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|
//
// primerdesigncommand.cpp
// Mothur
//
// Created by Sarah Westcott on 1/18/13.
// Copyright (c) 2013 Schloss Lab. All rights reserved.
//
#include "primerdesigncommand.h"
//**********************************************************************************************************************
vector<string> PrimerDesignCommand::setParameters(){
try {
CommandParameter plabel("label", "String", "", "", "", "", "","",false,false); parameters.push_back(plabel);
CommandParameter plist("list", "InputTypes", "", "", "none", "none", "none","summary-list",false,true,true); parameters.push_back(plist);
CommandParameter pfasta("fasta", "InputTypes", "", "", "none", "none", "none","",false,true, true); parameters.push_back(pfasta);
CommandParameter pname("name", "InputTypes", "", "", "NameCount", "none", "none","",false,false,true); parameters.push_back(pname);
CommandParameter pcount("count", "InputTypes", "", "", "NameCount", "none", "none","",false,false,true); parameters.push_back(pcount);
CommandParameter plength("length", "Number", "", "18", "", "", "","",false,false); parameters.push_back(plength);
CommandParameter pmintm("mintm", "Number", "", "-1", "", "", "","",false,false); parameters.push_back(pmintm);
CommandParameter pmaxtm("maxtm", "Number", "", "-1", "", "", "","",false,false); parameters.push_back(pmaxtm);
CommandParameter pprocessors("processors", "Number", "", "1", "", "", "","",false,false); parameters.push_back(pprocessors);
CommandParameter potunumber("otulabel", "String", "", "", "", "", "","",false,true,true); parameters.push_back(potunumber);
CommandParameter ppdiffs("pdiffs", "Number", "", "0", "", "", "","",false,false,true); parameters.push_back(ppdiffs);
CommandParameter pcutoff("cutoff", "Number", "", "100", "", "", "","",false,false); parameters.push_back(pcutoff);
CommandParameter pseed("seed", "Number", "", "0", "", "", "","",false,false); parameters.push_back(pseed);
CommandParameter pinputdir("inputdir", "String", "", "", "", "", "","",false,false); parameters.push_back(pinputdir);
CommandParameter poutputdir("outputdir", "String", "", "", "", "", "","",false,false); parameters.push_back(poutputdir);
abort = false; calledHelp = false;
vector<string> tempOutNames;
outputTypes["summary"] = tempOutNames;
outputTypes["fasta"] = tempOutNames;
outputTypes["list"] = tempOutNames;
vector<string> myArray;
for (int i = 0; i < parameters.size(); i++) { myArray.push_back(parameters[i].name); }
return myArray;
}
catch(exception& e) {
m->errorOut(e, "PrimerDesignCommand", "setParameters");
exit(1);
}
}
//**********************************************************************************************************************
string PrimerDesignCommand::getHelpString(){
try {
string helpString = "";
helpString += "The primer.design allows you to identify sequence fragments that are specific to particular OTUs.\n";
helpString += "The primer.design command parameters are: list, fasta, name, count, otulabel, cutoff, length, pdiffs, mintm, maxtm, processors and label.\n";
helpString += "The list parameter allows you to provide a list file and is required.\n";
helpString += "The fasta parameter allows you to provide a fasta file and is required.\n";
helpString += "The name parameter allows you to provide a name file associated with your fasta file.\n";
helpString += "The count parameter allows you to provide a count file associated with your fasta file.\n";
helpString += "The label parameter is used to indicate the label you want to use from your list file.\n";
helpString += "The otulabel parameter is used to indicate the otu you want to use from your list file. It is required.\n";
helpString += "The pdiffs parameter is used to specify the number of differences allowed in the primer. The default is 0.\n";
helpString += "The length parameter is used to indicate the length of the primer. The default is 18.\n";
helpString += "The mintm parameter is used to indicate minimum melting temperature.\n";
helpString += "The maxtm parameter is used to indicate maximum melting temperature.\n";
helpString += "The processors parameter allows you to indicate the number of processors you want to use. Default=1.\n";
helpString += "The cutoff parameter allows you set a percentage of sequences that support the base. For example: cutoff=97 would only return a sequence that only showed ambiguities for bases that were not supported by at least 97% of sequences.\n";
helpString += "The primer.desing command should be in the following format: primer.design(list=yourListFile, fasta=yourFastaFile, name=yourNameFile)\n";
helpString += "primer.design(list=final.an.list, fasta=final.fasta, name=final.names, label=0.03)\n";
return helpString;
}
catch(exception& e) {
m->errorOut(e, "PrimerDesignCommand", "getHelpString");
exit(1);
}
}
//**********************************************************************************************************************
string PrimerDesignCommand::getOutputPattern(string type) {
try {
string pattern = "";
if (type == "fasta") { pattern = "[filename],[distance],otu.cons.fasta"; }
else if (type == "summary") { pattern = "[filename],[distance],primer.summary"; }
else if (type == "list") { pattern = "[filename],pick,[extension]"; }
else { m->mothurOut("[ERROR]: No definition for type " + type + " output pattern.\n"); m->setControl_pressed(true); }
