5 * Created by westcott on 8/24/09.
6 * Copyright 2009 Schloss Lab. All rights reserved.
11 #include "ignoregaps.h"
12 #include "eachgapdist.h"
15 //***************************************************************************************************************
16 Ccode::Ccode(string filename, string o) {
17 fastafile = filename; outputDir = o;
18 distCalc = new eachGapDist();
19 decalc = new DeCalculator();
21 mapInfo = outputDir + getRootName(getSimpleName(fastafile)) + "mapinfo";
23 openOutputFile(mapInfo, out2);
25 out2 << "Place in masked, filtered and trimmed sequence\tPlace in original alignment" << endl;
28 //***************************************************************************************************************
33 //***************************************************************************************************************
34 void Ccode::printHeader(ostream& out) {
35 out << "For full window mapping info refer to " << mapInfo << endl << endl;
37 //***************************************************************************************************************
38 void Ccode::print(ostream& out) {
44 openOutputFileAppend(mapInfo, out2);
46 out2 << querySeq->getName() << endl;
47 for (it = spotMap.begin(); it!= spotMap.end(); it++) {
48 out2 << it->first << '\t' << it->second << endl;
51 out << querySeq->getName() << endl << endl << "Reference sequences used and distance to query:" << endl;
53 for (int j = 0; j < closest.size(); j++) {
54 out << closest[j].seq->getName() << '\t' << closest[j].dist << endl;
59 //window mapping info.
60 out << "Mapping information: ";
61 //you mask and did not filter
62 if ((seqMask != "") && (!filter)) { out << "mask and trim."; }
64 //you filtered and did not mask
65 if ((seqMask == "") && (filter)) { out << "filter and trim."; }
67 //you masked and filtered
68 if ((seqMask != "") && (filter)) { out << "mask, filter and trim."; }
70 out << endl << "Window\tStartPos\tEndPos" << endl;
72 for (int k = 0; k < windows.size()-1; k++) {
73 out << k+1 << '\t' << spotMap[windows[k]-it->first] << '\t' << spotMap[windows[k]-it->first+windowSizes] << endl;
76 out << windows.size() << '\t' << spotMap[windows[windows.size()-1]-it->first] << '\t' << spotMap[it->second-it->first-1] << endl;
78 out << "Window\tAvgQ\t(sdQ)\tAvgR\t(sdR)\tRatio\tAnova" << endl;
79 for (int k = 0; k < windows.size(); k++) {
80 float ds = averageQuery[k] / averageRef[k];
81 out << k+1 << '\t' << averageQuery[k] << '\t' << sdQuery[k] << '\t' << averageRef[k] << '\t'<< sdRef[k] << '\t' << ds << '\t' << anova[k] << endl;
87 /* F test for differences among variances.
88 * varQuery is expected to be higher or similar than varRef */
89 //float fs = varQuery[query] / varRef[query]; /* F-Snedecor, test for differences of variances */
93 //confidence limit, t - Student, anova
94 out << "Window\tConfidenceLimit\tt-Student\tAnova" << endl;
96 for (int k = 0; k < windows.size(); k++) {
98 if (isChimericConfidence[k]) { temp += "*\t"; }
99 else { temp += "\t"; }
101 if (isChimericTStudent[k]) { temp += "*\t"; }
102 else { temp += "\t"; }
104 if (isChimericANOVA[k]) { temp += "*\t"; }
105 else { temp += "\t"; }
107 out << k+1 << '\t' << temp << endl;
109 if (temp == "*\t*\t*\t") { results = true; }
114 mothurOut(querySeq->getName() + " was found have at least one chimeric window."); mothurOutEndLine();
118 for (int i = 0; i < closest.size(); i++) { delete closest[i].seq; }
122 catch(exception& e) {
123 errorOut(e, "Ccode", "print");
127 //***************************************************************************************************************
