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mipgen.cpp
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mipgen.cpp
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/*
Written by Evan Boyle, University of Washington
boylee [at] u.washington.edu
Copyright 2014, all rights reserved
*/
#include <map>
#include <string>
#include <fstream>
#include <iostream>
#include <iomanip>
#include <stdlib.h>
#include <vector>
#include <list>
#include <set>
#include <algorithm>
#include <sstream>
#include <ctype.h>
#include <errno.h>
#include <boost/shared_ptr.hpp>
#include <boost/lexical_cast.hpp>
#include <boost/algorithm/string.hpp>
#include "Featurev5.h"
#include "SVMipv4.h"
#include "PlusSVMipv4.h"
#include "MinusSVMipv4.h"
#include "svm.h"
using namespace std;
//LIBSVM identifiers
#define TOTAL_FEATURES 192
static string feature_mers [] = {"A","AA","AAA","AAC","AAG","AAT","AC","ACA","ACC","ACG","AG","AGA","AGC","AGG","AGT","AT","ATA","ATC","ATG","CAG","CG","CGG","G","GAC","GAG","GC","GCG","GG","GGC","GGG","GTG","TA","TAA","TAC","TAG","TC","TCC","TCG","TG","TGA","TGC","TGG","TTC","TTG"};
map<string,double> SVMipv4::junction_scores;
static int max_nr_attr = 64;
// comparison function for sorting BED records
bool compare_regions_to_scan (string a, string b)
{
if (a[0] == '>' || a[0] == '#') // header
return true;
else
{
string a_chr;
string b_chr;
if (a.substr(0,3) == "chr") a_chr = a.substr(3,a.find_first_of(" \t") - 3);
else a_chr = a.substr(0,a.find_first_of(" \t"));
if (b.substr(0,3) == "chr") b_chr = b.substr(3,b.find_first_of(" \t") - 3);
else b_chr = b.substr(0,b.find_first_of(" \t"));
if(a_chr != b_chr) return a_chr < b_chr;
else
{
int a_start_begin_index = a.find_first_of(" \t", 0);
a_start_begin_index = a.find_first_not_of(" \t", a_start_begin_index);
int a_start_size = a.find_first_of(" \t", a_start_begin_index) - a_start_begin_index;
if(a_start_size < 1)
{
a_start_size = a.length() - a_start_begin_index;
}
int a_start_position = boost::lexical_cast<int>(a.substr(a_start_begin_index, a_start_size));
int b_start_begin_index = b.find_first_of(" \t", 0);
b_start_begin_index = b.find_first_not_of(" \t", b_start_begin_index);
int b_start_size = b.find_first_of(" \t", b_start_begin_index) - b_start_begin_index;
int b_start_position = boost::lexical_cast<int>(b.substr(b_start_begin_index, b_start_size));
return a_start_position < b_start_position;
}
}
}
// mipgen object stores all relevant parameters, containers and functions for the purpose of MIP selection
class mipgen{
public:
//parameter and container setup
bool has_arm_length_option;
bool has_arm_length_sum_option;
bool double_tile_strands_separately;
bool double_tile;
map<string,string> args;
set<int> oligo_sizes;
map<int, list<int> > arm_lengths_by_sum;
list<Featurev5> features_to_scan;
int snp_load_count;
map<string, map<int, string> > chr_snp_positions;
map<int, map<string, set<int> > > unmappable_positions;
map<string, map<int, map<int, int> > > copy_chr_start_stop;
ofstream ALLMIPS;
ofstream COLLAPSEDMIPS;
ofstream PICKEDMIPS;
ofstream SNPMIPS;
ofstream PROGRESS;
ofstream GAPS;
ofstream DOUBLEGAPS;
ofstream MINUSGAPS;
ofstream DOUBLEMINUSGAPS;
set<int> arm_length_sum_set;
vector<char> lig_probe_bases;
vector<char> ext_probe_bases;
int bad_design_count;
map<string, map<string, set<int> > > chr_strand_pos_used_arm_bases;
map<int, map<string, boost::shared_ptr<SVMipv4> > > pos_strand_best_mip;
map<int, map<string, boost::shared_ptr<SVMipv4> > > scan_strand_best_mip;
map<int, map<string, list<boost::shared_ptr<SVMipv4> > > > scan_strand_mip_list;
boost::shared_ptr<SVMipv4> null_pointer;
int ext_tag_length;
int lig_tag_length;
string universal_middle_mip_seq;
string file_dir;
//variable and function declarations
int all_mip_counter;
int collapsed_mip_counter;
int picked_mip_counter;
int feature_counter;
int feature_flank;
int max_arm_copy;
int target_arm_copy;
double lower_score_limit;
double upper_score_limit;
string regions_to_scan;
string project_name;
string bwa_genome_index;
int max_mip_overlap;
int starting_mip_overlap;
int max_capture_size;
int min_capture_size;
int capture_increment;
// instantiates a mipgen object with default values for parameters not explicitly set
mipgen(int argc, char * argv[]) {
has_arm_length_option = false;
has_arm_length_sum_option = false;
double_tile_strands_separately = false;
double_tile = false;
snp_load_count = 0;
bad_design_count = 0;
all_mip_counter = 0;
collapsed_mip_counter = 0;
picked_mip_counter = 0;
feature_counter = 0;
size_t dir_end = string(argv[0]).find_last_of("/");
file_dir = dir_end == (size_t) -1 ? "" : string(argv[0]).substr(0, dir_end) + "/";
set_default_args();
string parse_status = parse_command_line(argc,argv);
if (parse_status.length() != 0)
{
cerr << parse_status << endl;
throw 1;
}
int a = system(args["-bwa"].c_str());
if (a != 256)
{