return pattern;
}
catch(exception& e) {
m->errorOut(e, "PrimerDesignCommand", "getOutputPattern");
exit(1);
}
}
//**********************************************************************************************************************
PrimerDesignCommand::PrimerDesignCommand(string option) : Command() {
try {
if(option == "help") { help(); abort = true; calledHelp = true; }
else if(option == "citation") { citation(); abort = true; calledHelp = true;}
else if(option == "category") { abort = true; calledHelp = true; }
else {
OptionParser parser(option, setParameters());
map<string,string> parameters = parser.getParameters();
ValidParameters validParameter;
namefile = validParameter.validFile(parameters, "name");
if (namefile == "not open") { abort = true; }
else if (namefile == "not found") { namefile = ""; }
else { current->setNameFile(namefile); }
countfile = validParameter.validFile(parameters, "count");
if (countfile == "not open") { countfile = ""; abort = true; }
else if (countfile == "not found") { countfile = ""; }
else { current->setCountFile(countfile); }
//get fastafile - it is required
fastafile = validParameter.validFile(parameters, "fasta");
if (fastafile == "not open") { fastafile = ""; abort=true; }
else if (fastafile == "not found") {
fastafile = current->getFastaFile();
if (fastafile != "") { m->mothurOut("Using " + fastafile + " as input file for the fasta parameter.\n"); }
else { m->mothurOut("You have no current fastafile and the fasta parameter is required.\n"); abort = true; }
}else { current->setFastaFile(fastafile); }
//get listfile - it is required
listfile = validParameter.validFile(parameters, "list");
if (listfile == "not open") { listfile = ""; abort=true; }
else if (listfile == "not found") {
listfile = current->getListFile();
if (listfile != "") { m->mothurOut("Using " + listfile + " as input file for the list parameter.\n"); }
else { m->mothurOut("You have no current listfile and the list parameter is required.\n"); abort = true; }
}else { current->setListFile(listfile); }
if ((namefile != "") && (countfile != "")) {
m->mothurOut("[ERROR]: you may only use one of the following: name or count.\n"); abort = true;
}
if (outputdir == ""){ outputdir = util.hasPath(listfile); }
string temp = validParameter.valid(parameters, "cutoff"); if (temp == "not found") { temp = "100"; }
util.mothurConvert(temp, cutoff);
temp = validParameter.valid(parameters, "pdiffs"); if (temp == "not found") { temp = "0"; }
util.mothurConvert(temp, pdiffs);
temp = validParameter.valid(parameters, "length"); if (temp == "not found") { temp = "18"; }
util.mothurConvert(temp, length);
temp = validParameter.valid(parameters, "mintm"); if (temp == "not found") { temp = "-1"; }
util.mothurConvert(temp, minTM);
temp = validParameter.valid(parameters, "maxtm"); if (temp == "not found") { temp = "-1"; }
util.mothurConvert(temp, maxTM);
otulabel = validParameter.valid(parameters, "otulabel"); if (otulabel == "not found") { otulabel = ""; }
if (otulabel == "") { m->mothurOut("[ERROR]: You must provide an OTU label, aborting.\n"); abort = true; }
temp = validParameter.valid(parameters, "processors"); if (temp == "not found"){ temp = current->getProcessors(); }
processors = current->setProcessors(temp);
label = validParameter.valid(parameters, "label");
if (label == "not found") { label = ""; m->mothurOut("You did not provide a label, I will use the first label in your inputfile.\n"); label=""; }
}
}
catch(exception& e) {
m->errorOut(e, "PrimerDesignCommand", "PrimerDesignCommand");
exit(1);
}
}
//**********************************************************************************************************************
int PrimerDesignCommand::execute(){
try {
if (abort) { if (calledHelp) { return 0; } return 2; }
long start = time(nullptr);
//////////////////////////////////////////////////////////////////////////////
// get file inputs //
//////////////////////////////////////////////////////////////////////////////
//reads list file and selects the label the users specified or the first label
ListVector* list = getListVector();
vector<string> binLabels = list->getLabels();
int binIndex = findIndex(otulabel, binLabels);
if (binIndex == -1) { m->mothurOut("[ERROR]: You selected an OTU label that is not in your in your list file, quitting.\n"); return 0; }
map<string, int> nameMap;
long long numSeqs; //used to sanity check the files. numSeqs = total seqs for namefile and uniques for count.
//list file should have all seqs if namefile was used to create it and only uniques in count file was used.
if (namefile != "") { unsigned long int temp; nameMap = util.readNames(namefile, temp); numSeqs = temp; }
else if (countfile != "") {
CountTable ct; ct.readTable(countfile, false, false);
numSeqs = ct.getNumUniqueSeqs();
nameMap = ct.getNameMap();
}else { numSeqs = list->getNumSeqs(); }
//sanity check
if (numSeqs != list->getNumSeqs()) {
if (namefile != "") { m->mothurOut("[ERROR]: Your list file contains " + toString(list->getNumSeqs()) + " sequences, and your name file contains " + toString(numSeqs) + " sequences, aborting. Do you have the correct files? Perhaps you forgot to include the name file when you clustered? \n"); }
else if (countfile != "") {
