try {

CommandParameter pdesign("design", "InputTypes", "", "", "none", "none", "none","",false,true,true); parameters.push_back(pdesign);

CommandParameter pshared("shared", "InputTypes", "", "", "none", "none", "none","summary",false,true,true); parameters.push_back(pshared);

CommandParameter pclass("class", "String", "", "", "", "", "","",false,false); parameters.push_back(pclass);

CommandParameter psubclass("subclass", "String", "", "", "", "", "","",false,false); parameters.push_back(psubclass);

CommandParameter plabel("label", "String", "", "", "", "", "","",false,false); parameters.push_back(plabel);

CommandParameter pclasses("classes", "String", "", "", "", "", "","",false,false); parameters.push_back(pclasses);

CommandParameter palpha("anova_alpha", "Number", "", "0.05", "", "", "","",false,false); parameters.push_back(palpha);

CommandParameter pwalpha("wilcoxon_alpha", "Number", "", "0.05", "", "", "","",false,false); parameters.push_back(pwalpha);

//every command must have inputdir and outputdir. This allows mothur users to redirect input and output files.

CommandParameter pinputdir("inputdir", "String", "", "", "", "", "","",false,false); parameters.push_back(pinputdir);

CommandParameter poutputdir("outputdir", "String", "", "", "", "", "","",false,false); parameters.push_back(poutputdir);

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, "LefseCommand", "setParameters");

exit(1);

}

try {

string helpString = "";

helpString += "The lefse command allows you to ....\n";

helpString += "The lefse command parameters are: shared, design, class, subclass, label, classes, wilcoxon_alpha, anova_alpha.\n";

helpString += "The class parameter is used to indicate the which category you would like used for the Kruskal Wallis analysis. If none is provided first category is used.\n";

helpString += "The subclass parameter is used to indicate the .....If none is provided second category is used, or if only one category subclass is ignored. \n";

helpString += "The classes parameter is used to indicate the classes you would like to use. Clases should be inputted in the following format: classes=label<value1|value2|value3>-label<value1|value2>. For example to include groups from treatment with value early or late and age= young or old. class=treatment<Early|Late>-age<young|old>.\n";

helpString += "The label parameter is used to indicate which distances in the shared file you would like to use. labels are separated by dashes.\n";

helpString += "The lefse command should be in the following format: lefse(shared=final.an.shared, design=final.design, class=treatment, subclass=age).\n";

return helpString;

}

catch(exception& e) {

m->errorOut(e, "LefseCommand", "getHelpString");

exit(1);

}

try {

string pattern = "";

if (type == "summary") { pattern = "[filename],[distance],lefse_summary"; }

else if (type == "kruskall-wallis") { pattern = "[filename],[distance],kruskall_wallis"; }

else if (type == "wilcoxon") { pattern = "[filename],[distance],wilcoxon"; }

else { m->mothurOut("[ERROR]: No definition for type " + type + " output pattern.\n"); m->control_pressed = true; }

return pattern;

}

catch(exception& e) {

m->errorOut(e, "LefseCommand", "getOutputPattern");

exit(1);

}

try {

abort = true; calledHelp = true;

setParameters();

vector<string> tempOutNames;

outputTypes["summary"] = tempOutNames;

outputTypes["kruskall-wallis"] = tempOutNames;

outputTypes["wilcoxon"] = tempOutNames;

}

catch(exception& e) {

m->errorOut(e, "LefseCommand", "LefseCommand");

exit(1);

}

try {

abort = false; calledHelp = false;

allLines = 1;

//allow user to run help

if(option == "help") { help(); abort = true; calledHelp = true; }

else if(option == "citation") { citation(); abort = true; calledHelp = true;}

else {

//valid paramters for this command

vector<string> myArray = setParameters();

OptionParser parser(option);

map<string,string> parameters = parser.getParameters();

ValidParameters validParameter;

map<string,string>::iterator it;

//check to make sure all parameters are valid for command

for (it = parameters.begin(); it != parameters.end(); it++) {

if (validParameter.isValidParameter(it->first, myArray, it->second) != true) { abort = true; }

}

vector<string> tempOutNames;

outputTypes["summary"] = tempOutNames;

outputTypes["kruskall-wallis"] = tempOutNames;

outputTypes["wilcoxon"] = tempOutNames;

