segment.cpp

/*
  This file is part of Vidjil <http://www.vidjil.org>
  Copyright (C) 2011, 2012, 2013, 2014, 2015, 2016 by Bonsai bioinformatics
  at CRIStAL (UMR CNRS 9189, Université Lille) and Inria Lille
  Contributors: 
      Mathieu Giraud <mathieu.giraud@vidjil.org>
      Mikaël Salson <mikael.salson@vidjil.org>
      Marc Duez <marc.duez@vidjil.org>

  "Vidjil" is free software: you can redistribute it and/or modify
  it under the terms of the GNU General Public License as published by
  the Free Software Foundation, either version 3 of the License, or
  (at your option) any later version.

  "Vidjil" is distributed in the hope that it will be useful,
  but WITHOUT ANY WARRANTY; without even the implied warranty of
  MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE.  See the
  GNU General Public License for more details.

  You should have received a copy of the GNU General Public License
  along with "Vidjil". If not, see <http://www.gnu.org/licenses/>
*/
#include <algorithm>    // std::sort
#include <cassert>
#include "segment.h"
#include "tools.h"
#include "affectanalyser.h"
#include <sstream>
#include <cstring>
#include <string>

#define NO_FORBIDDEN_ID (-1)

AlignBox::AlignBox(string _key) {
  key = _key;

  del_left = 0 ;
  start = 0 ;
  end = 0 ;
  del_right = 0 ;

  ref_nb = 0 ;
  ref = "";
  ref_label = "";
}

string AlignBox::getSequence(string sequence) {
  return sequence.substr(start, end-start+1);
}

void AlignBox::addToJson(json &seg) {

  seg[key] = ref_label;

  if (key == "5")
    {
      seg[key+"end"] = end;
      seg[key+"del"] = del_right;
    }
  else if (key == "3")
    {
      seg[key+"del"] = del_left;
      seg[key+"start"] = start;
    }
  else
    {
      seg[key+"delLeft"] = del_left;
      seg[key+"start"] = start;
      seg[key+"end"] = end;
      seg[key+"delRight"] = del_right;
    }
}

ostream &operator<<(ostream &out, const AlignBox &box)
{
  out << "[/" << box.del_left << " " ;
  out << "@" << box.start << " " ;
  out << box.ref_label << "(" << box.ref_nb << ") " ;
  out << "@" << box.end << " " ;
  out << box.del_right << "/]" ;

  return out ;
}

string codeFromBoxes(vector <AlignBox*> boxes, string sequence)
{
  string code = "";

  int n = boxes.size();

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

    if (i>0) {
      code += " " + string_of_int(boxes[i-1]->del_right) + "/"
        // From box_left->end + 1 to box_right->start - 1, both positions included
        + sequence.substr(boxes[i-1]->end + 1, boxes[i]->start - boxes[i-1]->end - 1)
        + "/" + string_of_int(boxes[i]->del_left) + " " ;
    }

    code += boxes[i]->ref_label ;
  }

  return code;
}


Segmenter::~Segmenter() {}

Sequence Segmenter::getSequence() const {
  Sequence s ;
  s.label_full = info ;
  s.label = label + " " + (reversed ? "-" : "+");
  s.sequence = revcomp(sequence, reversed);

  return s ;
}

string Segmenter::getJunction(int l) const {
  assert(isSegmented());

  int start = (getLeft() + getRight())/2 - l/2;
  
  if (start < 0 or start + l > (int)sequence.size())  // TODO: +l ou +l-1 ?
    return "" ;

  return getSequence().sequence.substr(start, l);
}

int Segmenter::getLeft() const {
  return box_V->end;
}
  
int Segmenter::getRight() const {
  return box_J->start;
}

int Segmenter::getLeftD() const {
  return box_D->start;
}
  
int Segmenter::getRightD() const {
  return box_D->end;
}

bool Segmenter::isReverse() const {
  return reversed;
}

bool Segmenter::isSegmented() const {
  return segmented;
}

bool Segmenter::isDSegmented() const {
  return dSegmented;
}


// E-values

void Segmenter::checkLeftRightEvaluesThreshold(double threshold, int strand)
{
  if (threshold == NO_LIMIT_VALUE)
    return ;

  if (evalue_left >= threshold && evalue_right >= threshold)
    because = UNSEG_TOO_FEW_ZERO ;
  else if ((strand == 1 ? evalue_left : evalue_right) >= threshold)
    because = UNSEG_TOO_FEW_V ;
  else if ((strand == 1 ? evalue_right : evalue_left) >= threshold)
    because = UNSEG_TOO_FEW_J ;
  else if (evalue >= threshold) // left and right are <= threshold, but their sum is > threshold
    because = UNSEG_TOO_FEW_ZERO ;
}