cerr << "load bwa" << endl;
throw 2;
}
if(args["-trf"] != "off")
{
a = system(args["-trf"].c_str());
if (a != 65280)
{
cerr << "TRF directory invalid" << endl;
throw 3;
}
}
parse_arg_values();
}
// sets default values for mipgen object
void set_default_args() {
args["-bwa"] = "bwa";
args["-feature_flank"] = "0";
args["-check_copy_number"] = "on";
args["-starting_mip_overlap"] = "0";
args["-double_tile_strand_unaware"] = "off";
args["-double_tile_strands_separately"] = "off";
args["-silent_mode"] = "off";
args["-tag_sizes"] = "5,0";
args["-trf"] = "off";
args["-tabix"] = "tabix";
args["-seal_both_strands"] = "off";
args["-half_seal_both_strands"] = "off";
args["-masked_arm_threshold"] = "0.5";
args["-snp_file"] = "<none>";
args["-capture_increment"] = "5";
args["-max_mip_overlap"] = "30";
args["-max_mip_overlap"] = "30";
args["-score_method"] = "logistic";
args["-lig_min_length"] = "18";
args["-ext_min_length"] = "16";
args["-max_arm_copy_product"] = "75";
args["-target_arm_copy"] = "20";
args["-bwa_threads"] = "1";
}
// turns parameters into their proper types for easy access by field
void parse_arg_values() {
feature_flank = boost::lexical_cast<int>(args["-feature_flank"]);
size_t tag_boundary = args["-tag_sizes"].find(",");
string ext_tag_string = args["-tag_sizes"].substr(0, tag_boundary);
string lig_tag_string = args["-tag_sizes"].substr(tag_boundary + 1);
ext_tag_length = boost::lexical_cast<int>(ext_tag_string);
lig_tag_length = boost::lexical_cast<int>(lig_tag_string);
max_arm_copy = boost::lexical_cast<int>(args["-max_arm_copy_product"]);
target_arm_copy = boost::lexical_cast<int>(args["-target_arm_copy"]);
string universal_constant_mip_seq = "CTTCAGCTTCCCGATATCCGACGGTAGTGT";
universal_middle_mip_seq = string(lig_tag_length, 'N') + universal_constant_mip_seq + string(ext_tag_length, 'N');
if (args["-double_tile_strand_unaware"] == "on")
{
double_tile = true;
}
if (args["-double_tile_strands_separately"] == "on")
{
double_tile_strands_separately = true;
}
if(args.find("-svr_optimal_score") == args.end())
args["-svr_optimal_score"] = "2.2";
if(args.find("-svr_priority_score") == args.end())
args["-svr_priority_score"] = "1.5";
if(args.find("-logistic_optimal_score") == args.end())
args["-logistic_optimal_score"] = "0.98";
if(args.find("-logistic_priority_score") == args.end())
args["-logistic_priority_score"] = "0.9";
if(args["-score_method"] != "logistic" && args["-score_method"] != "svr" && args["-score_method"] != "mixed")
{
cerr << "invalid scoring method given" << endl;
throw 4;
}
if (has_arm_length_option)
{
string input (args["-arm_lengths"]);
vector<string> input_parse;
boost::split(input_parse, input, boost::is_any_of(","));
for (size_t i = 0; i < input_parse.size(); i++)
{
size_t boundary = input_parse.at(i).find(":");
int extension_arm_length = boost::lexical_cast<int>(input_parse.at(i).substr(0, boundary));
int ligation_arm_length = boost::lexical_cast<int>(input_parse.at(i).substr(boundary + 1));
oligo_sizes.insert(extension_arm_length);
oligo_sizes.insert(ligation_arm_length);
arm_lengths_by_sum[extension_arm_length + ligation_arm_length].push_back(extension_arm_length);
}
}
if (has_arm_length_sum_option || !(has_arm_length_sum_option || has_arm_length_option))
{
string input;
if(has_arm_length_sum_option)
input = args["-arm_length_sums"];
else
input = "40,41,42,43,44,45";
vector<string> input_parse;
boost::split(input_parse, input, boost::is_any_of(","));
for (size_t i = 0; i<input_parse.size(); i++)
{
int arm_sum = boost::lexical_cast<int>(input_parse.at(i));
for (int ligation_arm_length = boost::lexical_cast<int>(args["-lig_min_length"]); ligation_arm_length <= arm_sum - boost::lexical_cast<int>(args["-ext_min_length"]) && ligation_arm_length <= 30; ligation_arm_length++)
{
int extension_arm_length = arm_sum - ligation_arm_length;
if (extension_arm_length <= 30)
{
arm_lengths_by_sum[arm_sum].push_back(extension_arm_length);
oligo_sizes.insert(extension_arm_length);
oligo_sizes.insert(ligation_arm_length);
}
}
arm_lengths_by_sum[arm_sum].sort();
}
}
lower_score_limit = args["-score_method"] == "svr" ? boost::lexical_cast<double>(args["-svr_priority_score"]) : boost::lexical_cast<double>(args["-logistic_priority_score"]);
upper_score_limit = args["-score_method"] == "svr" ? boost::lexical_cast<double>(args["-svr_optimal_score"]) : boost::lexical_cast<double>(args["-logistic_optimal_score"]);
regions_to_scan = args["-regions_to_scan"];
project_name = args["-project_name"];
bwa_genome_index = args["-bwa_genome_index"];
max_mip_overlap = boost::lexical_cast<int>(args["-max_mip_overlap"]);
starting_mip_overlap = boost::lexical_cast<int>(args["-starting_mip_overlap"]);
max_capture_size = boost::lexical_cast<int>(args["-max_capture_size"]);
min_capture_size = boost::lexical_cast<int>(args["-min_capture_size"]);
capture_increment = boost::lexical_cast<int>(args["-capture_increment"]);