128 int Ccode::getChimeras(Sequence* query) {
139 sumSquaredRef.clear();
140 sumSquaredQuery.clear();
142 averageQuery.clear();
144 isChimericConfidence.clear();
145 isChimericTStudent.clear();
146 isChimericANOVA.clear();
149 windowSizes = window;
155 //find closest matches to query
156 closest = findClosest(query, numWanted);
159 for (int i = 0; i < query->getAligned().length(); i++) { spotMap[i] = i; }
161 //mask sequences if the user wants to
163 decalc->setMask(seqMask);
165 decalc->runMask(query);
168 for (int i = 0; i < closest.size(); i++) { decalc->runMask(closest[i].seq); }
170 spotMap = decalc->getMaskMap();
174 vector<Sequence*> temp;
175 for (int i = 0; i < closest.size(); i++) { temp.push_back(closest[i].seq); }
176 temp.push_back(query);
178 createFilter(temp, 0.5);
180 for (int i = 0; i < temp.size(); i++) { runFilter(temp[i]); }
183 map<int, int> newMap;
186 for (int i = 0; i < filterString.length(); i++) {
187 if (filterString[i] == '1') {
189 newMap[spot] = spotMap[i];
196 //trim sequences - this follows ccodes remove_extra_gaps
197 trimSequences(query);
200 //windows are equivalent to words - ccode paper recommends windows are between 5% and 20% on alignment length().
201 //Our default will be 10% and we will warn if user tries to use a window above or below these recommendations
202 windows = findWindows();
205 //remove sequences that are more than 20% different and less than 0.5% different - may want to allow user to specify this later
206 removeBadReferenceSeqs(closest);
209 //find the averages for each querys references
210 getAverageRef(closest); //fills sumRef, averageRef, sumSquaredRef and refCombo.
211 getAverageQuery(closest, query); //fills sumQuery, averageQuery, sumSquaredQuery.
214 //find the averages for each querys references
215 findVarianceRef(); //fills varRef and sdRef also sets minimum error rate to 0.001 to avoid divide by 0.
218 //find the averages for the query
219 findVarianceQuery(); //fills varQuery and sdQuery also sets minimum error rate to 0.001 to avoid divide by 0.
221 determineChimeras(); //fills anova, isChimericConfidence, isChimericTStudent and isChimericANOVA.
225 catch(exception& e) {
226 errorOut(e, "Ccode", "getChimeras");
230 /***************************************************************************************************************/
231 //ccode algo says it does this to "Removes the initial and final gaps to avoid biases due to incomplete sequences."
232 void Ccode::trimSequences(Sequence* query) {
235 int frontPos = 0; //should contain first position in all seqs that is not a gap character
236 int rearPos = query->getAligned().length();
238 //********find first position in closest seqs that is a non gap character***********//
239 //find first position all query seqs that is a non gap character
240 for (int i = 0; i < closest.size(); i++) {
242 string aligned = closest[i].seq->getAligned();
245 //find first spot in this seq
246 for (int j = 0; j < aligned.length(); j++) {
247 if (isalpha(aligned[j])) {
253 //save this spot if it is the farthest
254 if (pos > frontPos) { frontPos = pos; }
257 //find first position all querySeq[query] that is a non gap character
258 string aligned = query->getAligned();
261 //find first spot in this seq
262 for (int j = 0; j < aligned.length(); j++) {
263 if (isalpha(aligned[j])) {
269 //save this spot if it is the farthest
270 if (pos > frontPos) { frontPos = pos; }
273 //********find last position in closest seqs that is a non gap character***********//
274 for (int i = 0; i < closest.size(); i++) {
276 string aligned = closest[i].seq->getAligned();