// Chevauchement

string Segmenter::removeChevauchement()
{
  assert(isSegmented());
  
  string chevauchement = "" ;

  if (box_V->end >= box_J->start)
    {
      int middle = (box_V->end + box_J->start) / 2 ;
      chevauchement = " !ov " + string_of_int (box_V->end - box_J->start + 1);
      box_V->end = middle ;
      box_J->start = middle+1 ;
    }

  return chevauchement ;
}

// Prettyprint


bool Segmenter::finishSegmentation() 
{
  assert(isSegmented());
  
  string seq = getSequence().sequence;
    
  seg_V = seq.substr(0, box_V->end+1) ;
  seg_N = seq.substr(box_V->end+1, box_J->start-box_V->end-1) ;  // Twice computed for FineSegmenter, but only once in KmerSegmenter !
  seg_J = seq.substr(box_J->start) ;
  box_D->start=0;
  box_D->end=0;

  info = "VJ \t" + string_of_int(FIRST_POS) + " " + info + " " + string_of_int(seq.size() - 1 + FIRST_POS) ;
  info += "\t" + code ;

  info = (reversed ? "- " : "+ ") + info ;

  return true ;
}

bool Segmenter::finishSegmentationD() 
{
  string seq = getSequence().sequence;

  seg_V = seq.substr(0, box_V->end+1) ; // From pos. 0 to box_V->end
  seg_J = seq.substr(box_J->start) ;
  seg_N = seq.substr(box_V->end+1, box_J->start-box_V->end-1) ;  // Twice computed for FineSegmenter, but only once in KmerSegmenter !
  seg_D  = seq.substr(box_D->start, box_D->end-box_D->start+1) ; // From Dstart to Dend
  
  info = "VDJ \t0 " + string_of_int(box_V->end) +
                " " + string_of_int(box_D->start) +
		" " + string_of_int(box_D->end) +
		" " + string_of_int(box_J->start) +
		" " + string_of_int(seq.size()-1+FIRST_POS) ;
		
  info += "\t" + code ;
  
  info = (reversed ? "- " : "+ ") + info ;

  return true ;
}

string Segmenter::getInfoLine() const
{
  string s = "" ;

  s += (segmented ? "" : "! ") + info ;
  s += " " + info_extra ;
  s += " " + segmented_germline->code ;
  s += " " + string(segmented_mesg[because]) ;

  if (evalue > NO_LIMIT_VALUE)
    s += " " + scientific_string_of_double(evalue);

  if (evalue_left > NO_LIMIT_VALUE)
    s += " " + scientific_string_of_double(evalue_left);
  if (evalue_right > NO_LIMIT_VALUE)
    s += "/" + scientific_string_of_double(evalue_right);

  if (CDR3start > 0)
    s += " {CDR3: " + string_of_int(CDR3start) + "(" + string_of_int(CDR3end-CDR3start+1) + ")" + string_of_int(CDR3end) + " "
      + "up"[CDR3productive] + " " + JUNCTIONaa + " " + CDR3nuc + "}";

  return s ;
}

string KmerSegmenter::getInfoLineWithAffects() const
{
   stringstream ss;

   ss << "# "
      << right << setw(3) << score << " "
      << left << setw(30)
      << getInfoLine() ;

   if (getSegmentationStatus() != UNSEG_TOO_SHORT)
     ss << getKmerAffectAnalyser()->toString();

   return ss.str();
}


ostream &operator<<(ostream &out, const Segmenter &s)
{
  out << ">" << s.label << " " ;
  out << s.getInfoLine() << endl;

  if (s.segmented)
    {
      out << s.seg_V << endl ;
      out << s.seg_N << endl ;
      out << s.seg_J << endl ;
    }
  else
    {
      out << s.getSequence().sequence << endl ;
    }

  return out ;
}


// KmerSegmenter (Cheap)