if (capture_increment == 0) capture_increment = 1; // Prevent any strange behavior associated with 0 value
}
// prints version and parameters for mipgen run
void print_header() {
PROGRESS.open((project_name + ".progress.txt").c_str());
if(!PROGRESS.is_open())
{
cerr << "progress file could not be opened" << endl;
throw 5;
}
PROGRESS << __FILE__ << endl << "last numbered version: 1.1" << endl;
PROGRESS << "contact: Evan Boyle\nemail: boylee@uw.edu\n";
for (map<string,string>::iterator it = args.begin(); it!= args.end(); it++)
{
PROGRESS << it->first << " " << it->second << endl;
}
}
// collects sequence from genome reference, checks for SNPs, copy number and tandem repeats and prepares for selection
void query_sequences(){
string feature_status = get_features_to_scan(); // creates Feature objects for targeted regions
if (feature_status.length() == 0)
{
cerr << "[mipgen] region file could not be opened" << endl;
throw 6;
}
PROGRESS << "successfully loaded features for mip design; retrieving chromosomal sequence\n";
cerr << "[mipgen] features loaded; retrieving chromosomal sequence\n";
if(args.find("-genome_dir") != args.end())
{
if (!(get_chr_fasta_sequence_from_genome_dir(args["-genome_dir"])))
{
cerr << "[mipgen] chromosome fasta not acquired" << endl;
throw 7;
}
}
else
{
int samtools_result = system("samtools");
if(samtools_result != 256) {
cerr << "[mipgen] load samtools or provide genome directory" << endl;
throw 8;
}
if (!(get_chr_fasta_sequence_using_samtools()))
{
cerr << "[mipgen] chromosome fasta not acquired" << endl;
throw 9;
}
}
if (!(get_masked_features_to_scan()))
{
cerr << "[mipgen] masked chromosome fasta not acquired; no repetitive bases?" << endl;
}
PROGRESS << "successfully acquired chromosomal data for mip design; accessing snp file ...\n";
cerr << "[mipgen] regions ready; accessing snp file\n";
map<string, map<int, string> > chr_snp_positions;
if(!(args["-snp_file"] == "<none>"))
load_snps(); // loads snp positions and alleles into memory using tabix
PROGRESS << "all " << snp_load_count << " snps loaded; generating files for bwa\n";
cerr << "[mipgen] all " << snp_load_count << " snps loaded; generating files for bwa\n";
string copy_status = check_copy_numbers(); // loads bad mip start positions into memory
if (copy_status.length() == 0)
{
cerr << "error with copy number analysis" << endl;
throw 11;
}
PROGRESS << copy_status;
find_copy();
PROGRESS << "bwa copy number analysis finished\n";
cerr << "[mipgen] bwa copy number analysis finished\n";
ALLMIPS.open((project_name + ".all_mips.txt").c_str());
if (ALLMIPS.is_open())
cerr << "[mipgen] file of all mips ready for write: " << project_name << ".all_mips.txt\n";
else
{
cerr << "[mipgen] all mips file could not be opened" << endl;
throw 12;
}
ALLMIPS << ">mip_key\t";
if(args["-score_method"] == "svr")
ALLMIPS << "svr";
else
ALLMIPS << "logistic";
ALLMIPS << "_score\tchr\text_probe_start\text_probe_stop\text_probe_copy\text_probe_sequence\tlig_probe_start\tlig_probe_stop\tlig_probe_copy\tlig_probe_sequence\tmip_scan_start_position\tmip_scan_stop_position\tscan_target_sequence\tmip_sequence\tfeature_start_position\tfeature_stop_position\tprobe_strand\tfailure_flags\tmip_name\n";
PROGRESS << "file of all mips ready for write: " << project_name << ".all_mips\n";
COLLAPSEDMIPS.open((project_name +".collapsed_mips.txt").c_str());
if (COLLAPSEDMIPS.is_open())
cerr << "[mipgen] file of collapsed mips ready for write: " << project_name << ".collapsed_mips.txt\n";
else
{
cerr << "[mipgen] file of collapsed mips could not be opened" << endl;
throw 13;
}
COLLAPSEDMIPS << ">mip_key\t";
if(args["-score_method"] == "svr")
COLLAPSEDMIPS << "svr";
else
COLLAPSEDMIPS << "logistic";
COLLAPSEDMIPS << "_score\tchr\text_probe_start\text_probe_stop\text_probe_copy\text_probe_sequence\tlig_probe_start\tlig_probe_stop\tlig_probe_copy\tlig_probe_sequence\tmip_scan_start_position\tmip_scan_stop_position\tscan_target_sequence\tmip_sequence\tfeature_start_position\tfeature_stop_position\tprobe_strand\tfailure_flags\tmip_name\n";
PROGRESS << "file of collapsed mips ready for write: " << project_name << ".collapsed_mips.txt\n";
PICKEDMIPS.open((project_name + ".picked_mips.txt").c_str());
if (!(PICKEDMIPS.is_open()))
throw 14;
PICKEDMIPS << ">mip_key\t";
if(args["-score_method"] == "logistic")
PICKEDMIPS << "logistic";
else
PICKEDMIPS << "svr";
PICKEDMIPS << "_score\tchr\text_probe_start\text_probe_stop\text_probe_copy\text_probe_sequence\tlig_probe_start\tlig_probe_stop\tlig_probe_copy\tlig_probe_sequence\tmip_scan_start_position\tmip_scan_stop_position\tscan_target_sequence\tmip_sequence\tfeature_start_position\tfeature_stop_position\tprobe_strand\tfailure_flags\tmip_name\n";
SNPMIPS.open((project_name + ".snp_mips.txt").c_str());
if (!(SNPMIPS.is_open()))
throw 15;
SNPMIPS << ">mip_key\t";
