samtools/bam2bcf_indel.c.pysam.c

   1 #include "pysam.h"
   2
   3 #include <assert.h>
   4 #include <ctype.h>
   5 #include <string.h>
   6 #include "bam.h"
   7 #include "bam2bcf.h"
   8 #include "kaln.h"
   9 #include "kprobaln.h"
  10 #include "khash.h"
  11 KHASH_SET_INIT_STR(rg)
  12
  13 #include "ksort.h"
  14 KSORT_INIT_GENERIC(uint32_t)
  15
  16 #define MINUS_CONST 0x10000000
  17 #define INDEL_WINDOW_SIZE 50
  18
  19 void *bcf_call_add_rg(void *_hash, const char *hdtext, const char *list)
  20 {
  21         const char *s, *p, *q, *r, *t;
  22         khash_t(rg) *hash;
  23         if (list == 0 || hdtext == 0) return _hash;
  24         if (_hash == 0) _hash = kh_init(rg);
  25         hash = (khash_t(rg)*)_hash;
  26         if ((s = strstr(hdtext, "@RG\t")) == 0) return hash;
  27         do {
  28                 t = strstr(s + 4, "@RG\t"); // the next @RG
  29                 if ((p = strstr(s, "\tID:")) != 0) p += 4;
  30                 if ((q = strstr(s, "\tPL:")) != 0) q += 4;
  31                 if (p && q && (t == 0 || (p < t && q < t))) { // ID and PL are both present
  32                         int lp, lq;
  33                         char *x;
  34                         for (r = p; *r && *r != '\t' && *r != '\n'; ++r); lp = r - p;
  35                         for (r = q; *r && *r != '\t' && *r != '\n'; ++r); lq = r - q;
  36                         x = calloc((lp > lq? lp : lq) + 1, 1);
  37                         for (r = q; *r && *r != '\t' && *r != '\n'; ++r) x[r-q] = *r;
  38                         if (strstr(list, x)) { // insert ID to the hash table
  39                                 khint_t k;
  40                                 int ret;
  41                                 for (r = p; *r && *r != '\t' && *r != '\n'; ++r) x[r-p] = *r;
  42                                 x[r-p] = 0;
  43                                 k = kh_get(rg, hash, x);
  44                                 if (k == kh_end(hash)) k = kh_put(rg, hash, x, &ret);
  45                                 else free(x);
  46                         } else free(x);
  47                 }
  48                 s = t;
  49         } while (s);
  50         return hash;
  51 }
  52
  53 void bcf_call_del_rghash(void *_hash)
  54 {
  55         khint_t k;
  56         khash_t(rg) *hash = (khash_t(rg)*)_hash;
  57         if (hash == 0) return;
  58         for (k = kh_begin(hash); k < kh_end(hash); ++k)
  59                 if (kh_exist(hash, k))
  60                         free((char*)kh_key(hash, k));
  61         kh_destroy(rg, hash);
  62 }
  63
  64 static int tpos2qpos(const bam1_core_t *c, const uint32_t *cigar, int32_t tpos, int is_left, int32_t *_tpos)
  65 {
  66         int k, x = c->pos, y = 0, last_y = 0;
  67         *_tpos = c->pos;
  68         for (k = 0; k < c->n_cigar; ++k) {
  69                 int op = cigar[k] & BAM_CIGAR_MASK;
  70                 int l = cigar[k] >> BAM_CIGAR_SHIFT;
  71                 if (op == BAM_CMATCH || op == BAM_CEQUAL || op == BAM_CDIFF) {
  72                         if (c->pos > tpos) return y;
  73                         if (x + l > tpos) {
  74                                 *_tpos = tpos;
  75                                 return y + (tpos - x);
  76                         }