KmerSegmenter::KmerSegmenter() { kaa = 0 ; }

KmerSegmenter::KmerSegmenter(Sequence seq, Germline *germline, double threshold, int multiplier)
{
  box_V = new AlignBox();
  box_D = new AlignBox();
  box_J = new AlignBox();

  CDR3start = -1;
  CDR3end = -1;

  label = seq.label ;
  sequence = seq.sequence ;
  info = "" ;
  info_extra = "seed";
  segmented = false;
  segmented_germline = germline ;
  system = germline->code; // useful ?
  reversed = false;
  because = NOT_PROCESSED ; // Cause of unsegmentation
  score = 0 ;
  evalue = NO_LIMIT_VALUE;
  evalue_left = NO_LIMIT_VALUE;
  evalue_right = NO_LIMIT_VALUE;

  int s = (size_t)germline->index->getS() ;
  int length = sequence.length() ;

  if (length < s) 
    {
      because = UNSEG_TOO_SHORT;
      kaa = NULL;
      return ;
    }
 
  kaa = new KmerAffectAnalyser(*(germline->index), sequence);
  
  // Check strand consistency among the affectations.
  int strand;
  int nb_strand[2] = {0,0};     // In cell 0 we'll put the number of negative
                                // strand, while in cell 1 we'll put the
                                // positives
  for (int i = 0; i < kaa->count(); i++) { 
    KmerAffect it = kaa->getAffectation(i);
    if (! it.isAmbiguous() && ! it.isUnknown()) {
      strand = affect_strand(it.affect);
      nb_strand[(strand + 1) / 2] ++; // (strand+1) / 2 → 0 if strand == -1; 1 if strand == 1
    }
  }

  score = nb_strand[0] + nb_strand[1] ; // Used only for non-segmented germlines

  reversed = (nb_strand[0] > nb_strand[1]) ;

  if ((germline->seg_method == SEG_METHOD_MAX12)
      || (germline->seg_method == SEG_METHOD_MAX1U))
    { // Pseudo-germline, MAX12 and MAX1U
      pair <KmerAffect, KmerAffect> max12 ;
      CountKmerAffectAnalyser ckaa(*(germline->index), sequence);


      set<KmerAffect> forbidden;
      forbidden.insert(KmerAffect::getAmbiguous());
      forbidden.insert(KmerAffect::getUnknown());

      if (germline->seg_method == SEG_METHOD_MAX12)
        // MAX12: two maximum k-mers (no unknown)
        {
          max12 = ckaa.max12(forbidden);

          if (max12.first.isUnknown() || max12.second.isUnknown())
            {
              because = UNSEG_TOO_FEW_ZERO ;
              return ;
            }
        }

      else
        // MAX1U: the maximum k-mers (no unknown) + unknown
        {
          CountKmerAffectAnalyser ckaa(*(germline->index), sequence);
          KmerAffect max = ckaa.max(forbidden);

          if (max.isUnknown())
            {
              because = UNSEG_TOO_FEW_ZERO ;
              return ;
            }
          max12 = make_pair(max, KmerAffect::getUnknown());
        }

      pair <KmerAffect, KmerAffect> before_after =  ckaa.sortLeftRight(max12);

      before = before_after.first ;
      after = before_after.second ;

      // This strand computation is only a heuristic, especially for chimera +/- reads
      // Anyway, it allows to gather such reads and their reverse complement into a unique window...
      // ... except when the read is quite different outside the window
      strand = reversed ? -1 : 1 ;
    }

  else
    { // Regular germline

  // Test on which strand we are, select the before and after KmerAffects
  if (nb_strand[0] == 0 && nb_strand[1] == 0) {
    because = UNSEG_TOO_FEW_ZERO ;
    return ;
  } else if (nb_strand[0] > RATIO_STRAND * nb_strand[1]) {
    strand = -1;
    before = KmerAffect(germline->affect_3, -1); 
    after = KmerAffect(germline->affect_5, -1);
  } else if (nb_strand[1] > RATIO_STRAND * nb_strand[0]) {
    strand = 1;
    before = KmerAffect(germline->affect_5, 1); 
    after = KmerAffect(germline->affect_3, 1);    
  } else {
    // Ambiguous information: we have positive and negative strands
    // and there is not enough difference to put them apart.
    if (nb_strand[0] + nb_strand[1] >= DETECT_THRESHOLD_STRAND)
      because = UNSEG_STRAND_NOT_CONSISTENT ;
    else
      because = UNSEG_TOO_FEW_ZERO ;
    return ;
  }

