void bounds(const Geometry& geo, vec3& lower, vec3& upper)
{
vec3 lbound(std::numeric_limits<float>::max());
vec3 ubound(-std::numeric_limits<float>::max());
for (auto& pos : geo.positions) {
lbound = min(pos, lbound);
ubound = max(pos, ubound);
}
lower = lbound;
upper = ubound;
}
int sputnik_ssr::find_primers(char* seq, int start, int end, int repeat_len) {
//int good_candidates = 0;
// This pattern is the repeating motif and shouldn't be found in the primer
char repeat[repeat_len + 1];
for (int i=start; i < start + repeat_len; i++) {
repeat[i - start] = seq[i];
}
repeat[repeat_len] = 0;
// Set locations for search
int fwd5begin = start - 90;
int fwd5end = start - 30;
if(fwd5begin < 1) { return 0; }
int rev5begin = end + 90;
int rev5end = end + 30;
if(rev5begin > (int) strlen(seq)) { return 0; }
revP->reverse_primer = TRUE;
revP->start_location_range_begin = rev5begin;
revP->start_location_range_end = rev5end;
revP->length_range_shortest = 20;
revP->length_range_longest = 30;
revP->optimum_primer_length = revP->length_range_shortest;
revP->optimum_Tm = 57;
//revP->seq_to_avoid = repeat;
revP->set_sequence_to_avoid(repeat);
int okrev = revP->generate_candidates(seq);
if(! okrev) {
return 0;
}
// First we calculate the basic cheap properties and sort the candidates
for(int i = 0; i < revP->candidates_found; i++) {
revP->hairpin(i);
revP->self_dimer(i);
revP->candidate[i].seqsim_matches = revP->blast_seq(i, seq);
}
revP->priority[0] = SELF_DIMER;
revP->priority[1] = SEQSIM_MATCH;
revP->priority[2] = LENGTH;
revP->priority[3] = SORT_END;
if(revP->rank_selection() == ERROR) {
return 0;
}
// Next we calculate temperature for the good ones, and resort.
for(int i = 0; i < revP->good_candidates; i++) {
revP->calculate_temperature(i);
}
revP->candidates_found = revP->good_candidates;
revP->priority[0] = TEMPERATURE;
revP->priority[1] = SORT_END;
if(revP->rank_selection() == ERROR) {
return 0;
}
int bestRev = 0;
int r_primer_end = revP->candidate[bestRev].location_5_prime_end;
char rev_flank[r_primer_end - (end + 1) + 1];
strncpy(rev_flank, seq + end, r_primer_end - end+1);
rev_flank[r_primer_end - end + 1] = '\0';
if(has_repeat(rev_flank)) {
return 0;
}
char *revseq = revP->candidate[bestRev].sequence;
int len_rev = strlen(revseq);
// set the properties we need for a forward primer
fwdP->reverse_primer = FALSE;
fwdP->start_location_range_begin = fwd5begin;
fwdP->start_location_range_end = fwd5end;
fwdP->length_range_shortest = lbound(len_rev, revP->length_range_shortest);
fwdP->length_range_longest = ubound(len_rev, revP->length_range_longest);
fwdP->optimum_primer_length = fwdP->length_range_shortest;
fwdP->optimum_Tm = revP->candidate[bestRev].annealing_temperature;
fwdP->required_GC_content = sequence_utils::GC_content(revseq);
fwdP->GC_tolerance = 0;
//fwdP->seq_to_avoid = repeat;
fwdP->set_sequence_to_avoid(repeat);
int okfwd = fwdP->generate_candidates(seq);
if(! okfwd) {
return 0;
}
// First we calculate the basic cheap properties and sort the candidates
for(int i = 0; i < fwdP->candidates_found; i++) {
fwdP->hairpin(i);
fwdP->self_dimer(i);
fwdP->primer_dimer_2(i, revseq);
fwdP->candidate[i].seqsim_matches = fwdP->blast_seq(i, seq);
}
