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bam2bcf_indel.c

#include <assert.h>
#include <ctype.h>
#include <string.h>
#include "bam.h"
#include "bam2bcf.h"
#include "ksort.h"
#include "kaln.h"
#include "kprobaln.h"
#include "khash.h"
KHASH_SET_INIT_STR(rg)

#define MINUS_CONST 0x10000000
#define INDEL_WINDOW_SIZE 50
#define MIN_SUPPORT_COEF 500

void *bcf_call_add_rg(void *_hash, const char *hdtext, const char *list)
{
      const char *s, *p, *q, *r, *t;
      khash_t(rg) *hash;
      if (list == 0 || hdtext == 0) return _hash;
      if (_hash == 0) _hash = kh_init(rg);
      hash = (khash_t(rg)*)_hash;
      if ((s = strstr(hdtext, "@RG\t")) == 0) return hash;
      do {
            t = strstr(s + 4, "@RG\t"); // the next @RG
            if ((p = strstr(s, "\tID:")) != 0) p += 4;
            if ((q = strstr(s, "\tPL:")) != 0) q += 4;
            if (p && q && (t == 0 || (p < t && q < t))) { // ID and PL are both present
                  int lp, lq;
                  char *x;
                  for (r = p; *r && *r != '\t' && *r != '\n'; ++r); lp = r - p;
                  for (r = q; *r && *r != '\t' && *r != '\n'; ++r); lq = r - q;
                  x = calloc((lp > lq? lp : lq) + 1, 1);
                  for (r = q; *r && *r != '\t' && *r != '\n'; ++r) x[r-q] = *r;
                  if (strstr(list, x)) { // insert ID to the hash table
                        khint_t k;
                        int ret;
                        for (r = p; *r && *r != '\t' && *r != '\n'; ++r) x[r-p] = *r;
                        x[r-p] = 0;
                        k = kh_get(rg, hash, x);
                        if (k == kh_end(hash)) k = kh_put(rg, hash, x, &ret);
                        else free(x);
                  } else free(x);
            }
            s = t;
      } while (s);
      return hash;
}

void bcf_call_del_rghash(void *_hash)
{
      khint_t k;
      khash_t(rg) *hash = (khash_t(rg)*)_hash;
      if (hash == 0) return;
      for (k = kh_begin(hash); k < kh_end(hash); ++k)
            if (kh_exist(hash, k))
                  free((char*)kh_key(hash, k));
      kh_destroy(rg, hash);
}

static int tpos2qpos(const bam1_core_t *c, const uint32_t *cigar, int32_t tpos, int is_left, int32_t *_tpos)
{
      int k, x = c->pos, y = 0, last_y = 0;
      *_tpos = c->pos;
      for (k = 0; k < c->n_cigar; ++k) {
            int op = cigar[k] & BAM_CIGAR_MASK;
            int l = cigar[k] >> BAM_CIGAR_SHIFT;
            if (op == BAM_CMATCH) {
                  if (c->pos > tpos) return y;
                  if (x + l > tpos) {
                        *_tpos = tpos;
                        return y + (tpos - x);
                  }
                  x += l; y += l;
                  last_y = y;
            } else if (op == BAM_CINS || op == BAM_CSOFT_CLIP) y += l;
            else if (op == BAM_CDEL || op == BAM_CREF_SKIP) {
                  if (x + l > tpos) {
                        *_tpos = is_left? x : x + l;
                        return y;
                  }
                  x += l;
            }
      }
      *_tpos = x;
      return last_y;
}
// FIXME: check if the inserted sequence is consistent with the homopolymer run
// l is the relative gap length and l_run is the length of the homopolymer on the reference
static inline int est_seqQ(const bcf_callaux_t *bca, int l, int l_run)
{
      int q, qh;
      q = bca->openQ + bca->extQ * (abs(l) - 1);
      qh = l_run >= 3? (int)(bca->tandemQ * (double)abs(l) / l_run + .499) : 1000;
      return q < qh? q : qh;
}

static inline int est_indelreg(int pos, const char *ref, int l, char *ins4)
{
      int i, j, max = 0, max_i = pos, score = 0;
      l = abs(l);
      for (i = pos + 1, j = 0; ref[i]; ++i, ++j) {
            if (ins4) score += (toupper(ref[i]) != "ACGTN"[(int)ins4[j%l]])? -10 : 1;
            else score += (toupper(ref[i]) != toupper(ref[pos+1+j%l]))? -10 : 1;
            if (score < 0) break;
            if (max < score) max = score, max_i = i;
