bam2bcf_indel.c

   1 #include <assert.h>
   2 #include <ctype.h>
   3 #include <string.h>
   4 #include "bam.h"
   5 #include "bam2bcf.h"
   6 #include "ksort.h"
   7 #include "kaln.h"
   8 #include "kprobaln.h"
   9 #include "khash.h"
  10 KHASH_SET_INIT_STR(rg)
  11
  12 #define MINUS_CONST 0x10000000
  13 #define INDEL_WINDOW_SIZE 50
  14
  15 void *bcf_call_add_rg(void *_hash, const char *hdtext, const char *list)
  16 {
  17         const char *s, *p, *q, *r, *t;
  18         khash_t(rg) *hash;
  19         if (list == 0 || hdtext == 0) return _hash;
  20         if (_hash == 0) _hash = kh_init(rg);
  21         hash = (khash_t(rg)*)_hash;
  22         if ((s = strstr(hdtext, "@RG\t")) == 0) return hash;
  23         do {
  24                 t = strstr(s + 4, "@RG\t"); // the next @RG
  25                 if ((p = strstr(s, "\tID:")) != 0) p += 4;
  26                 if ((q = strstr(s, "\tPL:")) != 0) q += 4;
  27                 if (p && q && (t == 0 || (p < t && q < t))) { // ID and PL are both present
  28                         int lp, lq;
  29                         char *x;
  30                         for (r = p; *r && *r != '\t' && *r != '\n'; ++r); lp = r - p;
  31                         for (r = q; *r && *r != '\t' && *r != '\n'; ++r); lq = r - q;
  32                         x = calloc((lp > lq? lp : lq) + 1, 1);
  33                         for (r = q; *r && *r != '\t' && *r != '\n'; ++r) x[r-q] = *r;
  34                         if (strstr(list, x)) { // insert ID to the hash table
  35                                 khint_t k;
  36                                 int ret;
  37                                 for (r = p; *r && *r != '\t' && *r != '\n'; ++r) x[r-p] = *r;
  38                                 x[r-p] = 0;
  39                                 k = kh_get(rg, hash, x);
  40                                 if (k == kh_end(hash)) k = kh_put(rg, hash, x, &ret);
  41                                 else free(x);
  42                         } else free(x);
  43                 }
  44                 s = t;
  45         } while (s);
  46         return hash;
  47 }
  48
  49 void bcf_call_del_rghash(void *_hash)
  50 {
  51         khint_t k;
  52         khash_t(rg) *hash = (khash_t(rg)*)_hash;
  53         if (hash == 0) return;
  54         for (k = kh_begin(hash); k < kh_end(hash); ++k)
  55                 if (kh_exist(hash, k))
  56                         free((char*)kh_key(hash, k));
  57         kh_destroy(rg, hash);
  58 }
  59
  60 static int tpos2qpos(const bam1_core_t *c, const uint32_t *cigar, int32_t tpos, int is_left, int32_t *_tpos)
  61 {
  62         int k, x = c->pos, y = 0, last_y = 0;
  63         *_tpos = c->pos;
  64         for (k = 0; k < c->n_cigar; ++k) {
  65                 int op = cigar[k] & BAM_CIGAR_MASK;
  66                 int l = cigar[k] >> BAM_CIGAR_SHIFT;
  67                 if (op == BAM_CMATCH) {
  68                         if (c->pos > tpos) return y;
  69                         if (x + l > tpos) {
  70                                 *_tpos = tpos;
  71                                 return y + (tpos - x);
  72                         }
  73                         x += l; y += l;
  74                         last_y = y;
  75                 } else if (op == BAM_CINS || op == BAM_CSOFT_CLIP) y += l;
  76                 else if (op == BAM_CDEL || op == BAM_CREF_SKIP) {
  77                         if (x + l > tpos) {
  78                                 *_tpos = is_left? x : x + l;
  79                                 return y;
  80                         }
  81                         x += l;
  82                 }
  83         }
  84         *_tpos = x;
  85         return last_y;
  86 }
  87 // FIXME: check if the inserted sequence is consistent with the homopolymer run
  88 // l is the relative gap length and l_run is the length of the homopolymer on the reference
  89 static inline int est_seqQ(const bcf_callaux_t *bca, int l, int l_run)
  90 {
  91         int q, qh;
  92         q = bca->openQ + bca->extQ * (abs(l) - 1);
  93         qh = l_run >= 3? (int)(bca->tandemQ * (double)abs(l) / l_run + .499) : 1000;
  94         return q < qh? q : qh;
  95 }
  96
  97 static inline int est_indelreg(int pos, const char *ref, int l, char *ins4)