fwdP->priority[0] = SELF_DIMER;
fwdP->priority[1] = F_DIMER;
fwdP->priority[2] = R_DIMER;
revP->priority[3] = SEQSIM_MATCH;
fwdP->priority[4] = LENGTH;
fwdP->priority[5] = SORT_END;
if(fwdP->rank_selection() == ERROR) {
return 0;
}
for(int i = 0; i < fwdP->good_candidates; i++) {
fwdP->calculate_temperature(i);
}
fwdP->candidates_found = fwdP->good_candidates;
fwdP->priority[0] = TEMPERATURE;
fwdP->priority[1] = SORT_END;
if(fwdP->rank_selection() == ERROR) {
return 0;
}
int bestFwd = 0;
int f_primer_end = fwdP->candidate[bestFwd].location_5_prime_end;
char fwd_flank[start - f_primer_end + 1];
strncpy(fwd_flank, seq + f_primer_end, start - f_primer_end);
fwd_flank[start - f_primer_end] = '\0';
if(has_repeat(fwd_flank)) {
return 0;
}
display_utils display;
char * product = NULL;
display.extract_product(seq, &fwdP->candidate[bestFwd], &revP->candidate[bestRev], product);
if (sequence_utils::nucleotide_content(ANYNUCLEOTIDE,product) > 0) {
//std::cout << "Product would contains Ns" << std::endl;
return 0;
}
if(fabs(revP->candidate[bestRev].annealing_temperature - fwdP->candidate[bestFwd].annealing_temperature) > MAX_TEMP_DIFF) {
return 0;
}
// Forward
std::cout << "-----------------------------------------------" << std::endl;
std::cout << "Forward primer" << std::endl;
std::cout << "Sequence: " << fwdP->candidate[bestFwd].sequence << std::endl;
std::cout << "Size: " << strlen(fwdP->candidate[bestFwd].sequence) << std::endl;
std::cout << "Fwdflank: "<< fwd_flank << std::endl;
std::cout << "Location 5',3': " << fwdP->candidate[bestFwd].location_5_prime_end + 1 << "," << fwdP->candidate[bestFwd].location_5_prime_end + strlen(fwdP->candidate[bestFwd].sequence) << std::endl;
std::cout << "Hairpin score: " << fwdP->candidate[bestFwd].hairpin << std::endl;
std::cout << "Self dimer score: " << fwdP->candidate[bestFwd].self_dimer << std::endl;
std::cout << "Seq sim score: " << fwdP->candidate[bestFwd].seqsim_matches << std::endl;
std::cout << "Temperature: " << fwdP->candidate[bestFwd].annealing_temperature << std::endl;
// Reverse
std::cout << "Reverse primer" << std::endl;
std::cout << "Sequence: " << revseq << std::endl;
std::cout << "Size: " << strlen(revseq) << std::endl;
std::cout << "Revflank: "<< rev_flank << std::endl;
std::cout << "Location 5',3': " << revP->candidate[bestRev].location_5_prime_end + 1 << "," << revP->candidate[bestRev].location_5_prime_end + 1 - strlen(revseq)<< std::endl;
std::cout << "Hairpin score: " << revP->candidate[bestRev].hairpin << std::endl;
std::cout << "Self dimer score: " << revP->candidate[bestRev].self_dimer << std::endl;
std::cout << "Seq sim score: " << revP->candidate[bestRev].seqsim_matches << std::endl;
std::cout << "Temperature: " << revP->candidate[bestRev].annealing_temperature << std::endl;
// Primer dimer
std::cout << "Pair details" << std::endl;
std::cout << "Primer dimer score (fwd): " << fwdP->candidate[bestFwd].forward_dimer << std::endl;
std::cout << "Primer dimer score (rev): " << fwdP->candidate[bestFwd].reverse_dimer << std::endl;
// Product
std::cout << "Product length: " << strlen(product) << std::endl;
std::cout << "Product: " << product << std::endl;
free(product);
std::cout << std::endl;
return 1;
}