    } // endif Pseudo-germline
 
  computeSegmentation(strand, before, after, threshold, multiplier);
}

KmerSegmenter::~KmerSegmenter() {
  if (kaa)
    delete kaa;

  delete box_V;
  delete box_D;
  delete box_J;
}

KmerMultiSegmenter::KmerMultiSegmenter(Sequence seq, MultiGermline *multigermline, ostream *out_unsegmented,
                                       double threshold, int nb_reads_for_evalue)
{
  bool found_seg = false ; // Found a segmentation
  double best_evalue_seg = NO_LIMIT_VALUE ; // Best evalue, segmented sequences
  int best_score_unseg = 0 ; // Best score, unsegmented sequences
  the_kseg = NULL;
  multi_germline = multigermline;
  threshold_nb_expected = threshold;

  // E-value multiplier
  int multiplier = multi_germline->germlines.size() * nb_reads_for_evalue;
  
  // Iterate over the germlines
  for (list<Germline*>::const_iterator it = multigermline->germlines.begin(); it != multigermline->germlines.end(); ++it)
    {
      Germline *germline = *it ;

      KmerSegmenter *kseg = new KmerSegmenter(seq, germline, threshold, multiplier);
      bool keep_seg = false;

      if (out_unsegmented)
        {
          // Debug, display k-mer affectation and segmentation result for this germline
          *out_unsegmented << kseg->getInfoLineWithAffects() << endl ;
        }

      // Always remember the first kseg
      if (the_kseg == NULL)
        keep_seg = true;
      
      if (kseg->isSegmented())
        {
          // Yes, it is segmented
          // Should we keep the kseg ?
          if (!found_seg || (kseg->evalue < best_evalue_seg))
            {
              keep_seg = true;
              best_evalue_seg = kseg->evalue ;

              found_seg = true;
            }
        }
      else
        {
          // It is not segmented
          // Should we keep the kseg (with the unsegmentation cause) ?
            if (kseg->score > best_score_unseg)
            {              
              best_score_unseg = kseg->score ;
              if (!found_seg)
                keep_seg = true;
            }
        }
      
      if (keep_seg) {
        if (the_kseg)
          delete the_kseg;
        the_kseg = kseg;
      } else {
        delete kseg;
      }
    } // end for (Germlines)
}

KmerMultiSegmenter::~KmerMultiSegmenter() {
  if (the_kseg)
    delete the_kseg;
}

void KmerSegmenter::computeSegmentation(int strand, KmerAffect before, KmerAffect after,
                                        double threshold, int multiplier) {
  // Try to segment, computing 'box_V->end' and 'box_J->start'
  // If not segmented, put the cause of unsegmentation in 'because'

  affect_infos max;
  max = kaa->getMaximum(before, after);

  // We did not find a good segmentation point
  if (!max.max_found) {
    // We labeled it detected if there were both enough affect_5 and enough affect_3
    bool detected_before = (max.nb_before_left + max.nb_before_right >= DETECT_THRESHOLD);
    bool detected_after = (max.nb_after_left + max.nb_after_right >= DETECT_THRESHOLD);

    if (detected_before && detected_after)
      because = UNSEG_AMBIGUOUS ;
    else if ((strand == 1 && detected_before) || (strand == -1 && detected_after))
      because = UNSEG_TOO_FEW_J ;
    else if ((strand == 1 && detected_after) || (strand == -1 && detected_before))
      because = UNSEG_TOO_FEW_V ;
    else
      because = UNSEG_TOO_FEW_ZERO ;

    return ;
  }


  // E-values
  pair <double, double> pvalues = kaa->getLeftRightProbabilityAtLeastOrAbove();
  evalue_left = pvalues.first * multiplier ;
  evalue_right = pvalues.second * multiplier ;
  evalue = evalue_left + evalue_right ;

  checkLeftRightEvaluesThreshold(threshold, strand);

  if (because != NOT_PROCESSED)
    return ;

