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clip_consensus_builder.cpp
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clip_consensus_builder.cpp
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#include <fstream>
#include <iostream>
#include <htslib/sam.h>
#include <htslib/kseq.h>
#include <unistd.h>
KSEQ_INIT(int, read)
#include "config.h"
#include "libs/cptl_stl.h"
#include "sam_utils.h"
#include "sw_utils.h"
config_t config;
std::mutex mtx;
std::string workdir;
std::vector<std::string> contig_id2name;
std::unordered_map<std::string, std::pair<char*, size_t> > chrs;
struct consensus_t {
bool left_clipped;
int contig_id, start, end;
std::string consensus;
int sc_reads;
consensus_t(bool left_clipped, int contig_id, int start, int end, const std::string& consensus, int sc_reads)
: left_clipped(left_clipped), contig_id(contig_id), start(start), end(end), consensus(consensus),
sc_reads(sc_reads) {}
char dir() { return left_clipped ? 'L' : 'R'; }
std::string name() {
std::stringstream ss;
ss << contig_id << "_" << start << "_" << end << "_" << dir() << "_" << sc_reads;
return ss.str();
}
int clipped_pos() const { return left_clipped ? start : end; }
};
std::vector<std::vector<consensus_t*> > consensus_vectors;
auto clip_start = [](bam1_t* r, bool left_clipped) {return left_clipped ? get_unclipped_start(r) : bam_endpos(r);};
auto clip_end = [](bam1_t* r, bool left_clipped) {return left_clipped ? r->core.pos : get_unclipped_end(r);};
std::string build_full_consensus_seq(std::vector<bam1_t*>& clipped, bool left_clipped) {
sort(clipped.begin(), clipped.end(), [&left_clipped](bam1_t* r1, bam1_t* r2) {
return clip_start(r1, left_clipped) < clip_start(r2, left_clipped);});
int first_clip_start = clip_start(clipped[0], left_clipped);
hts_pos_t last_clip_end = 0;
for (bam1_t* r : clipped) {
last_clip_end = std::max(last_clip_end, clip_end(r, left_clipped));
}
int clip_len = last_clip_end - first_clip_start;
if (clip_len < config.min_clip_len) return "";
char consensus[MAX_READ_SUPPORTED];
std::vector<int> clips_start, clips_end;
std::vector<const uint8_t*> read_seqs;
for (bam1_t* r : clipped) {
clips_start.push_back(clip_start(r, left_clipped)-first_clip_start);
clips_end.push_back(clip_end(r, left_clipped)-first_clip_start);
read_seqs.push_back(bam_get_seq(r));
}
int s = 0;
for (int i = 0; i < clip_len; i++) {
while (s < clipped.size() && clips_end[s] < i) s++;
int a = 0, c = 0, g = 0, t = 0;
for (int j = s; j < clipped.size() && clips_start[j] <= i; j++) {
if (clips_end[j] <= i) continue;
bam1_t* r = clipped[j];
int clip_start_in_read = left_clipped ? 0 : r->core.l_qseq - (get_unclipped_end(r)-bam_endpos(r));
uint8_t nucl = bam_seqi(read_seqs[j], clip_start_in_read + i-clips_start[j]);
if (nucl == 1) a++;
else if (nucl == 2) c++;
else if (nucl == 4) g++;
else if (nucl == 8) t++;
}
if (a+c+g+t == 0) {
return "";
} else if (a >= c && a >= g && a >= t) {
consensus[i] = 'A';
} else if (c >= a && c >= g && c >= t) {
consensus[i] = 'C';
} else if (g >= a && g >= c && g >= t) {
consensus[i] = 'G';
} else if (t >= a && t >= c && t >= g) {
consensus[i] = 'T';
}
}
consensus[clip_len] = '\0';
return std::string(consensus);
}
bool validate_clip(const consensus_t* consensus, StripedSmithWaterman::Aligner& aligner) {
std::pair<char *, size_t>& temp = chrs[contig_id2name[consensus->contig_id]];
char* chr = temp.first;
size_t chr_len = temp.second;
int region_len = consensus->consensus.length() * 1.1;
int region_start = consensus->left_clipped ? consensus->start-region_len : consensus->end;