277 int pos = aligned.length();
279 //find first spot in this seq
280 for (int j = aligned.length()-1; j >= 0; j--) {
281 if (isalpha(aligned[j])) {
287 //save this spot if it is the farthest
288 if (pos < rearPos) { rearPos = pos; }
291 //find last position all querySeqs[query] that is a non gap character
292 aligned = query->getAligned();
293 pos = aligned.length();
295 //find first spot in this seq
296 for (int j = aligned.length()-1; j >= 0; j--) {
297 if (isalpha(aligned[j])) {
303 //save this spot if it is the farthest
304 if (pos < rearPos) { rearPos = pos; }
307 //check to make sure that is not whole seq
308 if ((rearPos - frontPos - 1) <= 0) { mothurOut("Error, when I trim your sequences, the entire sequence is trimmed."); mothurOutEndLine(); exit(1); }
310 map<int, int> tempTrim;
311 tempTrim[frontPos] = rearPos;
313 //save trimmed locations
317 map<int, int> newMap;
320 for (int i = frontPos; i < rearPos; i++) {
322 newMap[spot] = spotMap[i];
327 catch(exception& e) {
328 errorOut(e, "Ccode", "trimSequences");
332 /***************************************************************************************************************/
333 vector<int> Ccode::findWindows() {
339 int length = it->second - it->first;
341 //default is wanted = 10% of total length
342 if (windowSizes > length) {
343 mothurOut("You have slected a window larger than your sequence length after all filters, masks and trims have been done. I will use the default 10% of sequence length.");
344 windowSizes = length / 10;
345 }else if (windowSizes == 0) { windowSizes = length / 10; }
346 else if (windowSizes > (length * 0.20)) {
347 mothurOut("You have selected a window that is larger than 20% of your sequence length. This is not recommended, but I will continue anyway."); mothurOutEndLine();
348 }else if (windowSizes < (length * 0.05)) {
349 mothurOut("You have selected a window that is smaller than 5% of your sequence length. This is not recommended, but I will continue anyway."); mothurOutEndLine();
352 //save starting points of each window
353 for (int m = it->first; m < (it->second-windowSizes); m+=windowSizes) { win.push_back(m); }
356 if (win[win.size()-1] < (it->first+length)) {
357 win.push_back(win[win.size()-1]+windowSizes); // ex. string length is 115, window is 25, without this you would get 0, 25, 50, 75
358 } //with this you would get 1,25,50,75,100
362 catch(exception& e) {
363 errorOut(e, "Ccode", "findWindows");
367 //***************************************************************************************************************
368 int Ccode::getDiff(string seqA, string seqB) {
373 for (int i = 0; i < seqA.length(); i++) {
374 //if you are both not gaps
375 //if (isalpha(seqA[i]) && isalpha(seqA[i])) {
377 if (seqA[i] != seqB[i]) {
378 int ok; /* ok=1 means equivalent base. Checks for degenerate bases */
380 /* the char in base_a and base_b have been checked and they are different */
381 if ((seqA[i] == 'N') && (seqB[i] != '-')) ok = 1;
382 else if ((seqB[i] == 'N') && (seqA[i] != '-')) ok = 1;
383 else if ((seqA[i] == 'Y') && ((seqB[i] == 'C') || (seqB[i] == 'T'))) ok = 1;
384 else if ((seqB[i] == 'Y') && ((seqA[i] == 'C') || (seqA[i] == 'T'))) ok = 1;
385 else if ((seqA[i] == 'R') && ((seqB[i] == 'G') || (seqB[i] == 'A'))) ok = 1;
386 else if ((seqB[i] == 'R') && ((seqA[i] == 'G') || (seqA[i] == 'A'))) ok = 1;
387 else if ((seqA[i] == 'S') && ((seqB[i] == 'C') || (seqB[i] == 'G'))) ok = 1;
388 else if ((seqB[i] == 'S') && ((seqA[i] == 'C') || (seqA[i] == 'G'))) ok = 1;
389 else if ((seqA[i] == 'W') && ((seqB[i] == 'T') || (seqB[i] == 'A'))) ok = 1;
