (C) 2017 INRIA
* Authors: R.Chikhi, G.Rizk, E.Drezen
*
* This program is free software: you can redistribute it and/or modify
* it under the terms of the GNU Affero General Public License as
* published by the Free Software Foundation, either version 3 of the
* License, or (at your option) any later version.
*
* This program is distributed in the hope that it will be useful,
* but WITHOUT ANY WARRANTY; without even the implied warranty of
* MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the
* GNU Affero General Public License for more details.
*
* You should have received a copy of the GNU Affero General Public License
* along with this program. If not, see <http://www.gnu.org/licenses/>.
*****************************************************************************/
/* merci does the mercy-kmer assembly trick of megahit
*
* IMPORTANT: really need to set k as the same as the one that was used for creating those contigs/unitigs,
* otherwise it will still run but the glue will make the machine run out of memory :/
*/
/********************************************************************************/
#include <gatb/gatb_core.hpp>
#include <iostream>
#include <fstream>
#include <string>
#include <set>
#include <unordered_map>
#include <iomanip> // for setprecision
const int max_delta = 5; // important parameter
using namespace std;
static void file_copy(string from, string to) // http://stackoverflow.com/questions/10195343/copy-a-file-in-a-sane-safe-and-efficient-way
{
std::ifstream src(from, std::ios::binary);
std::ofstream dst(to, std::ios::binary);
dst << src.rdbuf();
}
static void file_append(string dest, string with_file)
{
std::ofstream fout ( dest, std::ios_base::app);
std::ifstream f2 ( with_file);
fout << f2.rdbuf();
}
template <int span> using assembly_index_t = std::unordered_map<typename Kmer<span>::Type,uint64_t>;
typedef std::unordered_map<uint64_t,set<uint64_t>> connections_index_t ;
typedef std::unordered_map<uint64_t,set<string>> connections_t ;
template<int span>
void index_assembly(string assembly, int k, int nb_threads, bool verbose, assembly_index_t<span>& index)
{
if (verbose)
std::cout << "indexing assembly extremities" << std::endl;
class IndexAssembly
{
typedef typename Kmer<span>::ModelCanonical ModelCanon;
int k;
ModelCanon modelCanon;
uint64_t current_seq;
assembly_index_t<span> &index;
int _currentThreadIndex; // for later
bool debug;
public:
IndexAssembly(int k, assembly_index_t<span> &index) :
k(k), modelCanon(k), current_seq(0),
index(index), _currentThreadIndex(-1), debug(false)
{}
void operator() (const Sequence& sequence) {
const string seq = sequence.toString();
const string comment = sequence.getComment();
const bool no_link_left = comment.find("L:-") == string::npos;
const bool no_link_right = comment.find("L:+") == string::npos;
if (no_link_left)
{
const string kmerBegin = seq.substr(0, k );
const typename ModelCanon::Kmer kmmerBegin = modelCanon.codeSeed(kmerBegin.c_str(), Data::ASCII);
bool rc = modelCanon.toString(kmmerBegin.value()) != kmerBegin; // could be optimized
ExtremityInfo e (current_seq, rc, UNITIG_BEGIN); /* actually we dont know if its false/UNITIG_BEGIN or reverse/UNITIG_END but should think about it, for now, putting naive*/
index[kmmerBegin.value()] = e.pack();
if (debug) std::cout << "index left kmer " << modelCanon.toString(kmmerBegin.value()) << " of contig " << current_seq << std::endl;
}
if (no_link_right)
{
const string kmerEnd = seq.substr(seq.size() - k , k );
const typename ModelCanon::Kmer kmmerEnd = modelCanon.codeSeed(kmerEnd.c_str(), Data::ASCII);
bool rc = modelCanon.toString(kmmerEnd.value()) != kmerEnd; // could be optimized
ExtremityInfo e (current_seq, rc, UNITIG_END);
index[kmmerEnd.value()] = e.pack();
if (debug) std::cout << "index right kmer " << modelCanon.toString(kmmerEnd.value()) << " of contig " << current_seq << std::endl;
}
current_seq++;
}
};
IndexAssembly indexAssembly(k, index);
IBank *in = Bank::open (assembly);
auto it = in->iterator();
for (it->first (); !it->isDone(); it->next())
indexAssembly(it->item());
}
/* populates connections_index and connections
* may not insert if it detects that something is not right with respect to orientations:
* ctg1 ----------[kmer1]
* ctg2 ----------[kmer2]
* is only possible when read is
* [kmer1]---[kmer2 rc] or [kmer1 rc]---[kmer2],
* where rc is relative to the orientation of the kmer in ctg1/ctg2
*
* similarly,
* ctg1 ----------[kmer1]
* ctg2 [kmer2]---------
* is only possible when read is
* [kmer1]----[kmer2] or [kmer2 rc]----[kmer1 rc]]
*
* now, consider that kmer1 is either in its forward or reverse orientation with respect to the contig.