  77                         x += l; y += l;
  78                         last_y = y;
  79                 } else if (op == BAM_CINS || op == BAM_CSOFT_CLIP) y += l;
  80                 else if (op == BAM_CDEL || op == BAM_CREF_SKIP) {
  81                         if (x + l > tpos) {
  82                                 *_tpos = is_left? x : x + l;
  83                                 return y;
  84                         }
  85                         x += l;
  86                 }
  87         }
  88         *_tpos = x;
  89         return last_y;
  90 }
  91 // FIXME: check if the inserted sequence is consistent with the homopolymer run
  92 // l is the relative gap length and l_run is the length of the homopolymer on the reference
  93 static inline int est_seqQ(const bcf_callaux_t *bca, int l, int l_run)
  94 {
  95         int q, qh;
  96         q = bca->openQ + bca->extQ * (abs(l) - 1);
  97         qh = l_run >= 3? (int)(bca->tandemQ * (double)abs(l) / l_run + .499) : 1000;
  98         return q < qh? q : qh;
  99 }
 100
 101 static inline int est_indelreg(int pos, const char *ref, int l, char *ins4)
 102 {
 103         int i, j, max = 0, max_i = pos, score = 0;
 104         l = abs(l);
 105         for (i = pos + 1, j = 0; ref[i]; ++i, ++j) {
 106                 if (ins4) score += (toupper(ref[i]) != "ACGTN"[(int)ins4[j%l]])? -10 : 1;
 107                 else score += (toupper(ref[i]) != toupper(ref[pos+1+j%l]))? -10 : 1;
 108                 if (score < 0) break;
 109                 if (max < score) max = score, max_i = i;
 110         }
 111         return max_i - pos;
 112 }
 113
 114 int bcf_call_gap_prep(int n, int *n_plp, bam_pileup1_t **plp, int pos, bcf_callaux_t *bca, const char *ref,
 115                                           const void *rghash)
 116 {
 117         int i, s, j, k, t, n_types, *types, max_rd_len, left, right, max_ins, *score1, *score2, max_ref2;
 118         int N, K, l_run, ref_type, n_alt;
 119         char *inscns = 0, *ref2, *query, **ref_sample;
 120         khash_t(rg) *hash = (khash_t(rg)*)rghash;
 121         if (ref == 0 || bca == 0) return -1;
 122         // mark filtered reads
 123         if (rghash) {
 124                 N = 0;
 125                 for (s = N = 0; s < n; ++s) {
 126                         for (i = 0; i < n_plp[s]; ++i) {
 127                                 bam_pileup1_t *p = plp[s] + i;
 128                                 const uint8_t *rg = bam_aux_get(p->b, "RG");
 129                                 p->aux = 1; // filtered by default
 130                                 if (rg) {
 131                                         khint_t k = kh_get(rg, hash, (const char*)(rg + 1));
 132                                         if (k != kh_end(hash)) p->aux = 0, ++N; // not filtered
 133                                 }
 134                         }
 135                 }
 136                 if (N == 0) return -1; // no reads left
 137         }
 138         // determine if there is a gap
 139         for (s = N = 0; s < n; ++s) {
 140                 for (i = 0; i < n_plp[s]; ++i)
 141                         if (plp[s][i].indel != 0) break;
 142                 if (i < n_plp[s]) break;
 143         }
 144         if (s == n) return -1; // there is no indel at this position.