//if the user changes the input directory command factory will send this info to us in the output parameter

string inputDir = validParameter.validFile(parameters, "inputdir", false);

if (inputDir == "not found"){ inputDir = ""; }

else {

string path;

it = parameters.find("design");

//user has given a template file

if(it != parameters.end()){

path = m->hasPath(it->second);

//if the user has not given a path then, add inputdir. else leave path alone.

if (path == "") { parameters["desing"] = inputDir + it->second; }

}

it = parameters.find("shared");

//user has given a template file

if(it != parameters.end()){

path = m->hasPath(it->second);

//if the user has not given a path then, add inputdir. else leave path alone.

if (path == "") { parameters["shared"] = inputDir + it->second; }

}

}

//get shared file, it is required

sharedfile = validParameter.validFile(parameters, "shared", true);

if (sharedfile == "not open") { sharedfile = ""; abort = true; }

else if (sharedfile == "not found") {

//if there is a current shared file, use it

sharedfile = m->getSharedFile();

if (sharedfile != "") { m->mothurOut("Using " + sharedfile + " as input file for the shared parameter."); m->mothurOutEndLine(); }

else { m->mothurOut("You have no current sharedfile and the shared parameter is required."); m->mothurOutEndLine(); abort = true; }

}else { m->setSharedFile(sharedfile); }

//get shared file, it is required

designfile = validParameter.validFile(parameters, "design", true);

if (designfile == "not open") { designfile = ""; abort = true; }

else if (designfile == "not found") {

//if there is a current shared file, use it

designfile = m->getDesignFile();

if (designfile != "") { m->mothurOut("Using " + designfile + " as input file for the design parameter."); m->mothurOutEndLine(); }

else { m->mothurOut("You have no current design file and the design parameter is required."); m->mothurOutEndLine(); abort = true; }

}else { m->setDesignFile(designfile); }

//if the user changes the output directory command factory will send this info to us in the output parameter

outputDir = validParameter.validFile(parameters, "outputdir", false); if (outputDir == "not found"){

outputDir = m->hasPath(sharedfile); //if user entered a file with a path then preserve it

}

string label = validParameter.validFile(parameters, "label", false);

if (label == "not found") { label = ""; }

else {

if(label != "all") { m->splitAtDash(label, labels); allLines = 0; }

else { allLines = 1; }

}

mclass = validParameter.validFile(parameters, "class", false);

if (mclass == "not found") { mclass = ""; }

subclass = validParameter.validFile(parameters, "subclass", false);

if (subclass == "not found") { subclass = ""; }

classes = validParameter.validFile(parameters, "classes", false);

if (classes == "not found") { classes = ""; }

string temp = validParameter.validFile(parameters, "anova_alpha", false);

if (temp == "not found") { temp = "0.05"; }

m->mothurConvert(temp, anovaAlpha);

temp = validParameter.validFile(parameters, "wilcoxon_alpha", false);

if (temp == "not found") { temp = "0.05"; }

m->mothurConvert(temp, wilcoxonAlpha);

}

}

catch(exception& e) {

m->errorOut(e, "LefseCommand", "LefseCommand");

exit(1);

}

try {

if (abort == true) { if (calledHelp) { return 0; } return 2; }

DesignMap designMap(designfile);

//if user set classes set groups=those classes

if (classes != "") {

map<string, vector<string> > thisClasses = m->parseClasses(classes);

vector<string> groups = designMap.getNamesUnique(thisClasses);

if (groups.size() != 0) { m->setGroups(groups); }

else { m->mothurOut("[ERROR]: no groups meet your classes requirement, quitting.\n"); return 0; }

}

//if user did not select class use first column

if (mclass == "") { mclass = designMap.getDefaultClass(); m->mothurOut("\nYou did not provide a class, using " + mclass +".\n\n"); }

InputData input(sharedfile, "sharedfile");

vector<SharedRAbundVector*> lookup = input.getSharedRAbundVectors();

string lastLabel = lookup[0]->getLabel();

//if the users enters label "0.06" and there is no "0.06" in their file use the next lowest 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((lookup[0] != NULL) && ((allLines == 1) || (userLabels.size() != 0))) {

if (m->control_pressed) { for (int i = 0; i < lookup.size(); i++) { delete lookup[i]; } return 0; }

if(allLines == 1 || labels.count(lookup[0]->getLabel()) == 1){

m->mothurOut(lookup[0]->getLabel()); m->mothurOutEndLine();

process(lookup, designMap);

processedLabels.insert(lookup[0]->getLabel());

userLabels.erase(lookup[0]->getLabel());