  98 {
  99         int i, j, max = 0, max_i = pos, score = 0;
 100         l = abs(l);
 101         for (i = pos + 1, j = 0; ref[i]; ++i, ++j) {
 102                 if (ins4) score += (toupper(ref[i]) != "ACGTN"[(int)ins4[j%l]])? -10 : 1;
 103                 else score += (toupper(ref[i]) != toupper(ref[pos+1+j%l]))? -10 : 1;
 104                 if (score < 0) break;
 105                 if (max < score) max = score, max_i = i;
 106         }
 107         return max_i - pos;
 108 }
 109
 110 int bcf_call_gap_prep(int n, int *n_plp, bam_pileup1_t **plp, int pos, bcf_callaux_t *bca, const char *ref,
 111                                           const void *rghash)
 112 {
 113         extern void ks_introsort_uint32_t(int, uint32_t*);
 114         int i, s, j, k, t, n_types, *types, max_rd_len, left, right, max_ins, *score1, *score2, max_ref2;
 115         int N, K, l_run, ref_type, n_alt;
 116         char *inscns = 0, *ref2, *query, **ref_sample;
 117         khash_t(rg) *hash = (khash_t(rg)*)rghash;
 118         if (ref == 0 || bca == 0) return -1;
 119         // mark filtered reads
 120         if (rghash) {
 121                 N = 0;
 122                 for (s = N = 0; s < n; ++s) {
 123                         for (i = 0; i < n_plp[s]; ++i) {
 124                                 bam_pileup1_t *p = plp[s] + i;
 125                                 const uint8_t *rg = bam_aux_get(p->b, "RG");
 126                                 p->aux = 1; // filtered by default
 127                                 if (rg) {
 128                                         khint_t k = kh_get(rg, hash, (const char*)(rg + 1));
 129                                         if (k != kh_end(hash)) p->aux = 0, ++N; // not filtered
 130                                 }
 131                         }
 132                 }
 133                 if (N == 0) return -1; // no reads left
 134         }
 135         // determine if there is a gap
 136         for (s = N = 0; s < n; ++s) {
 137                 for (i = 0; i < n_plp[s]; ++i)
 138                         if (plp[s][i].indel != 0) break;
 139                 if (i < n_plp[s]) break;
 140         }
 141         if (s == n) return -1; // there is no indel at this position.
 142         for (s = N = 0; s < n; ++s) N += n_plp[s]; // N is the total number of reads
 143         { // find out how many types of indels are present
 144                 int m, n_alt = 0, n_tot = 0;
 145                 uint32_t *aux;
 146                 aux = calloc(N + 1, 4);
 147                 m = max_rd_len = 0;
 148                 aux[m++] = MINUS_CONST; // zero indel is always a type
 149                 for (s = 0; s < n; ++s) {
 150                         for (i = 0; i < n_plp[s]; ++i) {
 151                                 const bam_pileup1_t *p = plp[s] + i;
 152                                 if (rghash == 0 || p->aux == 0) {
 153                                         ++n_tot;
 154                                         if (p->indel != 0) {
 155                                                 ++n_alt;
 156                                                 aux[m++] = MINUS_CONST + p->indel;
 157                                         }
 158                                 }
 159                                 j = bam_cigar2qlen(&p->b->core, bam1_cigar(p->b));
 160                                 if (j > max_rd_len) max_rd_len = j;
 161                         }
 162                 }
 163                 ks_introsort(uint32_t, m, aux);
 164                 // squeeze out identical types
 165                 for (i = 1, n_types = 1; i < m; ++i)
 166                         if (aux[i] != aux[i-1]) ++n_types;
 167                 if (n_types == 1 || (double)n_alt / n_tot < bca->min_frac || n_alt < bca->min_support) { // then skip
 168                         free(aux); return -1;
 169                 }
 170                 types = (int*)calloc(n_types, sizeof(int));
 171                 t = 0;
 172                 types[t++] = aux[0] - MINUS_CONST;
 173                 for (i = 1; i < m; ++i)
 174                         if (aux[i] != aux[i-1])
 175                                 types[t++] = aux[i] - MINUS_CONST;
 176                 free(aux);
 177                 for (t = 0; t < n_types; ++t)