 145         for (s = N = 0; s < n; ++s) N += n_plp[s]; // N is the total number of reads
 146         { // find out how many types of indels are present
 147                 int m, n_alt = 0, n_tot = 0;
 148                 uint32_t *aux;
 149                 aux = calloc(N + 1, 4);
 150                 m = max_rd_len = 0;
 151                 aux[m++] = MINUS_CONST; // zero indel is always a type
 152                 for (s = 0; s < n; ++s) {
 153                         for (i = 0; i < n_plp[s]; ++i) {
 154                                 const bam_pileup1_t *p = plp[s] + i;
 155                                 if (rghash == 0 || p->aux == 0) {
 156                                         ++n_tot;
 157                                         if (p->indel != 0) {
 158                                                 ++n_alt;
 159                                                 aux[m++] = MINUS_CONST + p->indel;
 160                                         }
 161                                 }
 162                                 j = bam_cigar2qlen(&p->b->core, bam1_cigar(p->b));
 163                                 if (j > max_rd_len) max_rd_len = j;
 164                         }
 165                 }
 166                 ks_introsort(uint32_t, m, aux);
 167                 // squeeze out identical types
 168                 for (i = 1, n_types = 1; i < m; ++i)
 169                         if (aux[i] != aux[i-1]) ++n_types;
 170                 if (n_types == 1 || (double)n_alt / n_tot < bca->min_frac || n_alt < bca->min_support) { // then skip
 171                         free(aux); return -1;
 172                 }
 173                 if (n_types >= 64) {
 174                         free(aux);
 175                         if (bam_verbose >= 2)
 176                                 fprintf(pysamerr, "[%s] excessive INDEL alleles at position %d. Skip the position.\n", __func__, pos + 1);
 177                         return -1;
 178                 }
 179                 types = (int*)calloc(n_types, sizeof(int));
 180                 t = 0;
 181                 types[t++] = aux[0] - MINUS_CONST;
 182                 for (i = 1; i < m; ++i)
 183                         if (aux[i] != aux[i-1])
 184                                 types[t++] = aux[i] - MINUS_CONST;
 185                 free(aux);
 186                 for (t = 0; t < n_types; ++t)
 187                         if (types[t] == 0) break;
 188                 ref_type = t; // the index of the reference type (0)
 189         }
 190         { // calculate left and right boundary
 191                 left = pos > INDEL_WINDOW_SIZE? pos - INDEL_WINDOW_SIZE : 0;
 192                 right = pos + INDEL_WINDOW_SIZE;
 193                 if (types[0] < 0) right -= types[0];
 194                 // in case the alignments stand out the reference
 195                 for (i = pos; i < right; ++i)
 196                         if (ref[i] == 0) break;
 197                 right = i;
 198         }
 199         /* The following block fixes a long-existing flaw in the INDEL
 200          * calling model: the interference of nearby SNPs. However, it also
 201          * reduces the power because sometimes, substitutions caused by
 202          * indels are not distinguishable from true mutations. Multiple
 203          * sequence realignment helps to increase the power.
 204          */
 205         { // construct per-sample consensus
 206                 int L = right - left + 1, max_i, max2_i;
 207                 uint32_t *cns, max, max2;
 208                 char *ref0, *r;
 209                 ref_sample = calloc(n, sizeof(void*));
 210                 cns = calloc(L, 4);
 211                 ref0 = calloc(L, 1);
 212                 for (i = 0; i < right - left; ++i)
 213                         ref0[i] = bam_nt16_table[(int)ref[i+left]];
 214                 for (s = 0; s < n; ++s) {
 215                         r = ref_sample[s] = calloc(L, 1);
 216                         memset(cns, 0, sizeof(int) * L);
 217                         // collect ref and non-ref counts
 218                         for (i = 0; i < n_plp[s]; ++i) {
 219                                 bam_pileup1_t *p = plp[s] + i;
 220                                 bam1_t *b = p->b;
