4 #include "bam_maqcns.h"
7 KSORT_INIT_GENERIC(uint32_t)
9 #define INDEL_WINDOW_SIZE 50
10 #define INDEL_EXT_DEP 0.9
12 typedef struct __bmc_aux_t {
18 float esum[4], fsum[4];
23 char bam_nt16_nt4_table[] = { 4, 0, 1, 4, 2, 4, 4, 4, 3, 4, 4, 4, 4, 4, 4, 4 };
26 P(<b1,b2>) = \theta \sum_{i=1}^{N-1} 1/i
27 P(D|<b1,b2>) = \sum_{k=1}^{N-1} p_k 1/2 [(k/N)^n_2(1-k/N)^n_1 + (k/N)^n1(1-k/N)^n_2]
28 p_k = 1/k / \sum_{i=1}^{N-1} 1/i
30 static void cal_het(bam_maqcns_t *aa)
33 double sum_harmo; // harmonic sum
37 aa->lhet = (double*)calloc(256 * 256, sizeof(double));
39 for (k = 1; k <= aa->n_hap - 1; ++k)
41 for (n1 = 0; n1 < 256; ++n1) {
42 for (n2 = 0; n2 < 256; ++n2) {
43 long double sum = 0.0;
44 double lC = aa->is_soap? 0 : lgamma(n1+n2+1) - lgamma(n1+1) - lgamma(n2+1); // \binom{n1+n2}{n1}
45 for (k = 1; k <= aa->n_hap - 1; ++k) {
46 double pk = 1.0 / k / sum_harmo;
47 double log1 = log((double)k/aa->n_hap);
48 double log2 = log(1.0 - (double)k/aa->n_hap);
49 sum += pk * 0.5 * (expl(log1*n2) * expl(log2*n1) + expl(log1*n1) * expl(log2*n2));
51 aa->lhet[n1<<8|n2] = lC + logl(sum);
54 poly_rate = aa->het_rate * sum_harmo;
55 aa->q_r = -4.343 * log(2.0 * poly_rate / (1.0 - poly_rate));
58 /** initialize the helper structure */
59 static void cal_coef(bam_maqcns_t *aa)
62 long double sum_a[257], b[256], q_c[256], tmp[256], fk2[256];
65 // aa->lhet will be allocated and initialized
66 free(aa->fk); free(aa->coef);
68 aa->fk = (double*)calloc(256, sizeof(double));
69 aa->fk[0] = fk2[0] = 1.0;
70 for (n = 1; n != 256; ++n) {
71 aa->fk[n] = pow(aa->theta, n) * (1.0 - aa->eta) + aa->eta;
72 fk2[n] = aa->fk[n>>1]; // this is an approximation, assuming reads equally likely come from both strands
74 if (aa->is_soap) return;
75 aa->coef = (double*)calloc(256*256*64, sizeof(double));
76 lC = (double*)calloc(256 * 256, sizeof(double));
77 for (n = 1; n != 256; ++n)
78 for (k = 1; k <= n; ++k)
79 lC[n<<8|k] = lgamma(n+1) - lgamma(k+1) - lgamma(n-k+1);
80 for (q = 1; q != 64; ++q) {
81 double e = pow(10.0, -q/10.0);
83 double le1 = log(1.0-e);
84 for (n = 1; n != 256; ++n) {
85 double *coef = aa->coef + (q<<16|n<<8);
87 for (k = n; k >= 0; --k) { // a_k = \sum_{i=k}^n C^n_k \epsilon^k (1-\epsilon)^{n-k}
88 sum_a[k] = sum_a[k+1] + expl(lC[n<<8|k] + k*le + (n-k)*le1);
89 b[k] = sum_a[k+1] / sum_a[k];
90 if (b[k] > 0.99) b[k] = 0.99;
92 for (k = 0; k != n; ++k) // log(\bar\beta_{nk}(\bar\epsilon)^{f_k})
93 q_c[k] = -4.343 * fk2[k] * logl(b[k] / e);
94 for (k = 1; k != n; ++k) q_c[k] += q_c[k-1]; // \prod_{i=0}^k c_i