}

if ((m->anyLabelsToProcess(lookup[0]->getLabel(), userLabels, "") == true) && (processedLabels.count(lastLabel) != 1)) {

string saveLabel = lookup[0]->getLabel();

for (int i = 0; i < lookup.size(); i++) { delete lookup[i]; }

lookup = input.getSharedRAbundVectors(lastLabel);

m->mothurOut(lookup[0]->getLabel()); m->mothurOutEndLine();

process(lookup, designMap);

processedLabels.insert(lookup[0]->getLabel());

userLabels.erase(lookup[0]->getLabel());

//restore real lastlabel to save below

lookup[0]->setLabel(saveLabel);

}

lastLabel = lookup[0]->getLabel();

//prevent memory leak

for (int i = 0; i < lookup.size(); i++) { delete lookup[i]; lookup[i] = NULL; }

if (m->control_pressed) { return 0; }

//get next line to process

lookup = input.getSharedRAbundVectors();

}

if (m->control_pressed) { return 0; }

//output error messages about any remaining user labels

set<string>::iterator it;

bool needToRun = false;

for (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 + "."); m->mothurOutEndLine();

needToRun = true;

}else {

m->mothurOut(". Please refer to " + lastLabel + "."); m->mothurOutEndLine();

}

}

//run last label if you need to

if (needToRun == true) {

for (int i = 0; i < lookup.size(); i++) { if (lookup[i] != NULL) { delete lookup[i]; } }

lookup = input.getSharedRAbundVectors(lastLabel);

m->mothurOut(lookup[0]->getLabel()); m->mothurOutEndLine();

process(lookup, designMap);

for (int i = 0; i < lookup.size(); i++) { delete lookup[i]; }

}

//output files created by command

m->mothurOutEndLine();

m->mothurOut("Output File Names: "); m->mothurOutEndLine();

for (int i = 0; i < outputNames.size(); i++) { m->mothurOut(outputNames[i]); m->mothurOutEndLine(); }

m->mothurOutEndLine();

return 0;

}

catch(exception& e) {

m->errorOut(e, "LefseCommand", "execute");

exit(1);

}

try {

//run kruskal wallis on each otu

vector<int> significantOtuLabels = runKruskalWallis(lookup, designMap);

//check for subclass

if (subclass != "") { significantOtuLabels = runWilcoxon(lookup, designMap, significantOtuLabels); }

return 0;

}

catch(exception& e) {

m->errorOut(e, "LefseCommand", "process");

exit(1);

}

try {

map<string, string> variables;

variables["[filename]"] = outputDir + m->getRootName(m->getSimpleName(sharedfile));

variables["[distance]"] = lookup[0]->getLabel();

string outputFileName = getOutputFileName("kruskall-wallis",variables);

ofstream out;

m->openOutputFile(outputFileName, out);

outputNames.push_back(outputFileName); outputTypes["kruskall-wallis"].push_back(outputFileName);

out << "OTULabel\tKW\tPvalue\n";

vector<int> significantOtuLabels;

int numBins = lookup[0]->getNumBins();

//sanity check to make sure each treatment has a group in the shared file

set<string> treatments;

for (int j = 0; j < lookup.size(); j++) {

string group = lookup[j]->getGroup();

string treatment = designMap.get(group, mclass); //get value for this group in this category

treatments.insert(treatment);

}

if (treatments.size() < 2) { m->mothurOut("[ERROR]: need at least 2 things to classes to compare, quitting.\n"); m->control_pressed = true; }

LinearAlgebra linear;

for (int i = 0; i < numBins; i++) {

if (m->control_pressed) { break; }

vector<spearmanRank> values;

for (int j = 0; j < lookup.size(); j++) {

string group = lookup[j]->getGroup();

string treatment = designMap.get(group, mclass); //get value for this group in this category

spearmanRank temp(treatment, lookup[j]->getAbundance(i));

values.push_back(temp);

}

double pValue = 0.0;

double H = linear.calcKruskalWallis(values, pValue);

//output H and signifigance

out << m->currentBinLabels[i] << '\t' << H << '\t' << pValue << endl;

if (pValue < anovaAlpha) { significantOtuLabels.push_back(i); }

}

out.close();

return significantOtuLabels;

}

catch(exception& e) {

m->errorOut(e, "LefseCommand", "runKruskalWallis");

exit(1);

}

try {

vector<int> significantOtuLabels;

//if it exists and meets the following requirements run Wilcoxon

/*

1. Subclass members all belong to same main class

2. Number of groups in each subclass is the same

3. anything else??