      }
      return max_i - pos;
}

int bcf_call_gap_prep(int n, int *n_plp, bam_pileup1_t **plp, int pos, bcf_callaux_t *bca, const char *ref,
                                const void *rghash)
{
      extern void ks_introsort_uint32_t(int, uint32_t*);
      int i, s, j, k, t, n_types, *types, max_rd_len, left, right, max_ins, *score1, *score2;
      int N, K, l_run, ref_type, n_alt;
      char *inscns = 0, *ref2, *query;
      khash_t(rg) *hash = (khash_t(rg)*)rghash;
      if (ref == 0 || bca == 0) return -1;
      // mark filtered reads
      if (rghash) {
            N = 0;
            for (s = N = 0; s < n; ++s) {
                  for (i = 0; i < n_plp[s]; ++i) {
                        bam_pileup1_t *p = plp[s] + i;
                        const uint8_t *rg = bam_aux_get(p->b, "RG");
                        p->aux = 1; // filtered by default
                        if (rg) {
                              khint_t k = kh_get(rg, hash, (const char*)(rg + 1));
                              if (k != kh_end(hash)) p->aux = 0, ++N; // not filtered
                        }
                  }
            }
            if (N == 0) return -1; // no reads left
      }
      // determine if there is a gap
      for (s = N = 0; s < n; ++s) {
            for (i = 0; i < n_plp[s]; ++i)
                  if (plp[s][i].indel != 0) break;
            if (i < n_plp[s]) break;
      }
      if (s == n) return -1; // there is no indel at this position.
      for (s = N = 0; s < n; ++s) N += n_plp[s]; // N is the total number of reads
      { // find out how many types of indels are present
            int m, n_alt = 0, n_tot = 0;
            uint32_t *aux;
            aux = calloc(N + 1, 4);
            m = max_rd_len = 0;
            aux[m++] = MINUS_CONST; // zero indel is always a type
            for (s = 0; s < n; ++s) {
                  for (i = 0; i < n_plp[s]; ++i) {
                        const bam_pileup1_t *p = plp[s] + i;
                        if (rghash == 0 || p->aux == 0) {
                              ++n_tot;
                              if (p->indel != 0) {
                                    ++n_alt;
                                    aux[m++] = MINUS_CONST + p->indel;
                              }
                        }
                        j = bam_cigar2qlen(&p->b->core, bam1_cigar(p->b));
                        if (j > max_rd_len) max_rd_len = j;
                  }
            }
            ks_introsort(uint32_t, m, aux);
            // squeeze out identical types
            for (i = 1, n_types = 1; i < m; ++i)
                  if (aux[i] != aux[i-1]) ++n_types;
            if (n_types == 1 || n_alt * MIN_SUPPORT_COEF < n_tot) { // no indels or too few supporting reads
                  free(aux); return -1;
            }
            types = (int*)calloc(n_types, sizeof(int));
            t = 0;
            types[t++] = aux[0] - MINUS_CONST; 
            for (i = 1; i < m; ++i)
                  if (aux[i] != aux[i-1])
                        types[t++] = aux[i] - MINUS_CONST;
            free(aux);
            for (t = 0; t < n_types; ++t)
                  if (types[t] == 0) break;
            ref_type = t; // the index of the reference type (0)
            assert(n_types < 64);
      }
      { // calculate left and right boundary
            left = pos > INDEL_WINDOW_SIZE? pos - INDEL_WINDOW_SIZE : 0;
            right = pos + INDEL_WINDOW_SIZE;
            if (types[0] < 0) right -= types[0];
            // in case the alignments stand out the reference
            for (i = pos; i < right; ++i)
                  if (ref[i] == 0) break;
            right = i;
      }
      { // the length of the homopolymer run around the current position
            int c = bam_nt16_table[(int)ref[pos + 1]];
            if (c == 15) l_run = 1;
            else {
                  for (i = pos + 2; ref[i]; ++i)
                        if (bam_nt16_table[(int)ref[i]] != c) break;