 178                         if (types[t] == 0) break;
 179                 ref_type = t; // the index of the reference type (0)
 180                 assert(n_types < 64);
 181         }
 182         { // calculate left and right boundary
 183                 left = pos > INDEL_WINDOW_SIZE? pos - INDEL_WINDOW_SIZE : 0;
 184                 right = pos + INDEL_WINDOW_SIZE;
 185                 if (types[0] < 0) right -= types[0];
 186                 // in case the alignments stand out the reference
 187                 for (i = pos; i < right; ++i)
 188                         if (ref[i] == 0) break;
 189                 right = i;
 190         }
 191         /* The following block fixes a long-existing flaw in the INDEL
 192          * calling model: the interference of nearby SNPs. However, it also
 193          * reduces the power because sometimes, substitutions caused by
 194          * indels are not distinguishable from true mutations. Multiple
 195          * sequence realignment helps to increase the power.
 196          */
 197         { // construct per-sample consensus
 198                 int L = right - left + 1;
 199                 uint32_t *cns;
 200                 char *ref0, *r;
 201                 ref_sample = calloc(n, sizeof(void*));
 202                 cns = calloc(L, 4);
 203                 ref0 = calloc(L, 1);
 204                 for (i = 0; i < right - left; ++i)
 205                         ref0[i] = bam_nt16_table[(int)ref[i+left]];
 206                 for (s = 0; s < n; ++s) {
 207                         r = ref_sample[s] = calloc(L, 1);
 208                         memset(cns, 0, sizeof(int) * L);
 209                         // collect ref and non-ref counts
 210                         for (i = 0; i < n_plp[s]; ++i) {
 211                                 bam_pileup1_t *p = plp[s] + i;
 212                                 bam1_t *b = p->b;
 213                                 uint32_t *cigar = bam1_cigar(b);
 214                                 uint8_t *seq = bam1_seq(b);
 215                                 int x = b->core.pos, y = 0;
 216                                 for (k = 0; k < b->core.n_cigar; ++k) {
 217                                         int op = cigar[k]&0xf;
 218                                         int j, l = cigar[k]>>4;
 219                                         if (op == BAM_CMATCH) {
 220                                                 for (j = 0; j < l; ++j)
 221                                                         if (x + j >= left && x + j < right)
 222                                                                 cns[x+j-left] += (bam1_seqi(seq, y+j) == ref0[x+j-left])? 1 : 0x10000;
 223                                                 x += l; y += l;
 224                                         } else if (op == BAM_CDEL || op == BAM_CREF_SKIP) x += l;
 225                                         else if (op == BAM_CINS || op == BAM_CSOFT_CLIP) y += l;
 226                                 }
 227                         }
 228                         // determine the consensus
 229                         for (i = 0; i < right - left; ++i)
 230                                 r[i] = (cns[i] && (double)(cns[i]&0xffff) / ((cns[i]&0xffff)+(cns[i]>>16&0xffff)) < 0.7)? 15 : ref0[i];
 231 //                      for (i = 0; i < right - left; ++i) fputc("=ACMGRSVTWYHKDBN"[(int)r[i]], stderr); fputc('\n', stderr);
 232                 }
 233                 free(ref0); free(cns);
 234         }
 235         { // the length of the homopolymer run around the current position
 236                 int c = bam_nt16_table[(int)ref[pos + 1]];
 237                 if (c == 15) l_run = 1;
 238                 else {
 239                         for (i = pos + 2; ref[i]; ++i)
 240                                 if (bam_nt16_table[(int)ref[i]] != c) break;
 241                         l_run = i;
 242                         for (i = pos; i >= 0; --i)
 243                                 if (bam_nt16_table[(int)ref[i]] != c) break;
 244                         l_run -= i + 1;
 245                 }
 246         }
 247         // construct the consensus sequence
 248         max_ins = types[n_types - 1]; // max_ins is at least 0
 249         if (max_ins > 0) {