if(args["-score_method"] == "logistic")
SNPMIPS << "logistic";
else
SNPMIPS << "svr";
SNPMIPS << "_score\tchr\text_probe_start\text_probe_stop\text_probe_copy\text_probe_sequence\tlig_probe_start\tlig_probe_stop\tlig_probe_copy\tlig_probe_sequence\tmip_scan_start_position\tmip_scan_stop_position\tscan_target_sequence\tmip_sequence\tfeature_start_position\tfeature_stop_position\tprobe_strand\tfailure_flags\tmip_name\n";
}
// prints MIP data to files and selects a final MIP tiling
// this is the main process behind MIP design
void tile_regions()
{
for(map<int, list<int> >::iterator it = arm_lengths_by_sum.begin(); it != arm_lengths_by_sum.end(); it++)
arm_length_sum_set.insert(it->first);
Featurev5 * feature;
svm_model * model = svm_load_model((file_dir + "mipgen_svr.model").c_str());
vector<double> scoring_parameters;
for(list<Featurev5>::iterator it = features_to_scan.begin(); it != features_to_scan.end(); it++)
{
feature = &*it;
feature_counter++;
PROGRESS << "designing all mips for feature #" << feature_counter << endl;
cerr << "[mipgen] feature #" << feature_counter << endl;
string chr = feature->chr;
//cout << feature->start_position_flanked << "\t" << feature->stop_position_flanked << endl; //comment
feature->current_scan_start_position = feature->start_position_flanked - max_capture_size + *arm_length_sum_set.rbegin(); // build mips starting from when the start of the feature is as far as possible from the extension arm
if(feature->current_scan_start_position < 0) feature->current_scan_start_position = 0;
while (feature->current_scan_start_position < feature->stop_position_flanked) // continue until the feature stop position is the first base following the extension arm
{
feature->current_scan_start_position++;
double previous_best_score = 0;
for (int capture_size = max_capture_size; capture_size >= min_capture_size; capture_size -= capture_increment)
{
if (capture_size > feature->stop_position_flanked - feature->start_position_flanked + max_mip_overlap && capture_size - capture_increment >= min_capture_size) continue;
if (previous_best_score > upper_score_limit) continue;
for (set<int>::reverse_iterator rit = arm_length_sum_set.rbegin(); rit != arm_length_sum_set.rend(); rit++)
{
int arm_length_sum = *rit;
if (previous_best_score > upper_score_limit && arm_length_sum != *arm_length_sum_set.begin()) continue;
int previous_minus_score = 0;
int previous_plus_score = 0;
bool skip_ahead = false;
for(list<int>::iterator it2 = arm_lengths_by_sum[arm_length_sum].begin(); it2 != arm_lengths_by_sum[arm_length_sum].end(); it2++)
{
if (skip_ahead) continue;
int extension_arm_length = *it2;
int ligation_arm_length = arm_length_sum - extension_arm_length;
if(feature->current_scan_start_position - extension_arm_length <= 0 || feature->current_scan_start_position - ligation_arm_length <= 0) continue;
if(feature->current_scan_start_position + capture_size - extension_arm_length - 1 > feature->chromosomal_sequence_stop_position || feature->current_scan_start_position + capture_size - ligation_arm_length - 1 > feature->chromosomal_sequence_stop_position) continue;
//cout << extension_arm_length << ":" << capture_size << ":" << feature->current_scan_start_position << endl; // comment
boost::shared_ptr<PlusSVMipv4> current_plus_mip (new PlusSVMipv4 (
feature->chr,
feature->current_scan_start_position,
feature->current_scan_start_position + capture_size - arm_length_sum - 1,
extension_arm_length,
ligation_arm_length
));
boost::shared_ptr<MinusSVMipv4> current_minus_mip (new MinusSVMipv4 (
feature->chr,
feature->current_scan_start_position,
feature->current_scan_start_position + capture_size - arm_length_sum - 1,
extension_arm_length,
ligation_arm_length
));
current_plus_mip->set_scan_target_seq(feature->chromosomal_sequence.substr(current_plus_mip->scan_start_position - feature->chromosomal_sequence_start_position, current_plus_mip->scan_size));
current_minus_mip->set_scan_target_seq(feature->chromosomal_sequence.substr(current_minus_mip->scan_start_position - feature->chromosomal_sequence_start_position,current_minus_mip->scan_size));
// cout << "ready to design\n"; // comment
boost::shared_ptr<SVMipv4> designed_plus_mip;
designed_plus_mip = design_mip(feature, current_plus_mip);
if(args["-score_method"] == "logistic" || args["-score_method"] == "mixed")
designed_plus_mip->score = designed_plus_mip->get_score();
else if (args["-score_method"] == "svr")
{
designed_plus_mip->get_parameters(scoring_parameters, feature->long_range_content);
designed_plus_mip->score = predict_value(scoring_parameters, model);
}
all_mip_counter++;
scan_strand_mip_list[designed_plus_mip->scan_start_position]["+"].push_front(designed_plus_mip); // hashes with keys derived from strand and scan start position and values being arrays of mips at that position
if(args["-silent_mode"] != "on")