*/
static void
insert_connection(uint64_t previous_extremityinfo, uint64_t current_extremityinfo, connections_index_t &connections_index, connections_t &connections, int previous_pos, int current_pos, bool previous_rc, bool current_rc, const string &seq, int k, bool verbose)
{
/* there are two ways to write a connection and we need to write them both */
ExtremityInfo pe(previous_extremityinfo);
ExtremityInfo ce(current_extremityinfo);
if (pe.unitig == ce.unitig) return; // one case where we actually don't insert
if (pe.rc) previous_rc = !previous_rc; // haha i'm not proud of myself but i didn't carefully check that code. but it seems to make sense and no crashes anymore, so..
if (ce.rc) current_rc = !current_rc;
if (pe.pos == ce.pos && (previous_rc == current_rc)) return;
if (pe.pos != ce.pos && (previous_rc != current_rc)) return;
// at this points we don't need to care about rc anymore, we know that it's all good. somehow_merge() will somehow figure it out
uint64_t ce_packed = ce.pack_norc();
uint64_t pe_packed = pe.pack_norc();
connections_index[pe_packed].insert(ce_packed);
connections_index[ce_packed].insert(pe_packed);
// this the part of the read that is the connection sequence
string connection = seq.substr(previous_pos,current_pos-previous_pos+k);
connections[pe_packed].insert(connection);
connections[ce_packed].insert(connection);
//if (verbose) std::cout << "found connection between contig " << pe.unitig << " and contig " << ce.unitig << " : " << seq.substr(previous_pos,current_pos-previous_pos+k) << std::endl;
}
template<int span>
void se
10BC8
arch_reads(string reads, int k, int nb_threads, bool verbose, assembly_index_t<span>& assembly_index, connections_index_t &connections_index, connections_t &connections)
{
if (verbose)
std::cout << "searching reads for assembly extremities" << std::endl;
std::mutex insertMutex;
uint64_t nb_found_connections = 0;
class SearchReads
{
typedef typename Kmer<span>::ModelCanonical ModelCanon;
int k;
bool verbose;
ModelCanon modelCanon;
connections_index_t &connections_index;
connections_t &connections;
std::mutex &insertMutex;
assembly_index_t<span> &assembly_index;
int _currentThreadIndex; // for later
uint64_t &nb_found_connections;
public:
SearchReads(int k, bool verbose, assembly_index_t<span> &assembly_index, connections_index_t &connections_index, connections_t &connections, std::mutex &insertMutex, uint64_t &nb_found_connections) :
k(k), verbose(verbose), modelCanon(k), connections_index(connections_index), connections(connections), insertMutex(insertMutex),
assembly_index(assembly_index), _currentThreadIndex(-1), nb_found_connections(nb_found_connections)
{}
void operator() (/* not making it const because if itKmer */ Sequence& sequence) {
const string seq = sequence.toString();
const string comment = sequence.getComment();
typename Kmer<span>::ModelCanonical::Iterator itKmer (modelCanon);
itKmer.setData (sequence.getData());
int previous_pos = -1;
int current_pos = 0;
uint64_t previous_extremityinfo = 0;
bool previous_rc = false, current_rc = false;
//std::cout << "seq: " << seq << std::endl;
for (itKmer.first(); !itKmer.isDone(); itKmer.next())
{
typedef typename Kmer<span>::Type Type;
Type kmer = itKmer.item().value();
//std::cout << "testing kmer " << modelCanon.toString(kmer) << std::endl;
auto hit = assembly_index.find(kmer) ;
if (hit != assembly_index.end())
{
const string kmer_seq = seq.substr(current_pos,k);
if (kmer_seq.find("N") != string::npos)
continue;//discard kmers with N's
current_rc = modelCanon.toString(kmer) != kmer_seq;
uint64_t current_extremityinfo = hit->second;
if (previous_pos != -1 && current_pos - previous_pos < max_delta)
{
insertMutex.lock();
insert_connection(previous_extremityinfo, current_extremityinfo, connections_index, connections, previous_pos, current_pos, previous_rc, current_rc, seq, k, verbose);
nb_found_connections++;
insertMutex.unlock();
}
previous_pos = current_pos;
previous_rc = current_rc;
previous_extremityinfo = current_extremityinfo;
}
current_pos++;
}
}
};
SearchReads searchReads(k, verbose, assembly_index, connections_index, connections, insertMutex, nb_found_connections);
IBank *in = Bank::open (reads);
Dispatcher dispatcher (nb_threads);
dispatcher.iterate (in->iterator(), searchReads);
/*auto it = in->iterator(); // single threaded version
for (it->first (); !it->isDone(); it->next())
searchReads(it->item());
*/
if (verbose) std::cout << "found " << nb_found_connections << " connections" << std::endl;
}
// there are not so many places where i need a sequence revcomp. GraphUnitigs and here.