390 else if ((seqB[i] == 'W') && ((seqA[i] == 'T') || (seqA[i] == 'A'))) ok = 1;
391 else if ((seqA[i] == 'M') && ((seqB[i] == 'A') || (seqB[i] == 'C'))) ok = 1;
392 else if ((seqB[i] == 'M') && ((seqA[i] == 'A') || (seqA[i] == 'C'))) ok = 1;
393 else if ((seqA[i] == 'K') && ((seqB[i] == 'T') || (seqB[i] == 'G'))) ok = 1;
394 else if ((seqB[i] == 'K') && ((seqA[i] == 'T') || (seqA[i] == 'G'))) ok = 1;
395 else if ((seqA[i] == 'V') && ((seqB[i] == 'C') || (seqB[i] == 'A') || (seqB[i] == 'G'))) ok = 1;
396 else if ((seqB[i] == 'V') && ((seqA[i] == 'C') || (seqA[i] == 'A') || (seqA[i] == 'G'))) ok = 1;
397 else if ((seqA[i] == 'H') && ((seqB[i] == 'T') || (seqB[i] == 'A') || (seqB[i] == 'C'))) ok = 1;
398 else if ((seqB[i] == 'H') && ((seqA[i] == 'T') || (seqA[i] == 'A') || (seqA[i] == 'C'))) ok = 1;
399 else if ((seqA[i] == 'D') && ((seqB[i] == 'T') || (seqB[i] == 'A') || (seqB[i] == 'G'))) ok = 1;
400 else if ((seqB[i] == 'D') && ((seqA[i] == 'T') || (seqA[i] == 'A') || (seqA[i] == 'G'))) ok = 1;
401 else if ((seqA[i] == 'B') && ((seqB[i] == 'C') || (seqB[i] == 'T') || (seqB[i] == 'G'))) ok = 1;
402 else if ((seqB[i] == 'B') && ((seqA[i] == 'C') || (seqA[i] == 'T') || (seqA[i] == 'G'))) ok = 1;
403 else ok = 0; /* the bases are different and not equivalent */
405 //check if they are both blanks
406 if ((seqA[i] == '.') && (seqB[i] == '-')) ok = 1;
407 else if ((seqB[i] == '.') && (seqA[i] == '-')) ok = 1;
409 if (ok == 0) { numDiff++; }
417 catch(exception& e) {
418 errorOut(e, "Ccode", "getDiff");
422 //***************************************************************************************************************
423 //tried to make this look most like ccode original implementation
424 void Ccode::removeBadReferenceSeqs(vector<SeqDist>& seqs) {
427 vector< vector<int> > numDiffBases;
428 numDiffBases.resize(seqs.size());
430 for (int i = 0; i < numDiffBases.size(); i++) { numDiffBases[i].resize(seqs.size(),0); }
433 int length = it->second - it->first;
435 //calc differences from each sequence to everyother seq in the set
436 for (int i = 0; i < seqs.size(); i++) {
438 string seqA = seqs[i].seq->getAligned().substr(it->first, length);
440 //so you don't calc i to j and j to i since they are the same
441 for (int j = 0; j < i; j++) {
443 string seqB = seqs[j].seq->getAligned().substr(it->first, length);
446 int numDiff = getDiff(seqA, seqB);
448 numDiffBases[i][j] = numDiff;
449 numDiffBases[j][i] = numDiff;
453 //initailize remove to 0
454 vector<int> remove; remove.resize(seqs.size(), 0);
455 float top = ((20*length) / (float) 100);
456 float bottom = ((0.5*length) / (float) 100);
458 //check each numDiffBases and if any are higher than threshold set remove to 1 so you can remove those seqs from the closest set
459 for (int i = 0; i < numDiffBases.size(); i++) {
460 for (int j = 0; j < i; j++) {
461 //are you more than 20% different
462 if (numDiffBases[i][j] > top) { remove[j] = 1; }
463 //are you less than 0.5% different
464 if (numDiffBases[i][j] < bottom) { remove[j] = 1; }
470 //count seqs that are not going to be removed
471 for (int i = 0; i < remove.size(); i++) {
472 if (remove[i] == 0) { numSeqsLeft++; }
475 //if you have enough then remove bad ones
476 if (numSeqsLeft >= 3) {
477 vector<SeqDist> goodSeqs;
479 for (int i = 0; i < remove.size(); i++) {
480 if (remove[i] == 0) {
481 goodSeqs.push_back(seqs[i]);
487 }else { //warn, but dont remove any
488 mothurOut(querySeq->getName() + " does not have an adaquate number of reference sequences that are within 20% and 0.5% similarity. I will continue, but please check."); mothurOutEndLine();