 250                 int *inscns_aux = calloc(4 * n_types * max_ins, sizeof(int));
 251                 // count the number of occurrences of each base at each position for each type of insertion
 252                 for (t = 0; t < n_types; ++t) {
 253                         if (types[t] > 0) {
 254                                 for (s = 0; s < n; ++s) {
 255                                         for (i = 0; i < n_plp[s]; ++i) {
 256                                                 bam_pileup1_t *p = plp[s] + i;
 257                                                 if (p->indel == types[t]) {
 258                                                         uint8_t *seq = bam1_seq(p->b);
 259                                                         for (k = 1; k <= p->indel; ++k) {
 260                                                                 int c = bam_nt16_nt4_table[bam1_seqi(seq, p->qpos + k)];
 261                                                                 if (c < 4) ++inscns_aux[(t*max_ins+(k-1))*4 + c];
 262                                                         }
 263                                                 }
 264                                         }
 265                                 }
 266                         }
 267                 }
 268                 // use the majority rule to construct the consensus
 269                 inscns = calloc(n_types * max_ins, 1);
 270                 for (t = 0; t < n_types; ++t) {
 271                         for (j = 0; j < types[t]; ++j) {
 272                                 int max = 0, max_k = -1, *ia = &inscns_aux[(t*max_ins+j)*4];
 273                                 for (k = 0; k < 4; ++k)
 274                                         if (ia[k] > max)
 275                                                 max = ia[k], max_k = k;
 276                                 inscns[t*max_ins + j] = max? max_k : 4;
 277                         }
 278                 }
 279                 free(inscns_aux);
 280         }
 281         // compute the likelihood given each type of indel for each read
 282         max_ref2 = right - left + 2 + 2 * (max_ins > -types[0]? max_ins : -types[0]);
 283         ref2  = calloc(max_ref2, 1);
 284         query = calloc(right - left + max_rd_len + max_ins + 2, 1);
 285         score1 = calloc(N * n_types, sizeof(int));
 286         score2 = calloc(N * n_types, sizeof(int));
 287         bca->indelreg = 0;
 288         for (t = 0; t < n_types; ++t) {
 289                 int l, ir;
 290                 kpa_par_t apf1 = { 1e-4, 1e-2, 10 }, apf2 = { 1e-6, 1e-3, 10 };
 291                 apf1.bw = apf2.bw = abs(types[t]) + 3;
 292                 // compute indelreg
 293                 if (types[t] == 0) ir = 0;
 294                 else if (types[t] > 0) ir = est_indelreg(pos, ref, types[t], &inscns[t*max_ins]);
 295                 else ir = est_indelreg(pos, ref, -types[t], 0);
 296                 if (ir > bca->indelreg) bca->indelreg = ir;
 297 //              fprintf(stderr, "%d, %d, %d\n", pos, types[t], ir);
 298                 // realignment
 299                 for (s = K = 0; s < n; ++s) {
 300                         // write ref2
 301                         for (k = 0, j = left; j <= pos; ++j)
 302                                 ref2[k++] = bam_nt16_nt4_table[(int)ref_sample[s][j-left]];
 303                         if (types[t] <= 0) j += -types[t];
 304                         else for (l = 0; l < types[t]; ++l)
 305                                          ref2[k++] = inscns[t*max_ins + l];
 306                         for (; j < right && ref[j]; ++j)
 307                                 ref2[k++] = bam_nt16_nt4_table[(int)ref_sample[s][j-left]];
 308                         for (; k < max_ref2; ++k) ref2[k] = 4;
 309                         if (j < right) right = j;
 310                         // align each read to ref2
 311                         for (i = 0; i < n_plp[s]; ++i, ++K) {
 312                                 bam_pileup1_t *p = plp[s] + i;
 313                                 int qbeg, qend, tbeg, tend, sc;
 314                                 uint8_t *seq = bam1_seq(p->b);
 315                                 // FIXME: the following skips soft clips, but using them may be more sensitive.