m->mothurOut("[ERROR]: Your list file contains " + toString(list->getNumSeqs()) + " sequences, and your count file contains " + toString(numSeqs) + " unique sequences, aborting. Do you have the correct files? Perhaps you forgot to include the count file when you clustered? \n");
}
m->setControl_pressed(true);
}
if (m->getControl_pressed()) { delete list; return 0; }
//////////////////////////////////////////////////////////////////////////////
// process data //
//////////////////////////////////////////////////////////////////////////////
m->mothurOut("\nFinding consensus sequences for each otu...\n");
map<string, int> seq2Bin = getSequenceBinAssignments(list, nameMap);
vector<Sequence> conSeqs = createProcessesConSeqs(nameMap, seq2Bin, binLabels);
map<string, string> variables;
variables["[filename]"] = outputdir + util.getRootName(util.getSimpleName(listfile));
variables["[distance]"] = list->getLabel();
string consFastaFile = getOutputFileName("fasta", variables);
outputNames.push_back(consFastaFile); outputTypes["fasta"].push_back(consFastaFile);
ofstream out;
util.openOutputFile(consFastaFile, out);
for (int i = 0; i < conSeqs.size(); i++) { conSeqs[i].printSequence(out); }
out.close();
set<string> primers = getPrimer(conSeqs[binIndex]);
if (m->getControl_pressed()) { delete list; for (int i = 0; i < outputNames.size(); i++) { util.mothurRemove(outputNames[i]); } return 0; }
string consSummaryFile = getOutputFileName("summary", variables);
outputNames.push_back(consSummaryFile); outputTypes["summary"].push_back(consSummaryFile);
ofstream outSum;
util.openOutputFile(consSummaryFile, outSum);
outSum << "PrimerOtu: " << otulabel << " Members: " << list->get(binIndex) << endl << "Primers\tminTm\tmaxTm" << endl;
//find min and max melting points
vector<double> minTms;
vector<double> maxTms;
string primerString = "";
for (set<string>::iterator it = primers.begin(); it != primers.end();) {
double minTm, maxTm;
findMeltingPoint(*it, minTm, maxTm);
if (util.isEqual(minTM, -1) && util.isEqual(maxTM, -1)) { //user did not set min or max Tm so save this primer
minTms.push_back(minTm);
maxTms.push_back(maxTm);
outSum << *it << '\t' << minTm << '\t' << maxTm << endl;
it++;
}else if (util.isEqual(minTM, -1) && (maxTm <= maxTM)){ //user set max and no min, keep if below max
minTms.push_back(minTm);
maxTms.push_back(maxTm);
outSum << *it << '\t' << minTm << '\t' << maxTm << endl;
it++;
}else if (util.isEqual(maxTM, -1) && (minTm >= minTM)){ //user set min and no max, keep if above min
minTms.push_back(minTm);
maxTms.push_back(maxTm);
outSum << *it << '\t' << minTm << '\t' << maxTm << endl;
it++;
}else if ((maxTm <= maxTM) && (minTm >= minTM)) { //keep if above min and below max
minTms.push_back(minTm);
maxTms.push_back(maxTm);
outSum << *it << '\t' << minTm << '\t' << maxTm << endl;
it++;
}else { primers.erase(it++); } //erase because it didn't qualify
}
outSum << "\nOTU\tPrimer\tStart\tEnd\tLength\tMismatches\tminTm\tmaxTm\n";
outSum.close();
m->mothurOut("\nProcessing OTUs...\n");
//check each otu's conseq for each primer in otunumber
set<int> otuToRemove = createProcesses(consSummaryFile, minTms, maxTms, primers, conSeqs, binIndex, binLabels);
if (m->getControl_pressed()) { delete list; for (int i = 0; i < outputNames.size(); i++) { util.mothurRemove(outputNames[i]); } return 0; }
//print new list file
map<string, string> mvariables;
mvariables["[filename]"] = outputdir + util.getRootName(util.getSimpleName(listfile));
mvariables["[extension]"] = util.getExtension(listfile);
string newListFile = getOutputFileName("list", mvariables);
ofstream outListTemp;
util.openOutputFile(newListFile+".temp", outListTemp);
outListTemp << list->getLabel() << '\t' << (list->getNumBins()-otuToRemove.size());
string headers = "label\tnumOtus";
for (int j = 0; j < list->getNumBins(); j++) {
if (m->getControl_pressed()) { break; }
//good otus
if (otuToRemove.count(j) == 0) {
string bin = list->get(j);
if (bin != "") { outListTemp << '\t' << bin; headers += '\t' + binLabels[j]; }
}
}
outListTemp << endl;
outListTemp.close();
ofstream outList;
util.openOutputFile(newListFile, outList);
outList << headers << endl;
outList.close();
util.appendFiles(newListFile+".temp", newListFile);
util.mothurRemove(newListFile+".temp");
outputNames.push_back(newListFile); outputTypes["list"].push_back(newListFile);
delete list;
if (m->getControl_pressed()) { for (int i = 0; i < outputNames.size(); i++) { util.mothurRemove(outputNames[i]); } return 0; }
m->mothurOut("It took " + toString(time(nullptr) - start) + " secs to process " + toString(list->getNumBins()) + " OTUs.\n");
//output files created by command
m->mothurOut("\nOutput File Names: \n");
for (int i = 0; i < outputNames.size(); i++) { m->mothurOut(outputNames[i] +"\n"); } m->mothurOutEndLine();
return 0;
}
catch(exception& e) {
m->errorOut(e, "PrimerDesignCommand", "execute");
exit(1);
}
}
//********************************************************************/
//used http://www.biophp.org/minitools/melting_temperature/ as a reference to substitute degenerate bases
// in order to find the min and max Tm values.
//Tm = 64.9°C + 41°C x (number of G’s and C’s in the primer – 16.4)/N
/* A = adenine
* C = cytosine
* G = guanine
* T = thymine
* R = G A (purine)
* Y = T C (pyrimidine)
* K = G T (keto)
* M = A C (amino)
* S = G C (strong bonds)
* W = A T (weak bonds)
* B = G T C (all but A)
* D = G A T (all but C)
* H = A C T (all but G)
* V = G C A (all but T)
* N = A G C T (any) */