 221                                 uint32_t *cigar = bam1_cigar(b);
 222                                 uint8_t *seq = bam1_seq(b);
 223                                 int x = b->core.pos, y = 0;
 224                                 for (k = 0; k < b->core.n_cigar; ++k) {
 225                                         int op = cigar[k]&0xf;
 226                                         int j, l = cigar[k]>>4;
 227                                         if (op == BAM_CMATCH || op == BAM_CEQUAL || op == BAM_CDIFF) {
 228                                                 for (j = 0; j < l; ++j)
 229                                                         if (x + j >= left && x + j < right)
 230                                                                 cns[x+j-left] += (bam1_seqi(seq, y+j) == ref0[x+j-left])? 1 : 0x10000;
 231                                                 x += l; y += l;
 232                                         } else if (op == BAM_CDEL || op == BAM_CREF_SKIP) x += l;
 233                                         else if (op == BAM_CINS || op == BAM_CSOFT_CLIP) y += l;
 234                                 }
 235                         }
 236                         // determine the consensus
 237                         for (i = 0; i < right - left; ++i) r[i] = ref0[i];
 238                         max = max2 = 0; max_i = max2_i = -1;
 239                         for (i = 0; i < right - left; ++i) {
 240                                 if (cns[i]>>16 >= max>>16) max2 = max, max2_i = max_i, max = cns[i], max_i = i;
 241                                 else if (cns[i]>>16 >= max2>>16) max2 = cns[i], max2_i = i;
 242                         }
 243                         if ((double)(max&0xffff) / ((max&0xffff) + (max>>16)) >= 0.7) max_i = -1;
 244                         if ((double)(max2&0xffff) / ((max2&0xffff) + (max2>>16)) >= 0.7) max2_i = -1;
 245                         if (max_i >= 0) r[max_i] = 15;
 246                         if (max2_i >= 0) r[max2_i] = 15;
 247 //                      for (i = 0; i < right - left; ++i) fputc("=ACMGRSVTWYHKDBN"[(int)r[i]], pysamerr); fputc('\n', pysamerr);
 248                 }
 249                 free(ref0); free(cns);
 250         }
 251         { // the length of the homopolymer run around the current position
 252                 int c = bam_nt16_table[(int)ref[pos + 1]];
 253                 if (c == 15) l_run = 1;
 254                 else {
 255                         for (i = pos + 2; ref[i]; ++i)
 256                                 if (bam_nt16_table[(int)ref[i]] != c) break;
 257                         l_run = i;
 258                         for (i = pos; i >= 0; --i)
 259                                 if (bam_nt16_table[(int)ref[i]] != c) break;
 260                         l_run -= i + 1;
 261                 }
 262         }
 263         // construct the consensus sequence
 264         max_ins = types[n_types - 1]; // max_ins is at least 0
 265         if (max_ins > 0) {
 266                 int *inscns_aux = calloc(4 * n_types * max_ins, sizeof(int));
 267                 // count the number of occurrences of each base at each position for each type of insertion
 268                 for (t = 0; t < n_types; ++t) {
 269                         if (types[t] > 0) {
 270                                 for (s = 0; s < n; ++s) {
 271                                         for (i = 0; i < n_plp[s]; ++i) {
 272                                                 bam_pileup1_t *p = plp[s] + i;
 273                                                 if (p->indel == types[t]) {
 274                                                         uint8_t *seq = bam1_seq(p->b);
 275                                                         for (k = 1; k <= p->indel; ++k) {
 276                                                                 int c = bam_nt16_nt4_table[bam1_seqi(seq, p->qpos + k)];
 277                                                                 if (c < 4) ++inscns_aux[(t*max_ins+(k-1))*4 + c];
 278                                                         }
 279                                                 }
 280                                         }
 281                                 }