 316                                 // determine the start and end of sequences for alignment
 317                                 qbeg = tpos2qpos(&p->b->core, bam1_cigar(p->b), left,  0, &tbeg);
 318                                 qend = tpos2qpos(&p->b->core, bam1_cigar(p->b), right, 1, &tend);
 319                                 if (types[t] < 0) {
 320                                         int l = -types[t];
 321                                         tbeg = tbeg - l > left?  tbeg - l : left;
 322                                 }
 323                                 // write the query sequence
 324                                 for (l = qbeg; l < qend; ++l)
 325                                         query[l - qbeg] = bam_nt16_nt4_table[bam1_seqi(seq, l)];
 326                                 { // do realignment; this is the bottleneck
 327                                         const uint8_t *qual = bam1_qual(p->b), *bq;
 328                                         uint8_t *qq;
 329                                         qq = calloc(qend - qbeg, 1);
 330                                         bq = (uint8_t*)bam_aux_get(p->b, "ZQ");
 331                                         if (bq) ++bq; // skip type
 332                                         for (l = qbeg; l < qend; ++l) {
 333                                                 qq[l - qbeg] = bq? qual[l] + (bq[l] - 64) : qual[l];
 334                                                 if (qq[l - qbeg] > 30) qq[l - qbeg] = 30;
 335                                                 if (qq[l - qbeg] < 7) qq[l - qbeg] = 7;
 336                                         }
 337                                         sc = kpa_glocal((uint8_t*)ref2 + tbeg - left, tend - tbeg + abs(types[t]),
 338                                                                         (uint8_t*)query, qend - qbeg, qq, &apf1, 0, 0);
 339                                         l = (int)(100. * sc / (qend - qbeg) + .499); // used for adjusting indelQ below
 340                                         if (l > 255) l = 255;
 341                                         score1[K*n_types + t] = score2[K*n_types + t] = sc<<8 | l;
 342                                         if (sc > 5) {
 343                                                 sc = kpa_glocal((uint8_t*)ref2 + tbeg - left, tend - tbeg + abs(types[t]),
 344                                                                                 (uint8_t*)query, qend - qbeg, qq, &apf2, 0, 0);
 345                                                 l = (int)(100. * sc / (qend - qbeg) + .499);
 346                                                 if (l > 255) l = 255;
 347                                                 score2[K*n_types + t] = sc<<8 | l;
 348                                         }
 349                                         free(qq);
 350                                 }
 351 /*
 352                                 for (l = 0; l < tend - tbeg + abs(types[t]); ++l)
 353                                         fputc("ACGTN"[(int)ref2[tbeg-left+l]], stderr);
 354                                 fputc('\n', stderr);
 355                                 for (l = 0; l < qend - qbeg; ++l) fputc("ACGTN"[(int)query[l]], stderr);
 356                                 fputc('\n', stderr);
 357                                 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);
 358 */
 359                         }
 360                 }
 361         }
 362         free(ref2); free(query);
 363         { // compute indelQ
 364                 int *sc, tmp, *sumq;
 365                 sc   = alloca(n_types * sizeof(int));
 366                 sumq = alloca(n_types * sizeof(int));
 367                 memset(sumq, 0, sizeof(int) * n_types);
 368                 for (s = K = 0; s < n; ++s) {
 369                         for (i = 0; i < n_plp[s]; ++i, ++K) {
 370                                 bam_pileup1_t *p = plp[s] + i;
 371                                 int *sct = &score1[K*n_types], indelQ1, indelQ2, seqQ, indelQ;
 372                                 for (t = 0; t < n_types; ++t) sc[t] = sct[t]<<6 | t;
 373                                 for (t = 1; t < n_types; ++t) // insertion sort
 374                                         for (j = t; j > 0 && sc[j] < sc[j-1]; --j)
 375                                                 tmp = sc[j], sc[j] = sc[j-1], sc[j-1] = tmp;
 376                                 /* errmod_cal() assumes that if the call is wrong, the
 377                                  * likelihoods of other events are equal. This is about
 378                                  * right for substitutions, but is not desired for
 379                                  * indels. To reuse errmod_cal(), I have to make
 380                                  * compromise for multi-allelic indels.