int PrimerDesignCommand::findMeltingPoint(string primer, double& minTm, double& maxTm){
try {
string minTmprimer = primer;
string maxTmprimer = primer;
//find minimum Tm string substituting for degenerate bases
for (int i = 0; i < minTmprimer.length(); i++) {
minTmprimer[i] = toupper(minTmprimer[i]);
if (minTmprimer[i] == 'Y') { minTmprimer[i] = 'A'; }
else if (minTmprimer[i] == 'R') { minTmprimer[i] = 'A'; }
else if (minTmprimer[i] == 'W') { minTmprimer[i] = 'A'; }
else if (minTmprimer[i] == 'K') { minTmprimer[i] = 'A'; }
else if (minTmprimer[i] == 'M') { minTmprimer[i] = 'A'; }
else if (minTmprimer[i] == 'D') { minTmprimer[i] = 'A'; }
else if (minTmprimer[i] == 'V') { minTmprimer[i] = 'A'; }
else if (minTmprimer[i] == 'H') { minTmprimer[i] = 'A'; }
else if (minTmprimer[i] == 'B') { minTmprimer[i] = 'A'; }
else if (minTmprimer[i] == 'N') { minTmprimer[i] = 'A'; }
else if (minTmprimer[i] == 'S') { minTmprimer[i] = 'G'; }
}
//find maximum Tm string substituting for degenerate bases
for (int i = 0; i < maxTmprimer.length(); i++) {
maxTmprimer[i] = toupper(maxTmprimer[i]);
if (maxTmprimer[i] == 'Y') { maxTmprimer[i] = 'G'; }
else if (maxTmprimer[i] == 'R') { maxTmprimer[i] = 'G'; }
else if (maxTmprimer[i] == 'W') { maxTmprimer[i] = 'A'; }
else if (maxTmprimer[i] == 'K') { maxTmprimer[i] = 'G'; }
else if (maxTmprimer[i] == 'M') { maxTmprimer[i] = 'G'; }
else if (maxTmprimer[i] == 'D') { maxTmprimer[i] = 'G'; }
else if (maxTmprimer[i] == 'V') { maxTmprimer[i] = 'G'; }
else if (maxTmprimer[i] == 'H') { maxTmprimer[i] = 'G'; }
else if (maxTmprimer[i] == 'B') { maxTmprimer[i] = 'G'; }
else if (maxTmprimer[i] == 'N') { maxTmprimer[i] = 'G'; }
else if (maxTmprimer[i] == 'S') { maxTmprimer[i] = 'G'; }
}
int numGC = 0;
for (int i = 0; i < minTmprimer.length(); i++) {
if (minTmprimer[i] == 'G') { numGC++; }
else if (minTmprimer[i] == 'C') { numGC++; }
}
minTm = 64.9 + 41 * (numGC - 16.4) / (double) minTmprimer.length();
numGC = 0;
for (int i = 0; i < maxTmprimer.length(); i++) {
if (maxTmprimer[i] == 'G') { numGC++; }
else if (maxTmprimer[i] == 'C') { numGC++; }
}
maxTm = 64.9 + 41 * (numGC - 16.4) / (double) maxTmprimer.length();
return 0;
}
catch(exception& e) {
m->errorOut(e, "PrimerDesignCommand", "findMeltingPoint");
exit(1);
}
}
//********************************************************************/
//find all primers for the given sequence
set<string> PrimerDesignCommand::getPrimer(Sequence primerSeq){
try {
set<string> primers;
string rawSequence = primerSeq.getUnaligned();
for (int j = 0; j < rawSequence.length()-length; j++){
if (m->getControl_pressed()) { break; }
string primer = rawSequence.substr(j, length);
primers.insert(primer);
}
return primers;
}
catch(exception& e) {
m->errorOut(e, "PrimerDesignCommand", "getPrimer");
exit(1);
}
}
//********************************************************************/
/* A = adenine
* C = cytosine
* G = guanine
* T = thymine
* R = G A (purine)
* Y = T C (pyrimidine)
* K = G T (keto)
* M = A C (amino)
* S = G C (strong bonds)
* W = A T (weak bonds)
* B = G T C (all but A)
* D = G A T (all but C)
* H = A C T (all but G)
* V = G C A (all but T)
* N = A G C T (any) */
int countDiffs(string oligo, string seq, MothurOut* m){
try {
int length = oligo.length();
int countDiffs = 0;
for(int i=0;i<length;i++){
oligo[i] = toupper(oligo[i]);
seq[i] = toupper(seq[i]);
if(oligo[i] != seq[i]){
if(oligo[i] == 'A' && (seq[i] != 'A' && seq[i] != 'M' && seq[i] != 'R' && seq[i] != 'W' && seq[i] != 'D' && seq[i] != 'H' && seq[i] != 'V')) { countDiffs++; }
else if(oligo[i] == 'C' && (seq[i] != 'C' && seq[i] != 'Y' && seq[i] != 'M' && seq[i] != 'S' && seq[i] != 'B' && seq[i] != 'H' && seq[i] != 'V')) { countDiffs++; }
else if(oligo[i] == 'G' && (seq[i] != 'G' && seq[i] != 'R' && seq[i] != 'K' && seq[i] != 'S' && seq[i] != 'B' && seq[i] != 'D' && seq[i] != 'V')) { countDiffs++; }
else if(oligo[i] == 'T' && (seq[i] != 'T' && seq[i] != 'Y' && seq[i] != 'K' && seq[i] != 'W' && seq[i] != 'B' && seq[i] != 'D' && seq[i] != 'H')) { countDiffs++; }
else if((oligo[i] == '.' || oligo[i] == '-')) { countDiffs++; }
else if((oligo[i] == 'N' || oligo[i] == 'I') && (seq[i] == 'N')) { countDiffs++; }
else if(oligo[i] == 'R' && (seq[i] != 'A' && seq[i] != 'G')) { countDiffs++; }
else if(oligo[i] == 'Y' && (seq[i] != 'C' && seq[i] != 'T')) { countDiffs++; }
else if(oligo[i] == 'M' && (seq[i] != 'C' && seq[i] != 'A')) { countDiffs++; }
else if(oligo[i] == 'K' && (seq[i] != 'T' && seq[i] != 'G')) { countDiffs++; }
else if(oligo[i] == 'W' && (seq[i] != 'T' && seq[i] != 'A')) { countDiffs++; }
else if(oligo[i] == 'S' && (seq[i] != 'C' && seq[i] != 'G')) { countDiffs++; }
else if(oligo[i] == 'B' && (seq[i] != 'C' && seq[i] != 'T' && seq[i] != 'G')) { countDiffs++; }
else if(oligo[i] == 'D' && (seq[i] != 'A' && seq[i] != 'T' && seq[i] != 'G')) { countDiffs++; }
else if(oligo[i] == 'H' && (seq[i] != 'A' && seq[i] != 'T' && seq[i] != 'C')) { countDiffs++; }
else if(oligo[i] == 'V' && (seq[i] != 'A' && seq[i] != 'C' && seq[i] != 'G')) { countDiffs++; }
}
}
return countDiffs;
}
catch(exception& e) {
m->errorOut(e, "PrimerDesignCommand", "countDiffs");
exit(1);
}
}
//********************************************************************/
//search for a primer over the sequence string
bool findPrimer(string rawSequence, string primer, vector<int>& primerStart, vector<int>& primerEnd, vector<int>& mismatches, int length, int pdiffs, MothurOut* m){