 282                         }
 283                 }
 284                 // use the majority rule to construct the consensus
 285                 inscns = calloc(n_types * max_ins, 1);
 286                 for (t = 0; t < n_types; ++t) {
 287                         for (j = 0; j < types[t]; ++j) {
 288                                 int max = 0, max_k = -1, *ia = &inscns_aux[(t*max_ins+j)*4];
 289                                 for (k = 0; k < 4; ++k)
 290                                         if (ia[k] > max)
 291                                                 max = ia[k], max_k = k;
 292                                 inscns[t*max_ins + j] = max? max_k : 4;
 293                         }
 294                 }
 295                 free(inscns_aux);
 296         }
 297         // compute the likelihood given each type of indel for each read
 298         max_ref2 = right - left + 2 + 2 * (max_ins > -types[0]? max_ins : -types[0]);
 299         ref2  = calloc(max_ref2, 1);
 300         query = calloc(right - left + max_rd_len + max_ins + 2, 1);
 301         score1 = calloc(N * n_types, sizeof(int));
 302         score2 = calloc(N * n_types, sizeof(int));
 303         bca->indelreg = 0;
 304         for (t = 0; t < n_types; ++t) {
 305                 int l, ir;
 306                 kpa_par_t apf1 = { 1e-4, 1e-2, 10 }, apf2 = { 1e-6, 1e-3, 10 };
 307                 apf1.bw = apf2.bw = abs(types[t]) + 3;
 308                 // compute indelreg
 309                 if (types[t] == 0) ir = 0;
 310                 else if (types[t] > 0) ir = est_indelreg(pos, ref, types[t], &inscns[t*max_ins]);
 311                 else ir = est_indelreg(pos, ref, -types[t], 0);
 312                 if (ir > bca->indelreg) bca->indelreg = ir;
 313 //              fprintf(pysamerr, "%d, %d, %d\n", pos, types[t], ir);
 314                 // realignment
 315                 for (s = K = 0; s < n; ++s) {
 316                         // write ref2
 317                         for (k = 0, j = left; j <= pos; ++j)
 318                                 ref2[k++] = bam_nt16_nt4_table[(int)ref_sample[s][j-left]];
 319                         if (types[t] <= 0) j += -types[t];
 320                         else for (l = 0; l < types[t]; ++l)
 321                                          ref2[k++] = inscns[t*max_ins + l];
 322                         for (; j < right && ref[j]; ++j)
 323                                 ref2[k++] = bam_nt16_nt4_table[(int)ref_sample[s][j-left]];
 324                         for (; k < max_ref2; ++k) ref2[k] = 4;
 325                         if (j < right) right = j;
 326                         // align each read to ref2
 327                         for (i = 0; i < n_plp[s]; ++i, ++K) {
 328                                 bam_pileup1_t *p = plp[s] + i;
 329                                 int qbeg, qend, tbeg, tend, sc, kk;
 330                                 uint8_t *seq = bam1_seq(p->b);
 331                                 uint32_t *cigar = bam1_cigar(p->b);
 332                                 if (p->b->core.flag&4) continue; // unmapped reads
 333                                 // FIXME: the following loop should be better moved outside; nonetheless, realignment should be much slower anyway.
 334                                 for (kk = 0; kk < p->b->core.n_cigar; ++kk)
 335                                         if ((cigar[kk]&BAM_CIGAR_MASK) == BAM_CREF_SKIP) break;
 336                                 if (kk < p->b->core.n_cigar) continue;
 337                                 // FIXME: the following skips soft clips, but using them may be more sensitive.
 338                                 // determine the start and end of sequences for alignment
 339                                 qbeg = tpos2qpos(&p->b->core, bam1_cigar(p->b), left,  0, &tbeg);
 340                                 qend = tpos2qpos(&p->b->core, bam1_cigar(p->b), right, 1, &tend);
 341                                 if (types[t] < 0) {
 342                                         int l = -types[t];
 343                                         tbeg = tbeg - l > left?  tbeg - l : left;
 344                                 }
 345                                 // write the query sequence