 381                                  */
 382                                 if ((sc[0]&0x3f) == ref_type) {
 383                                         indelQ1 = (sc[1]>>14) - (sc[0]>>14);
 384                                         seqQ = est_seqQ(bca, types[sc[1]&0x3f], l_run);
 385                                 } else {
 386                                         for (t = 0; t < n_types; ++t) // look for the reference type
 387                                                 if ((sc[t]&0x3f) == ref_type) break;
 388                                         indelQ1 = (sc[t]>>14) - (sc[0]>>14);
 389                                         seqQ = est_seqQ(bca, types[sc[0]&0x3f], l_run);
 390                                 }
 391                                 tmp = sc[0]>>6 & 0xff;
 392                                 indelQ1 = tmp > 111? 0 : (int)((1. - tmp/111.) * indelQ1 + .499); // reduce indelQ
 393                                 sct = &score2[K*n_types];
 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                                 if ((sc[0]&0x3f) == ref_type) {
 399                                         indelQ2 = (sc[1]>>14) - (sc[0]>>14);
 400                                 } else {
 401                                         for (t = 0; t < n_types; ++t) // look for the reference type
 402                                                 if ((sc[t]&0x3f) == ref_type) break;
 403                                         indelQ2 = (sc[t]>>14) - (sc[0]>>14);
 404                                 }
 405                                 tmp = sc[0]>>6 & 0xff;
 406                                 indelQ2 = tmp > 111? 0 : (int)((1. - tmp/111.) * indelQ2 + .499);
 407                                 // pick the smaller between indelQ1 and indelQ2
 408                                 indelQ = indelQ1 < indelQ2? indelQ1 : indelQ2;
 409                                 p->aux = (sc[0]&0x3f)<<16 | seqQ<<8 | indelQ;
 410                                 sumq[sc[0]&0x3f] += indelQ < seqQ? indelQ : seqQ;
 411 //                              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);
 412                         }
 413                 }
 414                 // determine bca->indel_types[] and bca->inscns
 415                 bca->maxins = max_ins;
 416                 bca->inscns = realloc(bca->inscns, bca->maxins * 4);
 417                 for (t = 0; t < n_types; ++t)
 418                         sumq[t] = sumq[t]<<6 | t;
 419                 for (t = 1; t < n_types; ++t) // insertion sort
 420                         for (j = t; j > 0 && sumq[j] > sumq[j-1]; --j)
 421                                 tmp = sumq[j], sumq[j] = sumq[j-1], sumq[j-1] = tmp;
 422                 for (t = 0; t < n_types; ++t) // look for the reference type
 423                         if ((sumq[t]&0x3f) == ref_type) break;
 424                 if (t) { // then move the reference type to the first
 425                         tmp = sumq[t];
 426                         for (; t > 0; --t) sumq[t] = sumq[t-1];
 427                         sumq[0] = tmp;
 428                 }
 429                 for (t = 0; t < 4; ++t) bca->indel_types[t] = B2B_INDEL_NULL;
 430                 for (t = 0; t < 4 && t < n_types; ++t) {
 431                         bca->indel_types[t] = types[sumq[t]&0x3f];
 432                         memcpy(&bca->inscns[t * bca->maxins], &inscns[(sumq[t]&0x3f) * max_ins], bca->maxins);
 433                 }
 434                 // update p->aux
 435                 for (s = n_alt = 0; s < n; ++s) {
 436                         for (i = 0; i < n_plp[s]; ++i) {
 437                                 bam_pileup1_t *p = plp[s] + i;
 438                                 int x = types[p->aux>>16&0x3f];
 439                                 for (j = 0; j < 4; ++j)
 440                                         if (x == bca->indel_types[j]) break;
 441                                 p->aux = j<<16 | (j == 4? 0 : (p->aux&0xffff));
 442                                 if ((p->aux>>16&0x3f) > 0) ++n_alt;
 443 //                              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);
 444                         }
 445                 }
 446         }
 447         free(score1); free(score2);
 448         // free
 449         for (i = 0; i < n; ++i) free(ref_sample[i]);
 450         free(ref_sample);
 451         free(types); free(inscns);
 452         return n_alt > 0? 0 : -1;
 453 }