                  l_run = i;
                  for (i = pos; i >= 0; --i)
                        if (bam_nt16_table[(int)ref[i]] != c) break;
                  l_run -= i + 1;
            }
      }
      // construct the consensus sequence
      max_ins = types[n_types - 1]; // max_ins is at least 0
      if (max_ins > 0) {
            int *inscns_aux = calloc(4 * n_types * max_ins, sizeof(int));
            // count the number of occurrences of each base at each position for each type of insertion
            for (t = 0; t < n_types; ++t) {
                  if (types[t] > 0) {
                        for (s = 0; s < n; ++s) {
                              for (i = 0; i < n_plp[s]; ++i) {
                                    bam_pileup1_t *p = plp[s] + i;
                                    if (p->indel == types[t]) {
                                          uint8_t *seq = bam1_seq(p->b);
                                          for (k = 1; k <= p->indel; ++k) {
                                                int c = bam_nt16_nt4_table[bam1_seqi(seq, p->qpos + k)];
                                                if (c < 4) ++inscns_aux[(t*max_ins+(k-1))*4 + c];
                                          }
                                    }
                              }
                        }
                  }
            }
            // use the majority rule to construct the consensus
            inscns = calloc(n_types * max_ins, 1);
            for (t = 0; t < n_types; ++t) {
                  for (j = 0; j < types[t]; ++j) {
                        int max = 0, max_k = -1, *ia = &inscns_aux[(t*max_ins+j)*4];
                        for (k = 0; k < 4; ++k)
                              if (ia[k] > max)
                                    max = ia[k], max_k = k;
                        inscns[t*max_ins + j] = max? max_k : 4;
                  }
            }
            free(inscns_aux);
      }
      // compute the likelihood given each type of indel for each read
      ref2  = calloc(right - left + max_ins + 2, 1);
      query = calloc(right - left + max_rd_len + max_ins + 2, 1);
      score1 = calloc(N * n_types, sizeof(int));
      score2 = calloc(N * n_types, sizeof(int));
      bca->indelreg = 0;
      for (t = 0; t < n_types; ++t) {
            int l, ir;
            kpa_par_t apf1 = { 1e-4, 1e-2, 10 }, apf2 = { 1e-6, 1e-3, 10 };
            apf1.bw = apf2.bw = abs(types[t]) + 3;
            // compute indelreg
            if (types[t] == 0) ir = 0;
            else if (types[t] > 0) ir = est_indelreg(pos, ref, types[t], &inscns[t*max_ins]);
            else ir = est_indelreg(pos, ref, -types[t], 0);
            if (ir > bca->indelreg) bca->indelreg = ir;
//          fprintf(stderr, "%d, %d, %d\n", pos, types[t], ir);
            // write ref2
            for (k = 0, j = left; j <= pos; ++j)
                  ref2[k++] = bam_nt16_nt4_table[bam_nt16_table[(int)ref[j]]];
            if (types[t] <= 0) j += -types[t];
            else for (l = 0; l < types[t]; ++l)
                         ref2[k++] = inscns[t*max_ins + l];
            if (types[0] < 0) { // mask deleted sequences to avoid a particular error in the model.
                  int jj, tmp = types[t] >= 0? -types[0] : -types[0] + types[t];
                  for (jj = 0; jj < tmp && j < right && ref[j]; ++jj, ++j)
                        ref2[k++] = 4;
            }
            for (; j < right && ref[j]; ++j)
                  ref2[k++] = bam_nt16_nt4_table[bam_nt16_table[(int)ref[j]]];
            if (j < right) right = j;
            // align each read to ref2
            for (s = K = 0; s < n; ++s) {
                  for (i = 0; i < n_plp[s]; ++i, ++K) {
                        bam_pileup1_t *p = plp[s] + i;
                        int qbeg, qend, tbeg, tend, sc;
                        uint8_t *seq = bam1_seq(p->b);
                        // FIXME: the following skips soft clips, but using them may be more sensitive.