*/

vector<string> subclasses;

map<string, string> subclass2Class;

map<string, int> subclassCounts;

map<string, vector<int> > subClass2GroupIndex; //maps subclass name to vector of indexes in lookup from that subclass. old -> 1,2,3 means groups in location 1,2,3 of lookup are from old. Saves time below.

bool error = false;

for (int j = 0; j < lookup.size(); j++) {

string group = lookup[j]->getGroup();

string treatment = designMap.get(group, mclass); //get value for this group in this category

string thisSub = designMap.get(group, subclass);

map<string, string>::iterator it = subclass2Class.find(thisSub);

if (it == subclass2Class.end()) {

subclass2Class[thisSub] = treatment;

subclassCounts[thisSub] = 1;

vector<int> temp; temp.push_back(j);

subClass2GroupIndex[thisSub] = temp;

}

else {

subclassCounts[thisSub]++;

subClass2GroupIndex[thisSub].push_back(j);

if (it->second != treatment) {

error = true;

m->mothurOut("[ERROR]: subclass " + thisSub + " has members in " + it->second + " and " + treatment + ". Subclass members must be from the same class. Ignoring wilcoxon.\n");

}

}

}

if (error) { return significantOtuLabels; }

else { //check counts to make sure subclasses are the same size

set<int> counts;

for (map<string, int>::iterator it = subclassCounts.begin(); it != subclassCounts.end(); it++) { counts.insert(it->second); }

if (counts.size() > 1) { m->mothurOut("[ERROR]: subclasses must be the same size. Ignoring wilcoxon.\n");

return significantOtuLabels; }

}

int numBins = lookup[0]->getNumBins();

vector<compGroup> comp;

//find comparisons and fill comp

map<string, int>::iterator itB;

for(map<string, int>::iterator it=subclassCounts.begin();it!=subclassCounts.end();it++){

itB = it;itB++;

for(itB;itB!=subclassCounts.end();itB++){

compGroup temp(it->first,itB->first);

comp.push_back(temp);

}

}

int numComp = comp.size();

if (numComp < 2) { m->mothurOut("[ERROR]: Need at least 2 subclasses, Ignoring Wilcoxon.\n");

return significantOtuLabels; }

map<string, string> variables;

variables["[filename]"] = outputDir + m->getRootName(m->getSimpleName(sharedfile));

variables["[distance]"] = lookup[0]->getLabel();

string outputFileName = getOutputFileName("wilcoxon",variables);

ofstream out;

m->openOutputFile(outputFileName, out);

outputNames.push_back(outputFileName); outputTypes["wilcoxon"].push_back(outputFileName);

out << "OTULabel\tComparision\tWilcoxon\tPvalue\n";

LinearAlgebra linear;

for (int i = 0; i < numBins; i++) {

if (m->control_pressed) { break; }

if (m->inUsersGroups(i, bins)) { //flagged in Kruskal Wallis

bool sig = false;

//for each subclass comparision

for (int j = 0; j < numComp; j++) {

//fill x and y with this comparisons data

vector<double> x; vector<double> y;

//fill x and y

vector<int> xIndexes = subClass2GroupIndex[comp[j].group1]; //indexes in lookup for this subclass

for (int k = 0; k < xIndexes.size(); k++) { x.push_back(lookup[xIndexes[k]]->getAbundance(i)); }

vector<int> yIndexes = subClass2GroupIndex[comp[j].group2]; //indexes in lookup for this subclass

for (int k = 0; k < yIndexes.size(); k++) { y.push_back(lookup[yIndexes[k]]->getAbundance(i)); }

double pValue = 0.0;

double H = linear.calcWilcoxon(x, y, pValue);

//output H and signifigance

out << m->currentBinLabels[i] << '\t' << comp[j].getCombo() << '\t' << H << '\t' << pValue << endl;

//set sig - not sure how yet

}

if (sig) { significantOtuLabels.push_back(i); }

}

}

out.close();

return significantOtuLabels;

}

catch(exception& e) {

m->errorOut(e, "LefseCommand", "runWilcoxon");

exit(1);

}