static
char revcomp (char s) {
if (s == 'A') return 'T';
else if (s == 'C') return 'G';
else if (s == 'G') return 'C';
else if (s == 'T') return 'A';
else if (s == 'a') return 't';
else if (s == 'c') return 'g';
else if (s == 'g') return 'c';
else if (s == 't') return 'a';
return 'N';
}
static string revcomp (const string &s) {
string rc;
for (signed int i = s.length() - 1; i >= 0; i--) {rc += revcomp(s[i]);}
return rc;
}
/* naive, cause I'm lazy */
static bool
somehow_merge(string &seq, const string &extension, unsigned int k, bool verbose, int pos)
{
const string rev_extension = revcomp(extension);
const string extrem = seq.substr(pos,k);
const vector<string> extensions {extension, rev_extension};
for (auto e: extensions)
{
if (pos == 0)
{
/* [ ]---seq----
* -e-[ ]
*/
if (e.substr(e.size()-k) == extrem)
{
seq = e.substr(0,e.size()-k) + seq;
return true;
}
}
else
{
/* ----seq-----[ ]
* [ ]--e--
*/
if (e.substr(0,k) == extrem)
{
seq = seq + e.substr(k);
return true;
}
}
}
//std::cout << "oops, couldn't somehow merge " << seq << " with " << extension << std::endl;
/* yeah well, that's the following situation (all in forward orientation, no rc):
* assembly:
* [kmer1]--------
* [kmer2]--------
* read:
* --[kmer1]--[kmer2]--
* may occurs due to EC removal
*
*
* --------------[kmer2]-----
* /
* / EC
* /
* -----[kmer1]----
*
* so whenever this situation occurs, let's not glue again anyhow
*/
return false;
}
/* examines connections_index to see how contigs are connected to each other
* returns true for "compactable" nodes, those which have single out-neighbor and single-inneighbor in the connection graph
* oh indeed, this is a compaction problem all over again
* well no wonder why i'm using bglue after then
*
* unpure function: updates seqs_to_glue
*/
static bool maybe_merge(uint64_t packed, connections_index_t &connections_index, connections_t &connections, set<uint64_t> &seqs_to_glue, bool given_verbose, string &seq, int k, int pos)
{
bool verbose = given_verbose;
if (connections_index.find(packed) == connections_index.end())
return false;
ExtremityInfo current(packed);
set<uint64_t> &links = connections_index[packed];
if (links.size() != 1)
{
if (verbose) std::cout << "bad conn (" << (pos==UNITIG_BEGIN?"beg":"end") << "), contig: " << current.unitig << (current.pos==UNITIG_BEGIN?"beg":"end") << " connections: " << links.size() << std::endl;
return false;
}
ExtremityInfo other(*connections_index[packed].begin());
// check the incoming links of that other
if (connections_index[other.pack_norc()].size() != 1)
{
if (verbose) std::cout << "bad conn (" << (pos==UNITIG_BEGIN?"beg":"end") << "), other contig: " << other.unitig << (other.pos==UNITIG_BEGIN?"beg":"end") << " connections: " << connections_index[other.pack_norc()].size() << std::endl;
return false;
}
if (connections[packed].size() != 1) // may return false if it was previously cleared
return false;
// all good, we keep this one
// let's try to merge it
const string extension = *(connections[packed].begin());
if (!somehow_merge(seq, extension, k, verbose, pos==UNITIG_BEGIN?0:(seq.size()-k)))
return false;
// merge went okay!