                        // determine the start and end of sequences for alignment
                        qbeg = tpos2qpos(&p->b->core, bam1_cigar(p->b), left,  0, &tbeg);
                        qend = tpos2qpos(&p->b->core, bam1_cigar(p->b), right, 1, &tend);
                        if (types[t] < 0) {
                              int l = -types[t];
                              tbeg = tbeg - l > left?  tbeg - l : left;
                        }
                        // write the query sequence
                        for (l = qbeg; l < qend; ++l)
                              query[l - qbeg] = bam_nt16_nt4_table[bam1_seqi(seq, l)];
                        { // do realignment; this is the bottleneck
                              const uint8_t *qual = bam1_qual(p->b), *bq;
                              uint8_t *qq;
                              qq = calloc(qend - qbeg, 1);
                              bq = (uint8_t*)bam_aux_get(p->b, "ZQ");
                              if (bq) ++bq; // skip type
                              for (l = qbeg; l < qend; ++l) {
                                    qq[l - qbeg] = bq? qual[l] + (bq[l] - 64) : qual[l];
                                    if (qq[l - qbeg] > 30) qq[l - qbeg] = 30;
                                    if (qq[l - qbeg] < 7) qq[l - qbeg] = 7;
                              }
                              sc = kpa_glocal((uint8_t*)ref2 + tbeg - left, tend - tbeg + abs(types[t]),
                                                      (uint8_t*)query, qend - qbeg, qq, &apf1, 0, 0);
                              l = (int)(100. * sc / (qend - qbeg) + .499); // used for adjusting indelQ below
                              if (l > 255) l = 255;
                              score1[K*n_types + t] = score2[K*n_types + t] = sc<<8 | l;
                              if (sc > 5) {
                                    sc = kpa_glocal((uint8_t*)ref2 + tbeg - left, tend - tbeg + abs(types[t]),
                                                            (uint8_t*)query, qend - qbeg, qq, &apf2, 0, 0);
                                    l = (int)(100. * sc / (qend - qbeg) + .499);
                                    if (l > 255) l = 255;
                                    score2[K*n_types + t] = sc<<8 | l;
                              }
                              free(qq);
                        }
/*
                        for (l = 0; l < tend - tbeg + abs(types[t]); ++l)
                              fputc("ACGTN"[(int)ref2[tbeg-left+l]], stderr);
                        fputc('\n', stderr);
                        for (l = 0; l < qend - qbeg; ++l) fputc("ACGTN"[(int)query[l]], stderr);
                        fputc('\n', stderr);
                        fprintf(stderr, "pos=%d type=%d read=%d:%d name=%s qbeg=%d tbeg=%d score=%d\n", pos, types[t], s, i, bam1_qname(p->b), qbeg, tbeg, sc);
*/
                  }
            }
      }
      free(ref2); free(query);
      { // compute indelQ
            int *sc, tmp, *sumq;
            sc   = alloca(n_types * sizeof(int));
            sumq = alloca(n_types * sizeof(int));
            memset(sumq, 0, sizeof(int) * n_types);
            for (s = K = 0; s < n; ++s) {
                  for (i = 0; i < n_plp[s]; ++i, ++K) {
                        bam_pileup1_t *p = plp[s] + i;
                        int *sct = &score1[K*n_types], indelQ1, indelQ2, seqQ, indelQ;
                        for (t = 0; t < n_types; ++t) sc[t] = sct[t]<<6 | t;
                        for (t = 1; t < n_types; ++t) // insertion sort
                              for (j = t; j > 0 && sc[j] < sc[j-1]; --j)
                                    tmp = sc[j], sc[j] = sc[j-1], sc[j-1] = tmp;
                        /* errmod_cal() assumes that if the call is wrong, the
                         * likelihoods of other events are equal. This is about
                         * right for substitutions, but is not desired for
                         * indels. To reuse errmod_cal(), I have to make
                         * compromise for multi-allelic indels.