 346                                 for (l = qbeg; l < qend; ++l)
 347                                         query[l - qbeg] = bam_nt16_nt4_table[bam1_seqi(seq, l)];
 348                                 { // do realignment; this is the bottleneck
 349                                         const uint8_t *qual = bam1_qual(p->b), *bq;
 350                                         uint8_t *qq;
 351                                         qq = calloc(qend - qbeg, 1);
 352                                         bq = (uint8_t*)bam_aux_get(p->b, "ZQ");
 353                                         if (bq) ++bq; // skip type
 354                                         for (l = qbeg; l < qend; ++l) {
 355                                                 qq[l - qbeg] = bq? qual[l] + (bq[l] - 64) : qual[l];
 356                                                 if (qq[l - qbeg] > 30) qq[l - qbeg] = 30;
 357                                                 if (qq[l - qbeg] < 7) qq[l - qbeg] = 7;
 358                                         }
 359                                         sc = kpa_glocal((uint8_t*)ref2 + tbeg - left, tend - tbeg + abs(types[t]),
 360                                                                         (uint8_t*)query, qend - qbeg, qq, &apf1, 0, 0);
 361                                         l = (int)(100. * sc / (qend - qbeg) + .499); // used for adjusting indelQ below
 362                                         if (l > 255) l = 255;
 363                                         score1[K*n_types + t] = score2[K*n_types + t] = sc<<8 | l;
 364                                         if (sc > 5) {
 365                                                 sc = kpa_glocal((uint8_t*)ref2 + tbeg - left, tend - tbeg + abs(types[t]),
 366                                                                                 (uint8_t*)query, qend - qbeg, qq, &apf2, 0, 0);
 367                                                 l = (int)(100. * sc / (qend - qbeg) + .499);
 368                                                 if (l > 255) l = 255;
 369                                                 score2[K*n_types + t] = sc<<8 | l;
 370                                         }
 371                                         free(qq);
 372                                 }
 373 /*
 374                                 for (l = 0; l < tend - tbeg + abs(types[t]); ++l)
 375                                         fputc("ACGTN"[(int)ref2[tbeg-left+l]], pysamerr);
 376                                 fputc('\n', pysamerr);
 377                                 for (l = 0; l < qend - qbeg; ++l) fputc("ACGTN"[(int)query[l]], pysamerr);
 378                                 fputc('\n', pysamerr);
 379                                 fprintf(pysamerr, "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);
 380 */
 381                         }
 382                 }
 383         }
 384         free(ref2); free(query);
 385         { // compute indelQ
 386                 int *sc, tmp, *sumq;
 387                 sc   = alloca(n_types * sizeof(int));
 388                 sumq = alloca(n_types * sizeof(int));
 389                 memset(sumq, 0, sizeof(int) * n_types);
 390                 for (s = K = 0; s < n; ++s) {
 391                         for (i = 0; i < n_plp[s]; ++i, ++K) {
 392                                 bam_pileup1_t *p = plp[s] + i;
 393                                 int *sct = &score1[K*n_types], indelQ1, indelQ2, seqQ, indelQ;
 394                                 for (t = 0; t < n_types; ++t) sc[t] = sct[t]<<6 | t;
 395                                 for (t = 1; t < n_types; ++t) // insertion sort
 396                                         for (j = t; j > 0 && sc[j] < sc[j-1]; --j)
 397                                                 tmp = sc[j], sc[j] = sc[j-1], sc[j-1] = tmp;
 398                                 /* errmod_cal() assumes that if the call is wrong, the
 399                                  * likelihoods of other events are equal. This is about
 400                                  * right for substitutions, but is not desired for
 401                                  * indels. To reuse errmod_cal(), I have to make
 402                                  * compromise for multi-allelic indels.