   // There was a good segmentation point

   box_V->end = max.first_pos_max;
   box_J->start = max.last_pos_max + 1;
   if (strand == -1) {
     int tmp = sequence.size() - box_V->end - 1;
     box_V->end = sequence.size() - box_J->start - 1;
     box_J->start = tmp;
   }

  // Yes, it is segmented
  segmented = true;
  because = reversed ? SEG_MINUS : SEG_PLUS ;

  info = string_of_int(box_V->end + FIRST_POS) + " " + string_of_int(box_J->start + FIRST_POS)  ;

  // removeChevauchement is called once info was already computed: it is only to output info_extra
  info_extra += removeChevauchement();
  finishSegmentation();

  return ;
}

KmerAffectAnalyser *KmerSegmenter::getKmerAffectAnalyser() const {
  return kaa;
}

int Segmenter::getSegmentationStatus() const {
  return because;
}

void Segmenter::setSegmentationStatus(int status) {
  because = status;
  segmented = (status == SEG_PLUS || status == SEG_MINUS);
}

// FineSegmenter


string check_and_resolve_overlap(string seq, int seq_begin, int seq_end,
                                 AlignBox *box_left, AlignBox *box_right,
                                 Cost segment_cost)
{
  // Overlap size
  int overlap = box_left->end - box_right->start + 1;

  if (overlap > 0)
    {
      string seq_left = seq.substr(seq_begin, box_left->end - seq_begin + 1);
      string seq_right = seq.substr(box_right->start, seq_end - box_right->start + 1);

      int score_r[overlap+1];
      int score_l[overlap+1];
      
      //LEFT
      DynProg dp_l = DynProg(seq_left, box_left->ref,
			   DynProg::Local, segment_cost);
      score_l[0] = dp_l.compute();

      
      //RIGHT
      // reverse right sequence
      string ref_right=string(box_right->ref.rbegin(), box_right->ref.rend());
      seq_right=string(seq_right.rbegin(), seq_right.rend());


      DynProg dp_r = DynProg(seq_right, ref_right,
			   DynProg::Local, segment_cost);
      score_r[0] = dp_r.compute();



      int trim_l[overlap+1];
      int trim_r[overlap+1];

      for(int i=0; i<=overlap; i++) {
        score_l[i] = i < seq_left.size()  ? dp_l.best_score_on_i(seq_left.size()  - i, trim_l + i) : MINUS_INF ;
        score_r[i] = i < seq_right.size() ? dp_r.best_score_on_i(seq_right.size() - i, trim_r + i) : MINUS_INF ;
      }


// #define DEBUG_OVERLAP
#ifdef DEBUG_OVERLAP
      cout << dp_l ;
      cout << dp_r ;

      cout << "seq:" << seq_left << "\t\t" << seq_right << endl;
      cout << "ref:" << ref_left << "\t\t" << ref_right << endl;
      for(int i=0; i<=overlap; i++)
        cout << i << "  left: " << score_l[i] << "/" << trim_l[i] << "     right: " << score_r[i] << "/" << trim_r[i] << endl;
#endif

      int score = MINUS_INF;
      int best_i = 0 ;
      int best_j = 0 ;


      // Find (i, j), with i+j >= overlap,
      // maximizing score_l[j] + score_r[i]
      for (int i=0; i<=overlap; i++){
	for (int j=overlap-i; j<=overlap; j++){
          int score_ij = score_l[i] + score_r[j];

	  if (score_ij > score) {
            best_i = i ;
            best_j = j ;
            box_left->del_right = box_left->ref.size() - trim_l[i];
	    box_right->del_left = box_right->ref.size() - trim_r[j];
	    score = score_ij;
	  }
	}
      }

      box_left->end -= best_i ;
      box_right->start += best_j ;