if (region_start < 0) region_start = 0;
if (region_start+region_len >= chr_len) region_len = chr_len-region_start-1;
if (region_len <= 0) return true; // clipped at the end of a contig
for (int i = region_start; i <= region_start+region_len; i++) {
if (chr[i] == 'N') return false;
}
StripedSmithWaterman::Alignment alignemnt;
StripedSmithWaterman::Filter filter;
int mask_len = consensus->consensus.length()/2;
if (mask_len < 15) mask_len = 15; // suppress the warning
try {
aligner.Align(consensus->consensus.data(), chr+region_start, region_len, filter, &alignemnt, mask_len);
} catch (std::bad_alloc e) {
std::cout << "Sizes: " << consensus->consensus.size() << " " << region_len << std::endl;
}
if (alignemnt.query_end-alignemnt.query_begin >= consensus->consensus.length()*0.9
&& get_matches(alignemnt) >= consensus->consensus.length()*0.8
&& get_gaps(alignemnt) < alignemnt.query_end-alignemnt.query_begin*0.15) {
return false; // clip can be realigned well next to the anchor
} else {
return true;
}
}
consensus_t* build_clip_consensus(int contig_id, std::vector<bam1_t*>& clipped, bool left_clipped,
StripedSmithWaterman::Aligner& aligner) {
if (clipped.size() <= 2) return NULL;
std::string consensus = build_full_consensus_seq(clipped, left_clipped);
if (consensus == "") return NULL;
int first_clip_start = clip_start(clipped[0], left_clipped);
std::vector<bam1_t*> accepted_clips;
for (bam1_t* r : clipped) {
const uint8_t* read_seq = bam_get_seq(r);
int mm = 0;
int clip_start_in_consensus = clip_start(r, left_clipped)-first_clip_start;
int clip_end_in_consensus = clip_end(r, left_clipped)-first_clip_start;
int clip_start_in_read = left_clipped ? 0 : r->core.l_qseq - (get_unclipped_end(r)-bam_endpos(r));
for (int i = clip_start_in_consensus, j = clip_start_in_read; i < clip_end_in_consensus; i++, j++) {
if (consensus[i] != get_base(read_seq, j)) {
mm++;
}
}
if (mm < std::ceil(MAX_SEQ_ERROR * consensus.length())) {
accepted_clips.push_back(r);
}
}
clipped.swap(accepted_clips);
if (clipped.size() <= 2) {
clipped.clear();
return NULL;
} else {
hts_pos_t start = INT32_MAX, end = 0;
for (bam1_t* r : clipped) {
start = std::min(start, r->core.pos);
end = std::max(end, bam_endpos(r));
}
consensus = build_full_consensus_seq(clipped, left_clipped);
return new consensus_t(left_clipped, contig_id, start, end, consensus, clipped.size());
}
}
void build_clip_consensuses(int id, int contig_id) {
mtx.lock();
std::cout << "Building consensus for " << contig_id << "-CLIP.bam (" << contig_id2name[contig_id] << ")" << std::endl;
mtx.unlock();
StripedSmithWaterman::Aligner aligner(2,2,4,1,true);
std::vector<consensus_t*>& clip_consensuses = consensus_vectors[contig_id];
std::string bam_fname = workdir + "/workspace/" + std::to_string(contig_id) + "-CLIP.bam";
open_samFile_t* bam_file = open_samFile(bam_fname.c_str(), true);
hts_itr_t* iter = sam_itr_querys(bam_file->idx, bam_file->header, contig_id2name[contig_id].c_str());
bam1_t* read = bam_init1();
// divide soft-clipped reads into left-clipped and right-clipped
std::vector<bam1_t*> lc_reads, rc_reads;
while (sam_itr_next(bam_file->file, iter, read) >= 0) {
if (is_left_clipped(read, config.min_clip_len)) {
lc_reads.push_back(bam_dup1(read));
}
if (is_right_clipped(read, config.min_clip_len)) {
rc_reads.push_back(bam_dup1(read));
}
}
bam_destroy1(read);
close_samFile(bam_file);
hts_itr_destroy(iter);
auto lc_same_cluster = [](bam1_t* r1, bam1_t* r2) {return abs(r1->core.pos-r2->core.pos) <= config.max_sc_dist;};