ALLMIPS << print_details(feature, designed_plus_mip, all_mip_counter, false);
boost::shared_ptr<SVMipv4> designed_minus_mip;
designed_minus_mip = design_mip(feature, current_minus_mip);
if(args["-score_method"] == "logistic" || args["-score_method"] == "mixed")
designed_minus_mip->score = designed_minus_mip->get_score();
else if (args["-score_method"] == "svr")
{
designed_minus_mip->get_parameters(scoring_parameters, feature->long_range_content);
designed_minus_mip->score = predict_value(scoring_parameters, model);
}
all_mip_counter++;
scan_strand_mip_list[designed_minus_mip->scan_start_position]["-"].push_front(designed_minus_mip);
if(args["-silent_mode"] != "on")
ALLMIPS << print_details(feature, designed_minus_mip, all_mip_counter, false);
//cout << "design done!\n";
if (args["-score_method"] == "logistic" && args["-logistic_heuristic"] != "off" && designed_plus_mip->score < previous_plus_score && designed_minus_mip->score < previous_minus_score) skip_ahead = true;
previous_best_score = (designed_minus_mip->score > designed_plus_mip->score) ? designed_minus_mip->score : designed_plus_mip->score;
previous_minus_score = designed_minus_mip->score;
previous_plus_score = designed_plus_mip->score;
}
}
}
}
//cout << "condensing mips!\n";
condense_mips(feature);
//cout << "collapsing mips!\n";
collapse_mips(); // collapses mip space by choosing only one (best scoring) hopefully nonfailed mip for every position
if(args["-silent_mode"] != "on")
output_collapsed_mips(feature);
PROGRESS << "mips collapsed! picking mips...\n";
//cout << "mips collapsed! picking mips...\n";
if(args["-score_method"] == "mixed")
{
lower_score_limit = boost::lexical_cast<double>(args["-svr_priority_score"]);
upper_score_limit = boost::lexical_cast<double>(args["-svr_optimal_score"]);
}
pick_mips(feature, scoring_parameters, model);
if(args["-score_method"] == "mixed")
{
lower_score_limit = boost::lexical_cast<double>(args["-logistic_priority_score"]);
upper_score_limit = boost::lexical_cast<double>(args["-logistic_optimal_score"]);
}
//cout << "mips picked!\n";
scan_strand_mip_list.clear();
scan_strand_best_mip.clear();
pos_strand_best_mip.clear();
}
ALLMIPS.close();
COLLAPSEDMIPS.close();
PICKEDMIPS.close();
SNPMIPS.close();
PROGRESS << "mip picking complete:\n" << project_name << ".picked_mips.txt\n and \n" << project_name <<".snps_mips.txt\n";
cerr << "[mipgen] mip picking complete:\n" << project_name << ".picked_mips.txt\n and \n" << project_name << ".snp_mips.txt\n";
if (bad_design_count > 0)
{
PROGRESS << "WARNING: There are " << bad_design_count << " gaps in covering supplied regions\n";
cerr << "[mipgen] WARNING: There are " << bad_design_count << " gaps in covering supplied regions\n";
}
if(GAPS.is_open())
{
GAPS.close();
}
if(DOUBLEGAPS.is_open())
{
DOUBLEGAPS.close();
}
if(MINUSGAPS.is_open())
{
MINUSGAPS.close();
}
if(DOUBLEMINUSGAPS.is_open())
{
DOUBLEMINUSGAPS.close();
}
PROGRESS.close();
}
// uses BWA to find probe and target copy numbers
void find_copy ()
{
system((args["-bwa"] + " aln -t " + args["-bwa_threads"] + " " + bwa_genome_index + " " + project_name + ".oligo_copy_count.fq > " + project_name + ".oligo_copy_count.sai").c_str());
system((args["-bwa"] + " samse " + bwa_genome_index + " " + project_name + ".oligo_copy_count.sai " + project_name + ".oligo_copy_count.fq > " + project_name + ".oligo_copy_count.sam").c_str());
ifstream OLIGOSAM ((project_name + ".oligo_copy_count.sam").c_str());
if (OLIGOSAM.is_open()) cerr << "[mipgen] checking oligo copy" << endl;
else return;
string line;
while (OLIGOSAM.good())
{
getline(OLIGOSAM, line);
if (line[0] != '@' && line.length() > 1)
{
size_t chr_start = line.find("chr", 0) + 3;
size_t chr_stop = line.find(":", chr_start);
size_t start_start = chr_stop + 1;
size_t start_stop = line.find("-", start_start);
size_t stop_start = start_stop + 1;
size_t stop_stop = line.find("\t", stop_start);
string chr = line.substr(chr_start, chr_stop - chr_start);
int start = boost::lexical_cast<int>(line.substr(start_start, start_stop - start_start));
int stop = boost::lexical_cast<int>(line.substr(stop_start, stop_stop - stop_start));
size_t copy_start = line.find("X0:i:",0);
if (copy_start < line.length())
{
copy_start += 5;
size_t copy_stop = line.find("\t", copy_start);
int copy_count = boost::lexical_cast<int>((line.substr(copy_start, copy_stop - copy_start)));
copy_chr_start_stop[chr][start][stop] = copy_count;
}
else
{
copy_chr_start_stop[chr][start][stop] = 100;
}
}
}
OLIGOSAM.close();
}
// gets the basic information about potential mips, used for scoring purposes
boost::shared_ptr<SVMipv4> design_mip (Featurev5 * current_feature, boost::shared_ptr<SVMipv4> current_mip)
{
string chr = current_feature->chr;
current_mip->set_ext_probe_seq(current_feature->chromosomal_sequence.substr(current_mip->ext_probe_start - current_feature->chromosomal_sequence_start_position, current_mip->extension_arm_length));