#ifdef DEBUG_OVERLAP
      cout << "overlap: " << overlap << ", " << "best_overlap_split: " << score
           << "    left: " << best_i << "-" << box_left->del_right << " @" << box_left->end
           << "    right:" << best_j << "-" << box_right->del_left << " @" << box_right->start
           << endl;
#endif
    } // end if (overlap > 0)

  // From box_left->end + 1 to box_right->start - 1
  return seq.substr(box_left->end + 1, box_right->start - box_left->end - 1);
}

bool comp_pair (pair<int,int> i,pair<int,int> j)
{
  return ( i.first > j.first);
}


/**
 * Align a read against a collection of sequences, maximizing the alignment 'score'
 * @param read:         the read
 * @param rep:          a collection of reference sequences
 * @param reverse_ref:  if true, reverse the reference sequences (VkVk)
 * @param reverse_both: if true, reverse both the read and the reference sequences (J segment)
 * @param local:        if true, Local alignment (D segment), otherwise LocalEndWithSomeDeletions and onlyBottomTriangle (V and J segments)
 * @param box:          the AligBox to fill
 * @param segment_cost: the cost used by the dynamic programing
 * @post  box is filled
 */

void align_against_collection(string &read, Fasta &rep, int forbidden_rep_id,
                              bool reverse_ref, bool reverse_both, bool local,
                             AlignBox *box, Cost segment_cost)
{
  
  int best_score = MINUS_INF ;
  box->ref_nb = MINUS_INF ;
  int best_best_i = (int) string::npos ;
  int best_best_j = (int) string::npos ;
  int best_first_i = (int) string::npos ;
  int best_first_j = (int) string::npos ;

  vector<pair<int, int> > score_r;

  DynProg::DynProgMode dpMode = DynProg::LocalEndWithSomeDeletions;
  if (local==true) dpMode = DynProg::Local;

  // With reverse_ref, the read is reversed to prevent calling revcomp on each reference sequence
  string sequence_or_rc = revcomp(read, reverse_ref);
  
  for (int r = 0 ; r < rep.size() ; r++)
    {
      if (r == forbidden_rep_id)
        continue;

      DynProg dp = DynProg(sequence_or_rc, rep.sequence(r),
			   dpMode, // DynProg::SemiGlobalTrans, 
			   segment_cost, // DNA
			   reverse_both, reverse_both,
                          rep.read(r).marked_pos);

      bool onlyBottomTriangle = !local ;
      int score = dp.compute(onlyBottomTriangle, BOTTOM_TRIANGLE_SHIFT);
      
      if (local==true){ 
	dp.backtrack();
      }
      
      if (score > best_score)
	{
	  best_score = score ;
	  best_best_i = dp.best_i ;
	  best_best_j = dp.best_j ;
	  best_first_i = dp.first_i ;
	  best_first_j = dp.first_j ;
	  box->ref_nb = r ;
	  box->ref_label = rep.label(r) ;

          if (!local)
            dp.backtrack();
          box->marked_pos = dp.marked_pos_i ;
	}
	
	score_r.push_back(make_pair(score, r));

	// #define DEBUG_SEGMENT      

#ifdef DEBUG_SEGMENT	
	cout << rep.label(r) << " " << score << " " << dp.best_i << endl ;
#endif

    }
    sort(score_r.begin(),score_r.end(),comp_pair);

  box->ref = rep.sequence(box->ref_nb);
  box->del_right = reverse_both ? best_best_j : box->ref.size() - best_best_j - 1;
  box->del_left = best_first_j;
  box->start = best_first_i;
  
  box->score = score_r;

#ifdef DEBUG_SEGMENT	
  cout << "best: " << box->ref_label << " " << best_score ;
  cout << "del/del2/begin:" << (box->del_right) << "/" << (box->del_left) << "/" << (box->start) << endl;
  cout << endl;
#endif

  if (reverse_ref)
    // Why -1 here and +1 in dynprog.cpp /// best_i = m - best_i + 1 ;
    best_best_i = read.length() - best_best_i - 1 ;

  box->end = best_best_i ;
}

string format_del(int deletions)
{
  return deletions ? *"(" + string_of_int(deletions) + " del)" : "" ;
}