try {
bool foundAtLeastOne = false; //innocent til proven guilty
//look for exact match
if(rawSequence.length() < primer.length()) { return false; }
//search for primer
for (int j = 0; j < rawSequence.length()-length; j++){
if (m->getControl_pressed()) { return foundAtLeastOne; }
string rawChunk = rawSequence.substr(j, length);
int numDiff = countDiffs(primer, rawChunk, m);
if(numDiff <= pdiffs){
primerStart.push_back(j);
primerEnd.push_back(j+length);
mismatches.push_back(numDiff);
foundAtLeastOne = true;
}
}
return foundAtLeastOne;
}
catch(exception& e) {
m->errorOut(e, "PrimerDesignCommand", "findPrimer");
exit(1);
}
}
/**************************************************************************************************/
struct primerDesignData {
OutputWriter* summaryFile;
MothurOut* m;
int start;
int end;
int pdiffs, length, binIndex;
set<string> primers;
vector<double> minTms, maxTms;
set<int> otusToRemove;
vector<Sequence> consSeqs;
vector<string> binLabels;
int numBinsProcessed;
Utils util;
primerDesignData(){ delete summaryFile; }
primerDesignData(OutputWriter* sf, int st, int en, vector<double> min, vector<double> max, set<string> pri, vector<Sequence> seqs, int d, int otun, int l, vector<string> bl) {
summaryFile = sf;
m = MothurOut::getInstance();
start = st;
end = en;
pdiffs = d;
minTms = min;
maxTms = max;
primers = pri;
consSeqs = seqs;
binIndex = otun;
length = l;
binLabels = bl;
numBinsProcessed = 0;
}
};
//**********************************************************************************************************************
void driverPDesign(primerDesignData* params){
try {
for (int i = params->start; i < params->end; i++) {
if (params->m->getControl_pressed()) { break; }
if (i != (params->binIndex)) {
int primerIndex = 0;
string output = "";
for (set<string>::iterator it = params->primers.begin(); it != params->primers.end(); it++) {
vector<int> primerStarts;
vector<int> primerEnds;
vector<int> mismatches;
bool found = findPrimer(params->consSeqs[i].getUnaligned(), (*it), primerStarts, primerEnds, mismatches, params->length, params->pdiffs, params->m);
//if we found it report to the table
if (found) {
for (int j = 0; j < primerStarts.size(); j++) {
output += params->binLabels[i] + '\t' + *it + '\t' + toString(primerStarts[j]) + '\t' + toString(primerEnds[j]) + '\t' + toString(params->length) + '\t' + toString(mismatches[j]) + '\t' + toString(params->minTms[primerIndex]) + '\t' + toString(params->maxTms[primerIndex]) + '\n';
}
params->otusToRemove.insert(i);
}
primerIndex++;
}
params->summaryFile->write(output);
}
params->numBinsProcessed++;
if((params->numBinsProcessed) % 100 == 0){ params->m->mothurOutJustToScreen(toString(params->numBinsProcessed)+"\n"); }
}
if((params->numBinsProcessed) % 100 != 0){ params->m->mothurOutJustToScreen(toString(params->numBinsProcessed)+"\n"); }
}
catch(exception& e) {
params->m->errorOut(e, "PrimerDesignCommand", "driver");
exit(1);
}
}
/**************************************************************************************************/
set<int> PrimerDesignCommand::createProcesses(string newSummaryFile, vector<double>& minTms, vector<double>& maxTms, set<string>& primers, vector<Sequence>& conSeqs, int binIndex, vector<string>& binLabels) {
try {
//create array of worker threads
vector<std::thread*> workerThreads;
vector<primerDesignData*> data;
//sanity check
int numBins = conSeqs.size();
if (numBins < processors) { processors = numBins; }
//divide the otus between the processors
vector<linePair> lines;
int numOtusPerProcessor = numBins / processors;
for (int i = 0; i < processors; i++) {
int startIndex = i * numOtusPerProcessor;
int endIndex = (i+1) * numOtusPerProcessor;
if(i == (processors - 1)){ endIndex = numBins; }
lines.push_back(linePair(startIndex, endIndex));
}
auto synchronizedOutputSummaryFile = std::make_shared<SynchronizedOutputFile>(newSummaryFile, true); //open append
//Lauch worker threads
for (int i = 0; i < processors-1; i++) {
OutputWriter* threadSummaryWriter = new OutputWriter(synchronizedOutputSummaryFile);
primerDesignData* dataBundle = new primerDesignData(threadSummaryWriter, lines[i+1].start, lines[i+1].end, minTms, maxTms, primers, conSeqs, pdiffs, binIndex, length, binLabels);
data.push_back(dataBundle);
workerThreads.push_back(new std::thread(driverPDesign, dataBundle));
}
OutputWriter* threadSummaryWriter = new OutputWriter(synchronizedOutputSummaryFile);
primerDesignData* dataBundle = new primerDesignData(threadSummaryWriter, lines[0].start, lines[0].end, minTms, maxTms, primers, conSeqs, pdiffs, binIndex, length, binLabels);
driverPDesign(dataBundle);
set<int> otusToRemove = dataBundle->otusToRemove;
for (int i = 0; i < processors-1; i++) {
workerThreads[i]->join();
otusToRemove.insert(data[i]->otusToRemove.begin(), data[i]->otusToRemove.end());
delete data[i];
delete workerThreads[i];
}
delete dataBundle;
return otusToRemove;
}
catch(exception& e) {
m->errorOut(e, "PrimerDesignCommand", "createProcesses");
exit(1);
}
}
//**********************************************************************************************************************
int initializeCounts(vector< vector< vector<unsigned int> > >& counts, int length, int numBins, MothurOut* m){
try {
counts.clear();
//vector< vector< vector<unsigned int> > > counts - otu < spot_in_alignment < counts_for_A,T,G,C,Gap > > >