492 catch(exception& e) {
493 errorOut(e, "Ccode", "removeBadReferenceSeqs");
497 //***************************************************************************************************************
498 //makes copy of templateseq for filter
499 vector<SeqDist> Ccode::findClosest(Sequence* q, int numWanted) {
502 vector<SeqDist> topMatches;
504 Sequence query = *(q);
506 //calc distance to each sequence in template seqs
507 for (int i = 0; i < templateSeqs.size(); i++) {
509 Sequence ref = *(templateSeqs[i]);
512 distCalc->calcDist(query, ref);
513 float dist = distCalc->getDist();
517 temp.seq = new Sequence(templateSeqs[i]->getName(), templateSeqs[i]->getAligned());
520 topMatches.push_back(temp);
523 sort(topMatches.begin(), topMatches.end(), compareSeqDist);
525 for (int i = numWanted; i < topMatches.size(); i++) { delete topMatches[i].seq; }
527 topMatches.resize(numWanted);
532 catch(exception& e) {
533 errorOut(e, "Ccode", "findClosestSides");
537 /**************************************************************************************************/
538 //find the distances from each reference sequence to every other reference sequence for each window for this query
539 void Ccode::getAverageRef(vector<SeqDist> ref) {
542 vector< vector< vector<int> > > diffs; //diffs[0][1][2] is the number of differences between ref seq 0 and ref seq 1 at window 2.
544 //initialize diffs vector
545 diffs.resize(ref.size());
546 for (int i = 0; i < diffs.size(); i++) {
547 diffs[i].resize(ref.size());
548 for (int j = 0; j < diffs[i].size(); j++) {
549 diffs[i][j].resize(windows.size(), 0);
555 //find the distances from each reference sequence to every other reference sequence for each window for this query
556 for (int i = 0; i < ref.size(); i++) {
558 string refI = ref[i].seq->getAligned();
560 //j<i, so you don't find distances from i to j and then j to i.
561 for (int j = 0; j < i; j++) {
563 string refJ = ref[j].seq->getAligned();
565 for (int k = 0; k < windows.size(); k++) {
567 string refIWindowk, refJWindowk;
569 if (k < windows.size()-1) {
571 refIWindowk = refI.substr(windows[k], windowSizes);
572 refJWindowk = refJ.substr(windows[k], windowSizes);
573 }else { //last window may be smaller than rest - see findwindows
575 refIWindowk = refI.substr(windows[k], (it->second-windows[k]));
576 refJWindowk = refJ.substr(windows[k], (it->second-windows[k]));
580 int diff = getDiff(refIWindowk, refJWindowk);
582 //save differences in [i][j][k] and [j][i][k] since they are the same
583 diffs[i][j][k] = diff;
584 diffs[j][i][k] = diff;
592 //initialize sumRef for this query
593 sumRef.resize(windows.size(), 0);
594 sumSquaredRef.resize(windows.size(), 0);
595 averageRef.resize(windows.size(), 0);
597 //find the sum of the differences for hte reference sequences
598 for (int i = 0; i < diffs.size(); i++) {
599 for (int j = 0; j < i; j++) {
601 //increment this querys reference sequences combos
604 for (int k = 0; k < diffs[i][j].size(); k++) {
605 sumRef[k] += diffs[i][j][k];
606 sumSquaredRef[k] += (diffs[i][j][k]*diffs[i][j][k]);
613 //find the average of the differences for the references for each window
614 for (int i = 0; i < windows.size(); i++) {
615 averageRef[i] = sumRef[i] / (float) refCombo;
619 catch(exception& e) {
620 errorOut(e, "Ccode", "getAverageRef");
624 /**************************************************************************************************/
625 void Ccode::getAverageQuery (vector<SeqDist> ref, Sequence* query) {
628 vector< vector<int> > diffs; //diffs[1][2] is the number of differences between querySeqs[query] and ref seq 1 at window 2.