 403                                  */
 404                                 if ((sc[0]&0x3f) == ref_type) {
 405                                         indelQ1 = (sc[1]>>14) - (sc[0]>>14);
 406                                         seqQ = est_seqQ(bca, types[sc[1]&0x3f], l_run);
 407                                 } else {
 408                                         for (t = 0; t < n_types; ++t) // look for the reference type
 409                                                 if ((sc[t]&0x3f) == ref_type) break;
 410                                         indelQ1 = (sc[t]>>14) - (sc[0]>>14);
 411                                         seqQ = est_seqQ(bca, types[sc[0]&0x3f], l_run);
 412                                 }
 413                                 tmp = sc[0]>>6 & 0xff;
 414                                 indelQ1 = tmp > 111? 0 : (int)((1. - tmp/111.) * indelQ1 + .499); // reduce indelQ
 415                                 sct = &score2[K*n_types];
 416                                 for (t = 0; t < n_types; ++t) sc[t] = sct[t]<<6 | t;
 417                                 for (t = 1; t < n_types; ++t) // insertion sort
 418                                         for (j = t; j > 0 && sc[j] < sc[j-1]; --j)
 419                                                 tmp = sc[j], sc[j] = sc[j-1], sc[j-1] = tmp;
 420                                 if ((sc[0]&0x3f) == ref_type) {
 421                                         indelQ2 = (sc[1]>>14) - (sc[0]>>14);
 422                                 } else {
 423                                         for (t = 0; t < n_types; ++t) // look for the reference type
 424                                                 if ((sc[t]&0x3f) == ref_type) break;
 425                                         indelQ2 = (sc[t]>>14) - (sc[0]>>14);
 426                                 }
 427                                 tmp = sc[0]>>6 & 0xff;
 428                                 indelQ2 = tmp > 111? 0 : (int)((1. - tmp/111.) * indelQ2 + .499);
 429                                 // pick the smaller between indelQ1 and indelQ2
 430                                 indelQ = indelQ1 < indelQ2? indelQ1 : indelQ2;
 431                                 if (indelQ > 255) indelQ = 255;
 432                                 if (seqQ > 255) seqQ = 255;
 433                                 p->aux = (sc[0]&0x3f)<<16 | seqQ<<8 | indelQ; // use 22 bits in total
 434                                 sumq[sc[0]&0x3f] += indelQ < seqQ? indelQ : seqQ;
 435 //                              fprintf(pysamerr, "pos=%d read=%d:%d name=%s call=%d indelQ=%d seqQ=%d\n", pos, s, i, bam1_qname(p->b), types[sc[0]&0x3f], indelQ, seqQ);
 436                         }
 437                 }
 438                 // determine bca->indel_types[] and bca->inscns
 439                 bca->maxins = max_ins;
 440                 bca->inscns = realloc(bca->inscns, bca->maxins * 4);
 441                 for (t = 0; t < n_types; ++t)
 442                         sumq[t] = sumq[t]<<6 | t;
 443                 for (t = 1; t < n_types; ++t) // insertion sort
 444                         for (j = t; j > 0 && sumq[j] > sumq[j-1]; --j)
 445                                 tmp = sumq[j], sumq[j] = sumq[j-1], sumq[j-1] = tmp;
 446                 for (t = 0; t < n_types; ++t) // look for the reference type
 447                         if ((sumq[t]&0x3f) == ref_type) break;
 448                 if (t) { // then move the reference type to the first
 449                         tmp = sumq[t];
 450                         for (; t > 0; --t) sumq[t] = sumq[t-1];
 451                         sumq[0] = tmp;
 452                 }
 453                 for (t = 0; t < 4; ++t) bca->indel_types[t] = B2B_INDEL_NULL;
 454                 for (t = 0; t < 4 && t < n_types; ++t) {
 455                         bca->indel_types[t] = types[sumq[t]&0x3f];
 456                         memcpy(&bca->inscns[t * bca->maxins], &inscns[(sumq[t]&0x3f) * max_ins], bca->maxins);
 457                 }
 458                 // update p->aux
 459                 for (s = n_alt = 0; s < n; ++s) {
 460                         for (i = 0; i < n_plp[s]; ++i) {
 461                                 bam_pileup1_t *p = plp[s] + i;
 462                                 int x = types[p->aux>>16&0x3f];
 463                                 for (j = 0; j < 4; ++j)
 464                                         if (x == bca->indel_types[j]) break;
 465                                 p->aux = j<<16 | (j == 4? 0 : (p->aux&0xffff));
 466                                 if ((p->aux>>16&0x3f) > 0) ++n_alt;
 467 //                              fprintf(pysamerr, "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);
 468                         }
 469                 }
 470         }
 471         free(score1); free(score2);
 472         // free
 473         for (i = 0; i < n; ++i) free(ref_sample[i]);
 474         free(ref_sample);
 475         free(types); free(inscns);
 476         return n_alt > 0? 0 : -1;
 477 }