// to avoid redundancy, we delete the other
if (verbose) std::cout << "good conn (" << (pos==UNITIG_BEGIN?"beg":"end") << "): " << current.unitig << " connections: " << links.size() << std::endl;
if (verbose) std::cout << "clearing connections of contig " << other.unitig << std::endl;
connections[other.pack_norc()].clear();
seqs_to_glue.insert(other.pack_norc());
return true;
}
static void
parse_unitig_header(string header, float& mean_abundance)
{
bool debug = false;
if (debug) std::cout << "parsing unitig links for " << header << std::endl;
std::stringstream stream(header);
while(1) {
string tok;
stream >> tok;
if(!stream)
break;
if (tok.size() < 3)
// that's the id, skip it
continue;
string field = tok.substr(0,2);
if (field == "km")
{
mean_abundance = atof(tok.substr(tok.find_last_of(':')+1).c_str());
//std::cout << "unitig " << header << " mean abundance " << mean_abundance << std::endl;
}
}
}
void renumber_glue_file(string glue_filename, uint64_t nb_out_tigs)
{
{
std::ifstream infile(glue_filename);
std::ofstream outfile(glue_filename+".tmp");
std::string line;
uint64_t counter = 1;
while (std::getline(infile, line))
{
if (line[0] == '>')
{
size_t space_pos = line.find(' ');
/* // yolo
if (space_pos >= line.size())
{
std::cout << "error: no space in this glue file header (" << line << ") contact a developer." << std::endl;
exit(1);
}
*/
auto end_header = line.substr(space_pos);
string new_header = ">" + std::to_string(nb_out_tigs+counter) + end_header;
outfile << new_header << std::endl;
counter++;
}
F438
else
outfile << line << std::endl;
}
} // closes files
file_copy(glue_filename+".tmp",glue_filename);
System::file().remove (glue_filename+".tmp");
}
static void
extend_assembly_with_connections(const string assembly, int k, int nb_threads, bool verbose, connections_index_t &connections_index, connections_t &connections, BankFasta &out, BankFasta &glue)
{
if (verbose)
std::cout << "extending assembly with unambiguous connections" << std::endl;
IBank *in = Bank::open (assembly);
auto it = in->iterator();
uint64_t tig_index = 0;
set<uint64_t> seqs_to_glue;
for (it->first (); !it->isDone(); it->next())
{
const Sequence &sequence = it->item();
string seq = sequence.toString();
const string comment = sequence.getComment();
bool debug = false;
ExtremityInfo beg(tig_index, false, UNITIG_BEGIN);
ExtremityInfo end(tig_index, false, UNITIG_END);
bool lmark, rmark;
// see if the contig was already previously marked as one to glue
lmark = (seqs_to_glue.find(beg.pack_norc()) != seqs_to_glue.end());
rmark = (seqs_to_glue.find(end.pack_norc()) != seqs_to_glue.end());
if (debug) if (lmark || rmark) std::cout << "tig " << tig_index << " already marked with " << lmark << rmark << std::endl;
if ((!lmark) && maybe_merge(beg.pack_norc(), connections_index, connections, seqs_to_glue, debug, seq, k, UNITIG_BEGIN)) // order of evaluation matters, as check_connection is unpure
lmark = true;
if ((!rmark) && maybe_merge(end.pack_norc(), connections_index, connections, seqs_to_glue, debug, seq, k, UNITIG_END))
rmark = true;
if (debug) std::cout << "tig " << tig_index << " new mark " << lmark << rmark << std::endl;
Sequence s (Data::ASCII);
s.getData().setRef ((char*)seq.c_str(), seq.size());
s._comment = string(lmark?"1":"0")+string(rmark?"1":"0"); //We set the sequence comment.