630 //initialize diffs vector
631 diffs.resize(ref.size());
632 for (int j = 0; j < diffs.size(); j++) {
633 diffs[j].resize(windows.size(), 0);
638 string refQuery = query->getAligned();
640 //j<i, so you don't find distances from i to j and then j to i.
641 for (int j = 0; j < ref.size(); j++) {
643 string refJ = ref[j].seq->getAligned();
645 for (int k = 0; k < windows.size(); k++) {
647 string QueryWindowk, refJWindowk;
649 if (k < windows.size()-1) {
651 QueryWindowk = refQuery.substr(windows[k], windowSizes);
652 refJWindowk = refJ.substr(windows[k], windowSizes);
653 }else { //last window may be smaller than rest - see findwindows
655 QueryWindowk = refQuery.substr(windows[k], (it->second-windows[k]));
656 refJWindowk = refJ.substr(windows[k], (it->second-windows[k]));
660 int diff = getDiff(QueryWindowk, refJWindowk);
669 //initialize sumRef for this query
670 sumQuery.resize(windows.size(), 0);
671 sumSquaredQuery.resize(windows.size(), 0);
672 averageQuery.resize(windows.size(), 0);
674 //find the sum of the differences
675 for (int j = 0; j < diffs.size(); j++) {
676 for (int k = 0; k < diffs[j].size(); k++) {
677 sumQuery[k] += diffs[j][k];
678 sumSquaredQuery[k] += (diffs[j][k]*diffs[j][k]);
683 //find the average of the differences for the references for each window
684 for (int i = 0; i < windows.size(); i++) {
685 averageQuery[i] = sumQuery[i] / (float) ref.size();
688 catch(exception& e) {
689 errorOut(e, "Ccode", "getAverageQuery");
693 /**************************************************************************************************/
694 void Ccode::findVarianceRef() {
697 varRef.resize(windows.size(), 0);
698 sdRef.resize(windows.size(), 0);
701 for (int i = 0; i < windows.size(); i++) {
702 varRef[i] = (sumSquaredRef[i] - ((sumRef[i]*sumRef[i])/(float)refCombo)) / (float)(refCombo-1);
703 sdRef[i] = sqrt(varRef[i]);
705 //set minimum error rate to 0.001 - to avoid potential divide by zero - not sure if this is necessary but it follows ccode implementation
706 if (averageRef[i] < 0.001) { averageRef[i] = 0.001; }
707 if (sumRef[i] < 0.001) { sumRef[i] = 0.001; }
708 if (varRef[i] < 0.001) { varRef[i] = 0.001; }
709 if (sumSquaredRef[i] < 0.001) { sumSquaredRef[i] = 0.001; }
710 if (sdRef[i] < 0.001) { sdRef[i] = 0.001; }
714 catch(exception& e) {
715 errorOut(e, "Ccode", "findVarianceRef");
719 /**************************************************************************************************/
720 void Ccode::findVarianceQuery() {
722 varQuery.resize(windows.size(), 0);
723 sdQuery.resize(windows.size(), 0);
726 for (int i = 0; i < windows.size(); i++) {
727 varQuery[i] = (sumSquaredQuery[i] - ((sumQuery[i]*sumQuery[i])/(float) closest.size())) / (float) (closest.size()-1);
728 sdQuery[i] = sqrt(varQuery[i]);
730 //set minimum error rate to 0.001 - to avoid potential divide by zero - not sure if this is necessary but it follows ccode implementation
731 if (averageQuery[i] < 0.001) { averageQuery[i] = 0.001; }
732 if (sumQuery[i] < 0.001) { sumQuery[i] = 0.001; }
733 if (varQuery[i] < 0.001) { varQuery[i] = 0.001; }