for (int i = 0; i < numBins; i++) {
vector<unsigned int> temp; temp.resize(5, 0); //A,T,G,C,Gap
vector< vector<unsigned int> > temp2;
for (int j = 0; j < length; j++) {
temp2.push_back(temp);
}
counts.push_back(temp2);
}
return 0;
}
catch(exception& e) {
m->errorOut(e, "PrimerDesignCommand", "initializeCounts");
exit(1);
}
}
/**************************************************************************************************/
struct primerCountsData {
map<string, int> seq2Bin;
vector< vector< vector<unsigned int> > > counts; // - otu < spot_in_alignment < counts_for_A,T,G,C,Gap > > >
MothurOut* m;
long long start, end, count, total, alignedLength, numBins;
string fastafile;
Utils util;
map<string, int> nameMap;
vector<long long> otuCounts;
bool hasNameMap;
primerCountsData(){ }
primerCountsData(string ff, map<string, int> nmp, long long st, long long en, map<string, int> seq2B, int nb) {
fastafile = ff;
m = MothurOut::getInstance();
start = st;
end = en;
hasNameMap = false;
nameMap = nmp;
if (nameMap.size() != 0) { hasNameMap = true; }
seq2Bin = seq2B;
numBins = nb;
count = 0; total = 0;
}
};
//**********************************************************************************************************************
map<string, int> PrimerDesignCommand::getSequenceBinAssignments(ListVector* list, map<string, int>& nameMap){
try {
map<string, int> seq2Bin;
bool hasNameMap = false;
if (nameMap.size() != 0) { hasNameMap = true; }
for (int i = 0; i < list->getNumBins(); i++) {
string binNames = list->get(i);
vector<string> names;
util.splitAtComma(binNames, names);
//lets be smart and only map the unique names if a name or count file was given to save search time and memory
if (hasNameMap) {
for (int j = 0; j < names.size(); j++) {
map<string, int>::iterator itNames = nameMap.find(names[j]);
if (itNames != nameMap.end()) { //add name because its a unique one
seq2Bin[names[j]] = i;
}
}
}else { for (int j = 0; j < names.size(); j++) { seq2Bin[names[j]] = i; } } //map everyone
}
return seq2Bin;
}
catch(exception& e) {
m->errorOut(e, "PrimerDesignCommand", "getSequenceBinAssignments");
exit(1);
}
}
/**************************************************************************************************/
void driverGetCounts(primerCountsData* params){
try {
params->otuCounts.resize(params->numBins, 0);
params->alignedLength = 0;
ifstream in; params->util.openInputFile(params->fastafile, in);
in.seekg(params->start);
//adjust start if null strings
if (params->start == 0) { params->util.zapGremlins(in); gobble(in); }
bool done = false;
while (!done) {
if (params->m->getControl_pressed()) { break; }
Sequence seq(in); gobble(in);
if (seq.getName() != "") {
if (params->count == 0) { params->alignedLength = seq.getAligned().length(); initializeCounts(params->counts, params->alignedLength, params->numBins, params->m); }
else if (params->alignedLength != seq.getAligned().length()) {
params->m->mothurOut("[ERROR]: your sequences are not all the same length. primer.design requires sequences to be aligned.\n"); params->m->setControl_pressed(true); break;
}
int num = 1;
if (params->hasNameMap) {
map<string, int>::iterator itCount = params->nameMap.find(seq.getName());
if (itCount == params->nameMap.end()) { params->m->mothurOut("[ERROR]: " + seq.getName() + " is in your fasta file and not in your name or count file, aborting.\n"); params->m->setControl_pressed(true); break; }
else { params->total += itCount->second; num = itCount->second; }
}else { params->total++; }
//increment counts
map<string, int>::iterator itCount = params->seq2Bin.find(seq.getName());
if (itCount == params->seq2Bin.end()) { params->m->mothurOut("[ERROR]: " + seq.getName() + " is in your fasta file and not in your list file, aborting.\n"); params->m->setControl_pressed(true); break;
}else {
params->otuCounts[itCount->second] += num;
string aligned = seq.getAligned();
for (int i = 0; i < params->alignedLength; i++) {
char base = toupper(aligned[i]);
if (base == 'A') { params->counts[itCount->second][i][0]+=num; }
else if (base == 'T') { params->counts[itCount->second][i][1]+=num; }
else if (base == 'G') { params->counts[itCount->second][i][2]+=num; }
else if (base == 'C') { params->counts[itCount->second][i][3]+=num; }
else { params->counts[itCount->second][i][4]+=num; }
}
}
}
params->count++;
if((params->count) % 1000 == 0){ params->m->mothurOutJustToScreen(toString(params->count)+"\n"); }
#if defined NON_WINDOWS
unsigned long long pos = in.tellg();
if ((pos == -1) || (pos >= params->end)) { break; }
#else
if ((params->count == params->end) || (in.eof())) { break; }
#endif
}
if((params->count) % 1000 != 0){ params->m->mothurOutJustToScreen(toString(params->count)+"\n"); }
in.close();
}
catch(exception& e) {
params->m->errorOut(e, "PrimerDesignCommand", "driverGetCounts");
exit(1);
}
}
/**************************************************************************************************/
vector<Sequence> PrimerDesignCommand::createProcessesConSeqs(map<string, int>& nameMap, map<string, int>& seq2Bin, vector<string>& binLabels) {
try {
int numBins = binLabels.size();
vector<linePair> lines;
#if defined NON_WINDOWS
vector<double> positions; positions = util.divideFile(fastafile, processors);
for (int i = 0; i < (positions.size()-1); i++) { lines.push_back(linePair(positions[i], positions[(i+1)])); }
#else
long long numSeqs;