current_mip->set_lig_probe_seq(current_feature->chromosomal_sequence.substr(current_mip->lig_probe_start - current_feature->chromosomal_sequence_start_position, current_mip->ligation_arm_length));
current_mip->mip_seq = current_mip->lig_probe_sequence + universal_middle_mip_seq + current_mip->ext_probe_sequence;
string masked_ext_seq = current_feature->masked_chromosomal_sequence.substr(current_mip->ext_probe_start - current_feature->chromosomal_sequence_start_position, current_mip->extension_arm_length);
string masked_lig_seq = current_feature->masked_chromosomal_sequence.substr(current_mip->lig_probe_start - current_feature->chromosomal_sequence_start_position, current_mip->ligation_arm_length);
double ext_N_count = count(masked_ext_seq.begin(), masked_ext_seq.end(), 'N');
double lig_N_count = count(masked_lig_seq.begin(), masked_lig_seq.end(), 'N');
current_mip->arm_fraction_masked = (ext_N_count + lig_N_count) / (current_mip->ligation_arm_length + current_mip->extension_arm_length);
current_mip->ext_probe_copy = copy_chr_start_stop[current_mip->chr][current_mip->ext_probe_start][current_mip->ext_probe_stop];
current_mip->lig_probe_copy = copy_chr_start_stop[current_mip->chr][current_mip->lig_probe_start][current_mip->lig_probe_stop];
int capture_size = current_mip->scan_size + current_mip->extension_arm_length + current_mip->ligation_arm_length;
if ( !(unmappable_positions.empty())
&& !(unmappable_positions[capture_size].empty())
&& unmappable_positions[capture_size][chr].find(current_mip->get_mip_start()) != unmappable_positions[capture_size][chr].end()
&& args["-check_copy_number"] != "off"
)
{
current_mip->mapping_failed = '1';
// cout << "=(" << endl;
return current_mip;
}
if (current_mip->arm_fraction_masked > boost::lexical_cast<double>(args["-masked_arm_threshold"]))
{
current_mip->masking_failed = '1';
}
else
{
current_mip->masking_failed = '0';
}
for (int i = current_mip->ext_probe_start; i <= current_mip->ext_probe_stop; i++)
{
if (!(chr_snp_positions.empty()) && !(chr_snp_positions[chr].empty()) && chr_snp_positions[chr].find(i) != chr_snp_positions[chr].end())
{
bool flag = false; // used to track whether snp mip creation succeeded
string alleles = chr_snp_positions[chr][i];
current_mip->snp_count++;
string arm_sequence (current_mip->ext_probe_sequence);
ext_probe_bases.clear();
ext_probe_bases = vector<char> (arm_sequence.begin(), arm_sequence.end());
if (alleles.length() == 2 && alleles[0] != 'N' && alleles[1] != 'N' && alleles[0] != '-' && alleles[1] != '-') // only single nucleotide transitions and transversions are supported
{
int relative_ext_position;
if (current_mip->strand == "+")
{
relative_ext_position = i - current_mip->ext_probe_start;
}
else
{
relative_ext_position = current_mip->ext_probe_stop - i;
}
if (ext_probe_bases[relative_ext_position] == alleles[0])
{
ext_probe_bases[relative_ext_position] = alleles[1];
flag = true;
}
else // snp file might have allele on minus strand
{
char allele_1;
switch(alleles[0]) {
case 'A': allele_1 = 'T'; break;
case 'T': allele_1 = 'A'; break;
case 'G': allele_1 = 'C'; break;
case 'C': allele_1 = 'G'; break;
}
char allele_2;
switch(alleles[1]) {
case 'A': allele_2 = 'T'; break;
case 'T': allele_2 = 'A'; break;
case 'G': allele_2 = 'C'; break;
case 'C': allele_2 = 'G'; break;
}
if (ext_probe_bases[relative_ext_position] == allele_1)
{
ext_probe_bases[relative_ext_position] = allele_2;
flag = true;
}
}
}
if (flag)
{
current_mip->has_snp_mip = true;
current_mip->snp_ext_sequence = string(ext_probe_bases.begin(), ext_probe_bases.end());
current_mip->snp_lig_sequence = current_mip->lig_probe_sequence;
current_mip->snp_mip_sequence = current_mip->snp_lig_sequence + universal_middle_mip_seq + current_mip->snp_ext_sequence;
}
else // this will flag the necesary mip as having a snp with no alternate mip provided
{
current_mip->snp_failed = '1';
}
}
}
for (int i = current_mip->lig_probe_start; i <= current_mip->lig_probe_stop; i++)
{
if (!(chr_snp_positions[chr].empty()) && chr_snp_positions[chr].find(i) != chr_snp_positions[chr].end())
{
bool flag = false; // used to track whether snp mip creation succeeded
string alleles = chr_snp_positions[chr][i];
current_mip->snp_count++;
string arm_sequence (current_mip->lig_probe_sequence);
lig_probe_bases.clear();
lig_probe_bases = vector<char> (arm_sequence.begin(), arm_sequence.end());
if (alleles.length() == 2 && alleles[0] != 'N' && alleles[1] != 'N' && alleles[0] != '-' && alleles[1] != '-') // only single nucleotide transitions and transversions are supported
{
int relative_lig_position;
if (current_mip->strand == "+")
{
relative_lig_position = i - current_mip->lig_probe_start;
}
else
{
relative_lig_position = current_mip->lig_probe_stop - i;
}
if (lig_probe_bases[relative_lig_position] == alleles[0])