734 if (sumSquaredQuery[i] < 0.001) { sumSquaredQuery[i] = 0.001; }
735 if (sdQuery[i] < 0.001) { sdQuery[i] = 0.001; }
739 catch(exception& e) {
740 errorOut(e, "Ccode", "findVarianceQuery");
744 /**************************************************************************************************/
745 void Ccode::determineChimeras() {
748 isChimericConfidence.resize(windows.size(), false);
749 isChimericTStudent.resize(windows.size(), false);
750 isChimericANOVA.resize(windows.size(), false);
751 anova.resize(windows.size());
755 for (int i = 0; i < windows.size(); i++) {
757 //get confidence limits
758 float t = getT(closest.size()-1); //how many seqs you are comparing to this querySeq
759 float dsUpper = (averageQuery[i] + (t * sdQuery[i])) / averageRef[i];
760 float dsLower = (averageQuery[i] - (t * sdQuery[i])) / averageRef[i];
762 if ((dsUpper > 1.0) && (dsLower > 1.0) && (averageQuery[i] > averageRef[i])) { /* range does not include 1 */
763 isChimericConfidence[i] = true; /* significantly higher at P<0.05 */
768 int degreeOfFreedom = refCombo + closest.size() - 2;
769 float denomForT = (((refCombo-1) * varQuery[i] + (closest.size() - 1) * varRef[i]) / (float) degreeOfFreedom) * ((refCombo + closest.size()) / (float) (refCombo * closest.size())); /* denominator, without sqrt(), for ts calculations */
771 float ts = fabs((averageQuery[i] - averageRef[i]) / (sqrt(denomForT))); /* value of ts for t-student test */
772 t = getT(degreeOfFreedom);
774 if ((ts >= t) && (averageQuery[i] > averageRef[i])) {
775 isChimericTStudent[i] = true; /* significantly higher at P<0.05 */
779 float value1 = sumQuery[i] + sumRef[i];
780 float value2 = sumSquaredQuery[i] + sumSquaredRef[i];
781 float value3 = ((sumQuery[i]*sumQuery[i]) / (float) (closest.size())) + ((sumRef[i] * sumRef[i]) / (float) refCombo);
782 float value4 = (value1 * value1) / ( (float) (closest.size() + refCombo) );
783 float value5 = value2 - value4;
784 float value6 = value3 - value4;
785 float value7 = value5 - value6;
786 float value8 = value7 / ((float) degreeOfFreedom);
787 float anovaValue = value6 / value8;
789 float f = getF(degreeOfFreedom);
791 if ((anovaValue >= f) && (averageQuery[i] > averageRef[i])) {
792 isChimericANOVA[i] = true; /* significant P<0.05 */
795 if (isnan(anovaValue) || isinf(anovaValue)) { anovaValue = 0.0; }
797 anova[i] = anovaValue;
801 catch(exception& e) {
802 errorOut(e, "Ccode", "determineChimeras");
806 /**************************************************************************************************/
807 float Ccode::getT(int numseq) {
812 /* t-student critical values for different degrees of freedom and alpha 0.1 in one-tail tests (equivalent to 0.05) */
813 if (numseq > 120) tvalue = 1.645;
814 else if (numseq > 60) tvalue = 1.658;
815 else if (numseq > 40) tvalue = 1.671;
816 else if (numseq > 30) tvalue = 1.684;
817 else if (numseq > 29) tvalue = 1.697;
818 else if (numseq > 28) tvalue = 1.699;
819 else if (numseq > 27) tvalue = 1.701;
820 else if (numseq > 26) tvalue = 1.703;
821 else if (numseq > 25) tvalue = 1.706;
822 else if (numseq > 24) tvalue = 1.708;