s._comment += " ";
// add coverage information
float mean_abundance;
parse_unitig_header(comment,mean_abundance);
uint nb_kmers = seq.size() - k + 1;
for (uint i = 0; i < nb_kmers; i++)
s._comment += std::to_string((uint)mean_abundance) + " ";
if (lmark || rmark)
glue.insert(s);
else
{
s._comment = comment;
out.insert(s);
}
tig_index++;
}
}
template<int span>
void merci(int k, string reads, string assembly, int nb_threads, bool verbose)
{
if (verbose)
cout << "k: " << k << " threads: " << nb_threads << " reads: " << reads << " assembly: " << assembly << endl;
// make a copy of the assembly, and find links
string linked_assembly = assembly + ".linked";
file_copy(assembly, linked_assembly);
uint64_t nb_tigs = 0;
bool renumber_unitigs = true; // let's allow the input to be anything. Here it doesn't amtter much. We anyway renumber at the end
link_tigs<span>( linked_assembly, k, nb_threads, nb_tigs, verbose, renumber_unitigs);
// real trick here
// tigs of length exactly k are annoying, they need to be handled carefully with UNITIG_BOTH positions
// so to avoid that for now, I'm just going to do the rest of the program using (k-1)-mers, so that each tig has a clear beginning and end
// we lose one nucleotide of specificity, should i'm willing to accept that for now
k -= 1;
// index extremities of assembly that do not have links
assembly_index_t<span> index;
index_assembly<span>(linked_assembly, k, nb_threads, verbose, index);
// now pass the reads and find putative connections
connections_index_t connections_index; // associates a contig to a connection
connections_t connections; // a connection is basically a chunk of read linking two indexed kmers close to each other
search_reads<span>(reads,k,nb_threads,verbose, index, connections_index, connections);
// pass the assembly again and slightly extend some contigs with unambiguous connections
System::file().remove (assembly+".merci");
BankFasta out (assembly+".merci");
BankFasta glue(assembly+".glue.glue"); // bglue will glue the .glue.glue to .glue
extend_assembly_with_connections(assembly, k, nb_threads, verbose, connections_index, connections, out, glue);
glue.flush();
// glue what needs to be glued. magic, we're re-using bcalm code
bglue<span> (nullptr /*no storage*/, assembly+".glue", k, 0, nb_threads, false, verbose);
// renumber the .glue file just to avoid ID collision with .merci file
renumber_glue_file(assembly+".glue", nb_tigs );
// append glued to merci
out.flush();
file_append(assembly+".merci", assembly+".glue");
// bglue drop links so let's recreate them
k += 1;
file_copy(assembly+".merci", assembly+".merci.b4link");
renumber_unitigs = true; // here it's absolutely mandatory to renumber if we want the output to be processed by minia
link_tigs<span>( assembly+".merci", k, nb_threads, nb_tigs, verbose, false, renumber_unitigs);
}
class Merci : public gatb::core::tools::misc::impl::Tool
{
public:
/** Constructor. */
Merci () : Tool("merci")
{
#ifdef GIT_SHA1
std::cout << "Merci for Minia 3, git commit " << GIT_SHA1 << std::endl;
#endif
// reinit the parser to get rid of options added by the Tool class
setParser (new OptionsParser ("merci"));
OptionsParser* assemblyParser = new OptionsParser ("assembly");
assemblyParser->push_front (new OptionOneParam ("-reads", "input reads (fasta/fastq/compressed)", true));
assemblyParser->push_front (new OptionOneParam ("-assembly", "assembly to improve", true));
assemblyParser->push_front (new OptionOneParam (STR_KMER_SIZE, "kmer size", true));
assemblyParser->push_front (new OptionOneParam (STR_NB_CORES, "number of threads", false, "0")); // apparently its needed when there is no parser
assemblyParser->push_front (new OptionOneParam (STR_VERBOSE, "verbosity level", false, "1"));
getParser()->push_back (assemblyParser);
}
struct Parameter
{
string reads, assembly;
int k,t;
bool verbose;
};
template<size_t span>
struct MerciFunctor
{
void operator() (Parameter parameter)
{
merci<span>(parameter.k,parameter.reads,parameter.assembly,parameter.t,parameter.verbose);
}
};
/** \copydoc Tool::execute. */
void execute ()
{
/** we get the kmer size chosen by the end user. */
int kmerSize = getInput()->getInt (STR_KMER_SIZE);
string reads = getInput()->getStr ("-reads");
string assembly = getInput()->getStr ("-assembly");
int nb_threads = getInput()->getInt(STR_NB_CORES);
bool verbose = getInput()->getInt("-verbose") == 1;
/** We launch Merci with the correct Integer implementation according to the choosen kmer size. */
Integer::apply<MerciFunctor,Parameter> (kmerSize, Parameter{reads,assembly,kmerSize, nb_threads, verbose});
getInfo()->add (1, getTimeInfo().getProperties("time"));
}
private:
};
/********************************************************************************/
int main (int argc, char* argv[])
{
// We define a try/catch block in case some method fails (bad filename for instance)
try
{
/** We execute dsk. */
Merci().run (argc, argv);
}
catch (OptionFailure& e)
{
return e.displayErrors (cout);
}
catch (Exception& e)
{
cerr << "EXCEPTION: " << e.getMessage() << endl;
return EXIT_FAILURE;
}
return EXIT_SUCCESS;
}