auto rc_same_cluster = [](bam1_t* r1, bam1_t* r2) {return abs(bam_endpos(r1)-bam_endpos(r2)) <= config.max_sc_dist;};
std::vector<bam1_t*> curr_candidate_cluster;
if (!lc_reads.empty()) {
for (bam1_t* lc_read : lc_reads) {
if (!curr_candidate_cluster.empty() &&
!lc_same_cluster(curr_candidate_cluster[0], lc_read)) { // candidate cluster complete
consensus_t* consensus = build_clip_consensus(contig_id, curr_candidate_cluster, true, aligner);
curr_candidate_cluster.clear();
if (consensus != NULL) {
clip_consensuses.push_back(consensus);
}
}
curr_candidate_cluster.push_back(lc_read);
}
// process last cluster
consensus_t* consensus = build_clip_consensus(contig_id, curr_candidate_cluster, true, aligner);
curr_candidate_cluster.clear();
if (consensus != NULL) {
clip_consensuses.push_back(consensus);
}
}
sort(rc_reads.begin(), rc_reads.end(), [](bam1_t* r1, bam1_t* r2) {return bam_endpos(r1) < bam_endpos(r2);});
if (!rc_reads.empty()) {
for (bam1_t* rc_read : rc_reads) {
if (!curr_candidate_cluster.empty() &&
!rc_same_cluster(curr_candidate_cluster[0], rc_read)) { // candidate cluster complete
consensus_t* consensus = build_clip_consensus(contig_id, curr_candidate_cluster, false, aligner);
curr_candidate_cluster.clear();
if (consensus != NULL) {
clip_consensuses.push_back(consensus);
}
}
curr_candidate_cluster.push_back(rc_read);
}
// process last cluster
consensus_t* consensus = build_clip_consensus(contig_id, curr_candidate_cluster, false, aligner);
curr_candidate_cluster.clear();
if (consensus != NULL) {
clip_consensuses.push_back(consensus);
}
}
for (bam1_t* lc_read : lc_reads) {
bam_destroy1(lc_read);
}
for (bam1_t* rc_read : rc_reads) {
bam_destroy1(rc_read);
}
// remove non-validated clips
// clip_consensuses.erase(std::remove_if(clip_consensuses.begin(), clip_consensuses.end(),
// [&aligner](const consensus_t* c) {return !validate_clip(c, aligner);}),
// clip_consensuses.end());
// sort by clipping position
sort(clip_consensuses.begin(), clip_consensuses.end(), [](consensus_t* c1, consensus_t* c2) {
return c1->clipped_pos() < c2->clipped_pos();});
}
int main(int argc, char* argv[]) {
workdir = std::string(argv[1]);
std::string workspace = workdir + "/workspace/";
std::string reference_fname = std::string(argv[2]);
FILE* fastaf = fopen(reference_fname.c_str(), "r");
kseq_t *seq = kseq_init(fileno(fastaf));
while (kseq_read(seq) >= 0) {
chrs[std::string(seq->name.s)] = std::make_pair(new char[seq->seq.l+1], seq->seq.l);
strcpy(chrs[std::string(seq->name.s)].first, seq->seq.s);
}
kseq_destroy(seq);
fclose(fastaf);
config = parse_config(workdir + "/config.txt");
std::ifstream contig_map_fin(workdir + "/contig_map");
std::string contig_name; int contig_id;
while (contig_map_fin >> contig_name >> contig_id) {
contig_id2name.push_back(contig_name);
}
consensus_vectors.resize(contig_id2name.size());
ctpl::thread_pool thread_pool(config.threads);
std::vector<std::future<void> > futures;
for (int i = 0; i < contig_id2name.size(); i++) {
std::future<void> future = thread_pool.push(build_clip_consensuses, i);
futures.push_back(std::move(future));
}
thread_pool.stop(true);
for (int i = 0; i < futures.size(); i++) {
futures[i].get();
}
std::ofstream fa_out(workspace + "/CLIPS.fa");
for (int i = 0; i < contig_id2name.size(); i++) {
std::vector<consensus_t*>& cv = consensus_vectors[i];
for (consensus_t* consensus : cv) {
fa_out << ">" << consensus->name() << "\n" << consensus->consensus << "\n";
delete consensus;
}
}
fa_out.close();
for (auto& chr : chrs) {
delete[] chr.second.first;
}
}