823 else if (numseq > 23) tvalue = 1.711;
824 else if (numseq > 22) tvalue = 1.714;
825 else if (numseq > 21) tvalue = 1.717;
826 else if (numseq > 20) tvalue = 1.721;
827 else if (numseq > 19) tvalue = 1.725;
828 else if (numseq > 18) tvalue = 1.729;
829 else if (numseq > 17) tvalue = 1.734;
830 else if (numseq > 16) tvalue = 1.740;
831 else if (numseq > 15) tvalue = 1.746;
832 else if (numseq > 14) tvalue = 1.753;
833 else if (numseq > 13) tvalue = 1.761;
834 else if (numseq > 12) tvalue = 1.771;
835 else if (numseq > 11) tvalue = 1.782;
836 else if (numseq > 10) tvalue = 1.796;
837 else if (numseq > 9) tvalue = 1.812;
838 else if (numseq > 8) tvalue = 1.833;
839 else if (numseq > 7) tvalue = 1.860;
840 else if (numseq > 6) tvalue = 1.895;
841 else if (numseq > 5) tvalue = 1.943;
842 else if (numseq > 4) tvalue = 2.015;
843 else if (numseq > 3) tvalue = 2.132;
844 else if (numseq > 2) tvalue = 2.353;
845 else if (numseq > 1) tvalue = 2.920;
846 else if (numseq <= 1) {
847 mothurOut("Two or more reference sequences are required, your data will be flawed.\n"); mothurOutEndLine();
852 catch(exception& e) {
853 errorOut(e, "Ccode", "getT");
857 /**************************************************************************************************/
858 float Ccode::getF(int numseq) {
863 /* F-Snedecor critical values for v1=1 and different degrees of freedom v2 and alpha 0.05 */
864 if (numseq > 120) fvalue = 3.84;
865 else if (numseq > 60) fvalue = 3.92;
866 else if (numseq > 40) fvalue = 4.00;
867 else if (numseq > 30) fvalue = 4.08;
868 else if (numseq > 29) fvalue = 4.17;
869 else if (numseq > 28) fvalue = 4.18;
870 else if (numseq > 27) fvalue = 4.20;
871 else if (numseq > 26) fvalue = 4.21;
872 else if (numseq > 25) fvalue = 4.23;
873 else if (numseq > 24) fvalue = 4.24;
874 else if (numseq > 23) fvalue = 4.26;
875 else if (numseq > 22) fvalue = 4.28;
876 else if (numseq > 21) fvalue = 4.30;
877 else if (numseq > 20) fvalue = 4.32;
878 else if (numseq > 19) fvalue = 4.35;
879 else if (numseq > 18) fvalue = 4.38;
880 else if (numseq > 17) fvalue = 4.41;
881 else if (numseq > 16) fvalue = 4.45;
882 else if (numseq > 15) fvalue = 4.49;
883 else if (numseq > 14) fvalue = 4.54;
884 else if (numseq > 13) fvalue = 4.60;
885 else if (numseq > 12) fvalue = 4.67;
886 else if (numseq > 11) fvalue = 4.75;
887 else if (numseq > 10) fvalue = 4.84;
888 else if (numseq > 9) fvalue = 4.96;
889 else if (numseq > 8) fvalue = 5.12;
890 else if (numseq > 7) fvalue = 5.32;
891 else if (numseq > 6) fvalue = 5.59;
892 else if (numseq > 5) fvalue = 5.99;
893 else if (numseq > 4) fvalue = 6.61;
894 else if (numseq > 3) fvalue = 7.71;
895 else if (numseq > 2) fvalue = 10.1;
896 else if (numseq > 1) fvalue = 18.5;
897 else if (numseq > 0) fvalue = 161;
898 else if (numseq <= 0) {
899 mothurOut("Two or more reference sequences are required, your data will be flawed.\n"); mothurOutEndLine();
904 catch(exception& e) {
905 errorOut(e, "Ccode", "getF");
909 //***************************************************************************************************************