FineSegmenter::FineSegmenter(Sequence seq, Germline *germline, Cost segment_c,  double threshold, int multiplier)
{
  box_V = new AlignBox("5");
  box_D = new AlignBox("4");
  box_J = new AlignBox("3");

  segmented = false;
  dSegmented = false;
  because = NOT_PROCESSED ;
  segmented_germline = germline ;
  info_extra = "" ;
  label = seq.label ;
  sequence = seq.sequence ;
  segment_cost=segment_c;
  evalue = NO_LIMIT_VALUE;
  evalue_left = NO_LIMIT_VALUE;
  evalue_right = NO_LIMIT_VALUE;

  CDR3start = -1;
  CDR3end = -1;

  bool reverse_V = false ;
  bool reverse_J = false ;

  if ((germline->seg_method == SEG_METHOD_MAX12) || (germline->seg_method == SEG_METHOD_MAX1U))
    {
      // We check whether this sequence is segmented with MAX12 or MAX1U (with default e-value parameters)
      KmerSegmenter *kseg = new KmerSegmenter(seq, germline, THRESHOLD_NB_EXPECTED, 1);
      if (kseg->isSegmented())
        {
          reversed = kseg->isReverse();

          KmerAffect left = reversed ? KmerAffect(kseg->after, true) : kseg->before ;
          KmerAffect right = reversed ? KmerAffect(kseg->before, true) : kseg->after ;

          delete kseg ;

          reverse_V = (left.getStrand() == -1);
          reverse_J = (right.getStrand() == -1);

          code = "Unexpected ";

          code += left.toStringSigns() + germline->index->getLabel(left).basename;
          code += "/";
          code += right.toStringSigns() + germline->index->getLabel(right).basename;
          info_extra += " " + left.toString() + "/" + right.toString() + " (" + code + ")";

          if (germline->seg_method == SEG_METHOD_MAX1U)
            return ;

          germline->override_rep5_rep3_from_labels(left, right);
        }
      else
        {
          delete kseg ;
          return ;
        }
    }

  // Strand determination, with KmerSegmenter (with default e-value parameters)
  // Note that we use only the 'strand' component
  // When the KmerSegmenter fails, continue with positive strand
  // TODO: flag to force a strand / to test both strands ?

  KmerSegmenter *kseg = new KmerSegmenter(seq, germline, THRESHOLD_NB_EXPECTED, 1);
  reversed = kseg->isReverse();
  delete kseg ;
  
  sequence_or_rc = revcomp(sequence, reversed); // sequence, possibly reversed


  /* Segmentation */
  align_against_collection(sequence_or_rc, germline->rep_5, NO_FORBIDDEN_ID, reverse_V, reverse_V, false,
                                        box_V, segment_cost);

  align_against_collection(sequence_or_rc, germline->rep_3, NO_FORBIDDEN_ID, reverse_J, !reverse_J, false,
                                          box_J, segment_cost);

  // J was run with '!reverseJ', we copy the box informations from right to left
  // Should this directly be handled in align_against_collection() ?
  box_J->start = box_J->end ;
  box_J->del_left = box_J->del_right;

  /* E-values */
  evalue_left  = multiplier * sequence.size() * germline->rep_5.totalSize() * segment_cost.toPValue(box_V->score[0].first);
  evalue_right = multiplier * sequence.size() * germline->rep_3.totalSize() * segment_cost.toPValue(box_J->score[0].first);
  evalue = evalue_left + evalue_right ;

  /* Unsegmentation causes */
  if (box_V->end == (int) string::npos)
    {
      evalue_left = BAD_EVALUE ;
    }
      
  if (box_J->start == (int) string::npos)
    {
      evalue_right = BAD_EVALUE ;
    }

  checkLeftRightEvaluesThreshold(threshold, reversed ? -1 : 1);

  if (because != NOT_PROCESSED)
    {
      segmented = false;
      info = " @" + string_of_int (box_V->end + FIRST_POS) + "  @" + string_of_int(box_J->start + FIRST_POS) ;
      return ;
    }

  /* The sequence is segmented */
  segmented = true ;
  because = reversed ? SEG_MINUS : SEG_PLUS ;