                         */
                        if ((sc[0]&0x3f) == ref_type) {
                              indelQ1 = (sc[1]>>14) - (sc[0]>>14);
                              seqQ = est_seqQ(bca, types[sc[1]&0x3f], l_run);
                        } else {
                              for (t = 0; t < n_types; ++t) // look for the reference type
                                    if ((sc[t]&0x3f) == ref_type) break;
                              indelQ1 = (sc[t]>>14) - (sc[0]>>14);
                              seqQ = est_seqQ(bca, types[sc[0]&0x3f], l_run);
                        }
                        tmp = sc[0]>>6 & 0xff;
                        indelQ1 = tmp > 111? 0 : (int)((1. - tmp/111.) * indelQ1 + .499); // reduce indelQ
                        sct = &score2[K*n_types];
                        for (t = 0; t < n_types; ++t) sc[t] = sct[t]<<6 | t;
                        for (t = 1; t < n_types; ++t) // insertion sort
                              for (j = t; j > 0 && sc[j] < sc[j-1]; --j)
                                    tmp = sc[j], sc[j] = sc[j-1], sc[j-1] = tmp;
                        if ((sc[0]&0x3f) == ref_type) {
                              indelQ2 = (sc[1]>>14) - (sc[0]>>14);
                        } else {
                              for (t = 0; t < n_types; ++t) // look for the reference type
                                    if ((sc[t]&0x3f) == ref_type) break;
                              indelQ2 = (sc[t]>>14) - (sc[0]>>14);
                        }
                        tmp = sc[0]>>6 & 0xff;
                        indelQ2 = tmp > 111? 0 : (int)((1. - tmp/111.) * indelQ2 + .499);
                        // pick the smaller between indelQ1 and indelQ2
                        indelQ = indelQ1 < indelQ2? indelQ1 : indelQ2;
                        p->aux = (sc[0]&0x3f)<<16 | seqQ<<8 | indelQ;
                        sumq[sc[0]&0x3f] += indelQ < seqQ? indelQ : seqQ;
//                      fprintf(stderr, "pos=%d read=%d:%d name=%s call=%d q=%d\n", pos, s, i, bam1_qname(p->b), types[sc[0]&0x3f], indelQ);
                  }
            }
            // determine bca->indel_types[] and bca->inscns
            bca->maxins = max_ins;
            bca->inscns = realloc(bca->inscns, bca->maxins * 4);
            for (t = 0; t < n_types; ++t)
                  sumq[t] = sumq[t]<<6 | t;
            for (t = 1; t < n_types; ++t) // insertion sort
                  for (j = t; j > 0 && sumq[j] > sumq[j-1]; --j)
                        tmp = sumq[j], sumq[j] = sumq[j-1], sumq[j-1] = tmp;
            for (t = 0; t < n_types; ++t) // look for the reference type
                  if ((sumq[t]&0x3f) == ref_type) break;
            if (t) { // then move the reference type to the first
                  tmp = sumq[t];
                  for (; t > 0; --t) sumq[t] = sumq[t-1];
                  sumq[0] = tmp;
            }
            for (t = 0; t < 4; ++t) bca->indel_types[t] = B2B_INDEL_NULL;
            for (t = 0; t < 4 && t < n_types; ++t) {
                  bca->indel_types[t] = types[sumq[t]&0x3f];
                  memcpy(&bca->inscns[t * bca->maxins], &inscns[(sumq[t]&0x3f) * max_ins], bca->maxins);
            }
            // update p->aux
            for (s = n_alt = 0; s < n; ++s) {
                  for (i = 0; i < n_plp[s]; ++i) {
                        bam_pileup1_t *p = plp[s] + i;
                        int x = types[p->aux>>16&0x3f];
                        for (j = 0; j < 4; ++j)
                              if (x == bca->indel_types[j]) break;
                        p->aux = j<<16 | (j == 4? 0 : (p->aux&0xffff));
                        if ((p->aux>>16&0x3f) > 0) ++n_alt;
//                      fprintf(stderr, "X pos=%d read=%d:%d name=%s call=%d type=%d q=%d seqQ=%d\n", pos, s, i, bam1_qname(p->b), p->aux>>16&63, bca->indel_types[p->aux>>16&63], p->aux&0xff, p->aux>>8&0xff);
                  }
            }           
      }
      free(score1); free(score2);
      // free
      free(types); free(inscns);
      return n_alt > 0? 0 : -1;
}

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