vector<double> positions = util.setFilePosFasta(fastafile, numSeqs);
if (numSeqs < processors) { processors = numSeqs; m->mothurOut("Reducing processors to " + toString(numSeqs) + ".\n"); }
//figure out how many sequences you have to process
int numSeqsPerProcessor = numSeqs / processors;
for (int i = 0; i < processors; i++) {
int startIndex = i * numSeqsPerProcessor;
if(i == (processors - 1)){ numSeqsPerProcessor = numSeqs - i * numSeqsPerProcessor; }
lines.push_back(linePair(positions[startIndex], numSeqsPerProcessor));
}
#endif
//create array of worker threads
vector<std::thread*> workerThreads;
vector<primerCountsData*> data;
//Lauch worker threads
for (int i = 0; i < processors-1; i++) {
if (m->getControl_pressed()) { break; }
primerCountsData* dataBundle = new primerCountsData(fastafile, nameMap, lines[i+1].start, lines[i+1].end, seq2Bin, numBins);
data.push_back(dataBundle);
workerThreads.push_back(new std::thread(driverGetCounts, dataBundle));
}
primerCountsData* dataBundle = new primerCountsData(fastafile, nameMap, lines[0].start, lines[0].end, seq2Bin, numBins);
driverGetCounts(dataBundle);
vector< vector< vector<unsigned int> > > counts = dataBundle->counts;
vector<long long> otuCounts = dataBundle->otuCounts;
long long total = dataBundle->total;
int alignedLength = dataBundle->alignedLength;
delete dataBundle;
for (int i = 0; i < processors-1; i++) {
workerThreads[i]->join();
total += data[i]->total;
if (m->getControl_pressed()) { break; }
if (data[i]->alignedLength != alignedLength) { m->mothurOut("[ERROR]: your sequences are not all the same length. primer.design requires sequences to be aligned.\n"); m->setControl_pressed(true); }
for (int k = 0; k < numBins; k++) { //for each bin
for (int j = 0; j < data[i]->alignedLength; j++) { //for each position
for (int l = 0; l < 5; l++) { counts[k][j][l] += data[i]->counts[k][j][l]; } //for each base
}
otuCounts[k] += data[i]->otuCounts[k];
}
delete data[i];
delete workerThreads[i];
}
vector<Sequence> conSeqs;
if (m->getControl_pressed()) { return conSeqs; }
//build consensus seqs
for (int i = 0; i < counts.size(); i++) {
if (m->getControl_pressed()) { break; }
string otuLabel = binLabels[i];
string cons = "";
for (int j = 0; j < counts[i].size(); j++) { cons += getBase(counts[i][j], otuCounts[i]); }
Sequence consSeq(otuLabel, cons);
conSeqs.push_back(consSeq);
}
if (m->getControl_pressed()) { conSeqs.clear(); return conSeqs; }
return conSeqs;
}
catch(exception& e) {
m->errorOut(e, "PrimerDesignCommand", "createProcessesConSeqs");
exit(1);
}
}
//***************************************************************************************************************
char PrimerDesignCommand::getBase(vector<unsigned int> counts, int size){ //A,T,G,C,Gap
try{
/* A = adenine
* C = cytosine
* G = guanine
* T = thymine
* R = G A (purine)
* Y = T C (pyrimidine)
* K = G T (keto)
* M = A C (amino)
* S = G C (strong bonds)
* W = A T (weak bonds)
* B = G T C (all but A)
* D = G A T (all but C)
* H = A C T (all but G)
* V = G C A (all but T)
* N = A G C T (any) */
char conBase = 'N';
//zero out counts that don't make the cutoff
float percentage = (100.0 - cutoff) / 100.0;
for (int i = 0; i < counts.size(); i++) {
float countPercentage = counts[i] / (float) size;
if (countPercentage < percentage) { counts[i] = 0; }
}
//any
if ((counts[0] != 0) && (counts[1] != 0) && (counts[2] != 0) && (counts[3] != 0) && (counts[4] != 0)) { conBase = 'n'; }
//any no gap
else if ((counts[0] != 0) && (counts[1] != 0) && (counts[2] != 0) && (counts[3] != 0) && (counts[4] == 0)) { conBase = 'N'; }
//all but T
else if ((counts[0] != 0) && (counts[1] == 0) && (counts[2] != 0) && (counts[3] != 0) && (counts[4] != 0)) { conBase = 'v'; }
//all but T no gap
else if ((counts[0] != 0) && (counts[1] == 0) && (counts[2] != 0) && (counts[3] != 0) && (counts[4] == 0)) { conBase = 'V'; }
//all but G
else if ((counts[0] != 0) && (counts[1] != 0) && (counts[2] == 0) && (counts[3] != 0) && (counts[4] != 0)) { conBase = 'h'; }
//all but G no gap
else if ((counts[0] != 0) && (counts[1] != 0) && (counts[2] == 0) && (counts[3] != 0) && (counts[4] == 0)) { conBase = 'H'; }
//all but C
else if ((counts[0] != 0) && (counts[1] != 0) && (counts[2] != 0) && (counts[3] == 0) && (counts[4] != 0)) { conBase = 'd'; }
//all but C no gap
else if ((counts[0] != 0) && (counts[1] != 0) && (counts[2] != 0) && (counts[3] == 0) && (counts[4] == 0)) { conBase = 'D'; }
//all but A
else if ((counts[0] == 0) && (counts[1] != 0) && (counts[2] != 0) && (counts[3] != 0) && (counts[4] != 0)) { conBase = 'b'; }
//all but A no gap
else if ((counts[0] == 0) && (counts[1] != 0) && (counts[2] != 0) && (counts[3] != 0) && (counts[4] == 0)) { conBase = 'B'; }
//W = A T (weak bonds)
else if ((counts[0] != 0) && (counts[1] != 0) && (counts[2] == 0) && (counts[3] == 0) && (counts[4] != 0)) { conBase = 'w'; }
//W = A T (weak bonds) no gap
else if ((counts[0] != 0) && (counts[1] != 0) && (counts[2] == 0) && (counts[3] == 0) && (counts[4] == 0)) { conBase = 'W'; }
//S = G C (strong bonds)
else if ((counts[0] == 0) && (counts[1] == 0) && (counts[2] != 0) && (counts[3] != 0) && (counts[4] != 0)) { conBase = 's'; }
//S = G C (strong bonds) no gap
else if ((counts[0] == 0) && (counts[1] == 0) && (counts[2] != 0) && (counts[3] != 0) && (counts[4] == 0)) { conBase = 'S'; }
//M = A C (amino)