    //overlap VJ
  seg_N = check_and_resolve_overlap(sequence_or_rc, 0, sequence_or_rc.length(),
                                    box_V, box_J, segment_cost);

  // Why could this happen ?
      if (box_J->start>=(int) sequence.length())
	  box_J->start=sequence.length()-1;

  // seg_N will be recomputed in finishSegmentation()

  boxes.clear();
  boxes.push_back(box_V);
  boxes.push_back(box_J);
  code = codeFromBoxes(boxes, sequence_or_rc);

  info = string_of_int(box_V->end + FIRST_POS) + " " + string_of_int(box_J->start + FIRST_POS) ;
  finishSegmentation();
}

bool FineSegmenter::FineSegmentD(Germline *germline,
                                 AlignBox *box_Y, AlignBox *box_DD, AlignBox *box_Z,
                                 int forbidden_id,
                                 int extend_DD_on_Y, int extend_DD_on_Z,
                                 double evalue_threshold, int multiplier){

    // Create a zone where to look for D, adding some nucleotides on both sides
    int l = box_Y->end - extend_DD_on_Y;
    if (l<0) 
      l=0 ;

    int r = box_Z->start + extend_DD_on_Z;

    string seq = getSequence().sequence; // segmented sequence, possibly rev-comped

    if (r > (int) seq.length())
      r = seq.length();
      
    string str = seq.substr(l, r-l);

    // Align
    align_against_collection(str, germline->rep_4, forbidden_id, false, false, true,
                                           box_DD, segment_cost);

    box_DD->start += l ;
    box_DD->end += l ;

    float evalue_D = multiplier * (r-l) * germline->rep_4.totalSize() * segment_cost.toPValue(box_DD->score[0].first);

    if (evalue_D > evalue_threshold)
      return false;

    int save_box_Y_end = box_Y->end ;
    int save_box_Y_del_right = box_Y->del_right ;
    int save_box_Z_del_left = box_Z->del_left;
    int save_box_Z_start = box_Z->start ;
    
    //overlap VD
    seg_N1 = check_and_resolve_overlap(seq, 0, box_DD->end,
                                       box_Y, box_DD, segment_cost);
    
    //overlap DJ
    seg_N2 = check_and_resolve_overlap(seq, box_DD->start, seq.length(),
                                       box_DD, box_Z, segment_cost);

    // Realign D to see whether the score is enough
    DynProg dp = DynProg(box_DD->getSequence(seq), box_DD->ref,
                         DynProg::SemiGlobal, segment_cost, false, false);
    int score_new = dp.compute();

    float evalue_DD_new = multiplier * (box_DD->end - box_DD->start + 1) * box_DD->ref.size() * segment_cost.toPValue(score_new);

    if (evalue_DD_new > evalue_threshold)
      {
        // Restore box_Y and box_Z
        box_Y->end =  save_box_Y_end;
        box_Y->del_right = save_box_Y_del_right;
        box_Z->del_left = save_box_Z_del_left;
        box_Z->start = save_box_Z_start;

        return false ;
      }

    return true;
}

void FineSegmenter::FineSegmentD(Germline *germline, bool several_D,
                                 double evalue_threshold, int multiplier){

  if (segmented){

    dSegmented = FineSegmentD(germline,
                              box_V, box_D, box_J,
                              NO_FORBIDDEN_ID,
                              EXTEND_D_ZONE, EXTEND_D_ZONE,
                              evalue_threshold, multiplier);

    if (!dSegmented)
      return ;

#define DD_MIN_SEARCH 5

    boxes.clear();
    boxes.push_back(box_V);

    if (several_D && (box_D->start - box_V->end >= DD_MIN_SEARCH))
      {
        AlignBox *box_D1 = new AlignBox("4a");

        bool d1 = FineSegmentD(germline,
                               box_V, box_D1, box_D,
                               box_D->ref_nb,
                               EXTEND_D_ZONE, 0,
                               evalue_threshold, multiplier);

        if (d1)
          boxes.push_back(box_D1);
        else
          delete box_D1;
      }

    boxes.push_back(box_D);

    if (several_D && (box_J->start - box_D->end >= DD_MIN_SEARCH))
      {