{
lig_probe_bases[relative_lig_position] = alleles[1];
flag = true;
}
else // snp file might have allele on minus strand
{
char allele_1;
switch(alleles[0]) {
case 'A': allele_1 = 'T'; break;
case 'T': allele_1 = 'A'; break;
case 'G': allele_1 = 'C'; break;
case 'C': allele_1 = 'G'; break;
}
char allele_2;
switch(alleles[1]) {
case 'A': allele_2 = 'T'; break;
case 'T': allele_2 = 'A'; break;
case 'G': allele_2 = 'C'; break;
case 'C': allele_2 = 'G'; break;
}
if (lig_probe_bases[relative_lig_position] == allele_1)
{
lig_probe_bases[relative_lig_position] = allele_2;
flag = true;
}
}
}
if (flag)
{
current_mip->has_snp_mip = true;
current_mip->snp_ext_sequence = current_mip->ext_probe_sequence;
current_mip->snp_lig_sequence = string(lig_probe_bases.begin(), lig_probe_bases.end());
current_mip->snp_mip_sequence = current_mip->snp_lig_sequence + universal_middle_mip_seq + current_mip->snp_ext_sequence;
}
else // this will flag the necesary mip as having a snp with no alternate mip provided
{
current_mip->snp_failed = '1';
}
}
}
if (current_mip->snp_count > 1)
current_mip->snp_failed = '1';
return current_mip;
}
// prints standard MIP design file record
string print_details (Featurev5 * feature, boost::shared_ptr<SVMipv4> mip, int mip_index, bool minor)// used throughout the program to standardize output of MIP features
{
stringstream ss;
ss << mip->chr << ":";
ss << (mip->strand == "+" ? mip->ext_probe_start : mip->lig_probe_start) << "-";
ss << (mip->strand == "+" ? mip->lig_probe_stop : mip->ext_probe_stop) << "/";
ss << mip->extension_arm_length << ",";
ss << mip->ligation_arm_length << "/";
ss << mip->strand << "\t";
ss << mip->score << "\t";
ss << mip->chr << "\t";
ss << mip->ext_probe_start << "\t";
ss << mip->ext_probe_stop << "\t";
ss << mip->ext_probe_copy << "\t";
ss << mip->ext_probe_sequence << "\t";
ss << mip->lig_probe_start << "\t";
ss << mip->lig_probe_stop << "\t";
ss << mip->lig_probe_copy << "\t";
ss << mip->lig_probe_sequence << "\t";
ss << mip->scan_start_position << "\t";
ss << mip->scan_stop_position << "\t";
ss << mip->scan_target_sequence << "\t";
ss << mip->mip_seq << "\t";
ss << feature->start_position - 1 << "\t";
ss << feature->stop_position << "\t";
ss << mip->strand << "\t";
ss << mip->mapping_failed << mip->snp_failed << mip->masking_failed << "\t";
ss << feature->label << "_" << setw(4) << setfill('0') << mip_index << (mip->snp_count == 1 ? ("_SNP_" + string(minor ? "b" : "a")) : "") << "\n";
return ss.str();
}
// uses bwa to check that targets map uniquely
string check_copy_numbers()
{
ofstream BWAFQ ((project_name + ".all_sequences.fq").c_str());
ofstream ARMSFQ ((project_name + ".oligo_copy_count.fq").c_str());
if (!(BWAFQ.is_open()) || !(ARMSFQ.is_open()))
{
cerr << "[mipgen] copy_check files could not be opened" << endl;
return "";
}
Featurev5 * feature;
for (list<Featurev5>::iterator it = features_to_scan.begin(); it != features_to_scan.end(); it++)
{
feature = &*it;
string chr = feature->chr;
for (int capture_size = max_capture_size; capture_size >= min_capture_size; capture_size -= capture_increment)
{
int current_mip_start = feature->start_position_flanked - capture_size;
while (current_mip_start < feature->stop_position_flanked)
{
if(current_mip_start > 0 && current_mip_start + capture_size - 1 <= feature->chromosomal_sequence_stop_position)
{
BWAFQ << "@" << capture_size << "_" << feature->chr << "_" << current_mip_start << endl;
BWAFQ << feature->chromosomal_sequence.substr(current_mip_start - feature->chromosomal_sequence_start_position, capture_size) << endl;
BWAFQ << "+\n";
for (int i = 0; i < capture_size; i++) BWAFQ << "#";
BWAFQ << endl;
}
current_mip_start++;
}
}
for (set<int>::iterator it = oligo_sizes.begin(); it != oligo_sizes.end(); it++)
{
int oligo_size = *it;
for(unsigned int relative_start_position = 0; relative_start_position < feature->chromosomal_sequence.length() - oligo_size; relative_start_position++)
{
ARMSFQ << "@chr" << chr << ":" << (feature->chromosomal_sequence_start_position + relative_start_position) << "-" << (feature->chromosomal_sequence_start_position + relative_start_position + oligo_size - 1) << endl;
ARMSFQ << feature->chromosomal_sequence.substr(relative_start_position, oligo_size) << endl;
ARMSFQ << "+\n";
for (int i=0; i < oligo_size; i++) ARMSFQ << "#";
ARMSFQ << endl;
}
}
}
BWAFQ.close();
ARMSFQ.close();
system((args["-bwa"] + " aln -t " + args["-bwa_threads"] + " " + bwa_genome_index + " " + project_name + ".all_sequences.fq > " + project_name + ".all_sequences.sai").c_str());
system((args["-bwa"] + " samse " + bwa_genome_index + " " + project_name + ".all_sequences.sai " + project_name +".all_sequences.fq > " + project_name + ".all_sequences.sam").c_str());
int bad_site_counter = 0;
ifstream CAPTURESAM ((project_name + ".all_sequences.sam").c_str());