else if ((counts[0] != 0) && (counts[1] == 0) && (counts[2] == 0) && (counts[3] != 0) && (counts[4] != 0)) { conBase = 'm'; }
//M = A C (amino) no gap
else if ((counts[0] != 0) && (counts[1] == 0) && (counts[2] == 0) && (counts[3] != 0) && (counts[4] == 0)) { conBase = 'M'; }
//K = G T (keto)
else if ((counts[0] == 0) && (counts[1] != 0) && (counts[2] != 0) && (counts[3] == 0) && (counts[4] != 0)) { conBase = 'k'; }
//K = G T (keto) no gap
else if ((counts[0] == 0) && (counts[1] != 0) && (counts[2] != 0) && (counts[3] == 0) && (counts[4] == 0)) { conBase = 'K'; }
//Y = T C (pyrimidine)
else if ((counts[0] == 0) && (counts[1] != 0) && (counts[2] == 0) && (counts[3] != 0) && (counts[4] != 0)) { conBase = 'y'; }
//Y = T C (pyrimidine) no gap
else if ((counts[0] == 0) && (counts[1] != 0) && (counts[2] == 0) && (counts[3] != 0) && (counts[4] == 0)) { conBase = 'Y'; }
//R = G A (purine)
else if ((counts[0] != 0) && (counts[1] == 0) && (counts[2] != 0) && (counts[3] == 0) && (counts[4] != 0)) { conBase = 'r'; }
//R = G A (purine) no gap
else if ((counts[0] != 0) && (counts[1] == 0) && (counts[2] != 0) && (counts[3] == 0) && (counts[4] == 0)) { conBase = 'R'; }
//only A
else if ((counts[0] != 0) && (counts[1] == 0) && (counts[2] == 0) && (counts[3] == 0) && (counts[4] != 0)) { conBase = 'a'; }
//only A no gap
else if ((counts[0] != 0) && (counts[1] == 0) && (counts[2] == 0) && (counts[3] == 0) && (counts[4] == 0)) { conBase = 'A'; }
//only T
else if ((counts[0] == 0) && (counts[1] != 0) && (counts[2] == 0) && (counts[3] == 0) && (counts[4] != 0)) { conBase = 't'; }
//only T no gap
else if ((counts[0] == 0) && (counts[1] != 0) && (counts[2] == 0) && (counts[3] == 0) && (counts[4] == 0)) { conBase = 'T'; }
//only G
else if ((counts[0] == 0) && (counts[1] == 0) && (counts[2] != 0) && (counts[3] == 0) && (counts[4] != 0)) { conBase = 'g'; }
//only G no gap
else if ((counts[0] == 0) && (counts[1] == 0) && (counts[2] != 0) && (counts[3] == 0) && (counts[4] == 0)) { conBase = 'G'; }
//only C
else if ((counts[0] == 0) && (counts[1] == 0) && (counts[2] == 0) && (counts[3] != 0) && (counts[4] != 0)) { conBase = 'c'; }
//only C no gap
else if ((counts[0] == 0) && (counts[1] == 0) && (counts[2] == 0) && (counts[3] != 0) && (counts[4] == 0)) { conBase = 'C'; }
//only gap
else if ((counts[0] == 0) && (counts[1] == 0) && (counts[2] == 0) && (counts[3] == 0) && (counts[4] != 0)) { conBase = '-'; }
//cutoff removed all counts
else if ((counts[0] == 0) && (counts[1] == 0) && (counts[2] == 0) && (counts[3] == 0) && (counts[4] == 0)) { conBase = 'N'; }
else{ m->mothurOut("[ERROR]: cannot find consensus base.\n"); }
return conBase;
}
catch(exception& e) {
m->errorOut(e, "PrimerDesignCommand", "getBase");
exit(1);
}
}
//**********************************************************************************************************************
ListVector* PrimerDesignCommand::getListVector(){
try {
InputData input(listfile, "list", nullVector);
ListVector* list = input.getListVector();
string lastLabel = list->getLabel();
if (label == "") { label = lastLabel; return list; }
//if the users enters label "0.06" and there is no "0.06" in their file use the next lowest label.
set<string> labels; labels.insert(label);
set<string> processedLabels;
set<string> userLabels = labels;
//as long as you are not at the end of the file or done wih the lines you want
while((list != nullptr) && (userLabels.size() != 0)) {
if (m->getControl_pressed()) { return list; }
if(labels.count(list->getLabel()) == 1){
processedLabels.insert(list->getLabel());
userLabels.erase(list->getLabel());
break;
}
if ((util.anyLabelsToProcess(list->getLabel(), userLabels, "") ) && (processedLabels.count(lastLabel) != 1)) {
string saveLabel = list->getLabel();
delete list;
list = input.getListVector(lastLabel);
processedLabels.insert(list->getLabel());
userLabels.erase(list->getLabel());
//restore real lastlabel to save below
list->setLabel(saveLabel);
break;
}
lastLabel = list->getLabel();
//get next line to process
//prevent memory leak
delete list;
list = input.getListVector();
}
if (m->getControl_pressed()) { return list; }
//output error messages about any remaining user labels
bool needToRun = false;
for (set<string>::iterator it = userLabels.begin(); it != userLabels.end(); it++) {
m->mothurOut("Your file does not include the label " + *it);
if (processedLabels.count(lastLabel) != 1) { m->mothurOut(". I will use " + lastLabel + ".\n"); needToRun = true; }
else { m->mothurOut(". Please refer to " + lastLabel + ".\n"); }
}
//run last label if you need to
if (needToRun ) {
delete list;
list = input.getListVector(lastLabel);
}
return list;
}
catch(exception& e) {
m->errorOut(e, "PrimerDesignCommand", "getListVector");
exit(1);
}
}
//**********************************************************************************************************************
int PrimerDesignCommand::findIndex(string binLabel, vector<string> binLabels){
try {
int index = -1;
for (int i = 0; i < binLabels.size(); i++){
if (m->getControl_pressed()) { return index; }
if (util.isLabelEquivalent(binLabel, binLabels[i])) { index = i; break; }
}
return index;
}
catch(exception& e) {
m->errorOut(e, "PrimerDesignCommand", "findIndex");
exit(1);
}
}
//**********************************************************************************************************************
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