if (CAPTURESAM.is_open()) cerr << "[mipgen] sam file opened" << endl;
else return "";
string line;
bool header = true;
while (CAPTURESAM.good())
{
getline(CAPTURESAM, line);
if(header && line.find("X0:i:", 0) >= line.length() - 1) continue;
else if (header) header = false;
unsigned int line_len = line.length();
if(line_len < 2) continue;
if(line.find("X0:i:1",0) >= line_len - 1 || line.find("X1:i:0",0) >= line_len - 1) // first regex: ignore header lines. second regex: reject non-unique mappings. third regex: reject mappings that are non-unique with 1 bp edits
{
string size_chr_and_pos = line.substr(0, line.find("\t"));
size_t sep1 = size_chr_and_pos.find("_", 0);
size_t sep2 = size_chr_and_pos.rfind("_");
int size = boost::lexical_cast<int>(size_chr_and_pos.substr(0, sep1));
string chr = size_chr_and_pos.substr(sep1 + 1, sep2 - sep1 - 1);
int pos = boost::lexical_cast<int>(size_chr_and_pos.substr(sep2 + 1));
unmappable_positions[size][chr].insert(pos);
bad_site_counter++;
}
}
CAPTURESAM.close();
stringstream i;
i << bad_site_counter;
return (i.str() + " ambiguously mapping start positions must be avoided\n");
}
// acquires, prints and load snp data into memory
void load_snps()
{
string query = "";
int first_position = features_to_scan.front().start_position_flanked - 500;
int last_position = features_to_scan.front().stop_position_flanked + 500;
string current_chr = features_to_scan.front().chr;
string previous_chr;
string start_str;
string stop_str;
Featurev5 * feature;
for (list<Featurev5>::iterator it = features_to_scan.begin(); it != features_to_scan.end(); it++)
{
feature = &*it;
previous_chr = current_chr;
current_chr = feature->chr;
int access_start = feature->start_position_flanked - 500;
int access_stop = feature->stop_position_flanked + 500;
if (previous_chr == current_chr && access_stop >= last_position && access_start <= last_position)
{
last_position = access_stop;
}
else
{
stringstream start_ss;
stringstream stop_ss;
start_ss << first_position;
start_str = start_ss.str();
stop_ss << last_position;
stop_str = stop_ss.str();
query.append(previous_chr + ":" + start_str + "-" + stop_str + " ");
//cout << query << endl;
first_position = access_start;
last_position = access_stop;
}
}
stringstream start_ss;
stringstream stop_ss;
start_ss << first_position;
stop_ss << last_position;
start_str = start_ss.str();
stop_str = stop_ss.str();
query.append( current_chr + ":" + start_str + "-" + stop_str + " ");
//PROGRESS << "tabix query: " << endl << query << endl;
int tabix_test = system(args["-tabix"].c_str());
if(tabix_test != 256)
{
cerr << "[mipgen] tabix not loaded" << endl;
throw 16;
}
if(args["-snp_file"] != "<none>")
{
system((args["-tabix"] + " " + args["-snp_file"] + " " + query + " > " + project_name + ".local_snp_data.vcf").c_str()) ;
parse_vcf(project_name + ".local_snp_data.vcf");
}
}
// reads a VCF and stores SNP positions in memory for evasion purposes
void parse_vcf(string vcf_file)
{
string line;
ifstream SNPS (vcf_file.c_str());
if(SNPS.is_open())
{
while (SNPS.good())
{
getline(SNPS, line);
if(line.length() < 2 or line[0] == '#') continue;
int start_position = 0;
int stop_position = line.find_first_of(" \t", 0);
string chr = line.substr(start_position, stop_position);
start_position = stop_position + 1;
stop_position = line.find_first_of(" \t", start_position);
//cout << line.substr(start_position, stop_position - start_position) << endl;
int position = boost::lexical_cast<int>(line.substr(start_position, stop_position - start_position));
start_position = stop_position + 1;
stop_position = line.find_first_of(" \t", start_position);
start_position = stop_position + 1;
stop_position = line.find_first_of(" \t", start_position);
string ref_allele = line.substr(start_position, stop_position - start_position);
start_position = stop_position + 1;
stop_position = line.find_first_of(" \t", start_position);
string alt_allele = line.substr(start_position, stop_position - start_position);
string alleles_content = ref_allele + alt_allele;
if (ref_allele.length() > 1) // indel
{
for (unsigned int i = 1; i < ref_allele.length(); i++)
{
chr_snp_positions[chr][position + i] = alleles_content;
}
}
else
{
chr_snp_positions[chr][position] = alleles_content;
}
snp_load_count++;
}
SNPS.close();
}
else
{
cerr << "[mipgen] VCF file could not be opened" << endl;
}
}
// loads feature details into memory
string get_features_to_scan()
{
string details;
list<string> regions_to_scan_contents;
ifstream FH (regions_to_scan.c_str());
if(FH.is_open()) cerr << "[mipgen] success on opening region file" << endl;
else return "";
while(FH.good())
{
getline(FH,details);
boost::trim(details);
// cout << details << endl;
if (details.length() > 1 && details[0] != '#')
{
regions_to_scan_contents.push_back(details);
}
}
FH.close();
regions_to_scan_contents.sort(compare_regions_to_scan);
vector<string> bed_fields;