95 for (k = 0; k <= n; ++k) { // powl() in 64-bit mode seems broken on my Mac OS X 10.4.9
96 tmp[k] = -4.343 * logl(1.0 - expl(fk2[k] * logl(b[k])));
97 coef[k] = (k? q_c[k-1] : 0) + tmp[k]; // this is the final c_{nk}
104 bam_maqcns_t *bam_maqcns_init()
107 bm = (bam_maqcns_t*)calloc(1, sizeof(bam_maqcns_t));
108 bm->aux = (bmc_aux_t*)calloc(1, sizeof(bmc_aux_t));
109 bm->het_rate = 0.001;
117 void bam_maqcns_prepare(bam_maqcns_t *bm)
119 cal_coef(bm); cal_het(bm);
122 void bam_maqcns_destroy(bam_maqcns_t *bm)
125 free(bm->lhet); free(bm->fk); free(bm->coef); free(bm->aux->info);
126 free(bm->aux); free(bm);
129 glf1_t *bam_maqcns_glfgen(int _n, const bam_pileup1_t *pl, uint8_t ref_base, bam_maqcns_t *bm)
132 int i, j, k, w[8], c, n;
133 glf1_t *g = (glf1_t*)calloc(1, sizeof(glf1_t));
134 float p[16], min_p = 1e30;
137 g->ref_base = ref_base;
138 if (_n == 0) return g;
140 // construct aux array
141 if (bm->aux->max < _n) {
143 kroundup32(bm->aux->max);
144 bm->aux->info = (uint32_t*)realloc(bm->aux->info, 4 * bm->aux->max);
146 for (i = n = 0; i < _n; ++i) {
147 const bam_pileup1_t *p = pl + i;
148 uint32_t q, x = 0, qq;
149 if (p->is_del || (p->b->core.flag&BAM_FUNMAP)) continue;
150 q = (uint32_t)bam1_qual(p->b)[p->qpos];
151 x |= (uint32_t)bam1_strand(p->b) << 18 | q << 8 | p->b->core.qual;
152 if (p->b->core.qual < q) q = p->b->core.qual;
154 qq = bam1_seqi(bam1_seq(p->b), p->qpos);
155 q = bam_nt16_nt4_table[qq? qq : ref_base];
156 if (!p->is_del && q < 4) x |= 1 << 21 | q << 16;
157 bm->aux->info[n++] = x;
159 ks_introsort(uint32_t, n, bm->aux->info);
160 // generate esum and fsum
161 b = (glf_call_aux_t*)calloc(1, sizeof(glf_call_aux_t));
162 for (k = 0; k != 8; ++k) w[k] = 0;
164 for (j = n - 1; j >= 0; --j) { // calculate esum and fsum
165 uint32_t info = bm->aux->info[j];
167 if (info>>24 < 4 && (info>>8&0x3f) != 0) info = 4<<24 | (info&0xffffff);
170 b->esum[k&3] += bm->fk[w[k]] * (info>>24);
171 b->fsum[k&3] += bm->fk[w[k]];
172 if (w[k] < 0xff) ++w[k];
175 tmp = (int)(info&0xff) < bm->cap_mapQ? (int)(info&0xff) : bm->cap_mapQ;
178 b->rms_mapQ = (uint8_t)(sqrt((double)rms / n) + .499);
180 for (j = c = 0; j != 4; ++j) c += b->c[j];
182 for (j = 0; j != 4; ++j) b->c[j] = (int)(254.0 * b->c[j] / c + 0.5);
183 for (j = c = 0; j != 4; ++j) c += b->c[j];
186 // generate likelihood
187 for (j = 0; j != 4; ++j) {
191 for (k = 0, tmp1 = tmp3 = 0.0, tmp2 = 0; k != 4; ++k) {
192 if (j == k) continue;
193 tmp1 += b->esum[k]; tmp2 += b->c[k]; tmp3 += b->fsum[k];
196 bar_e = (int)(tmp1 / tmp3 + 0.5);
197 if (bar_e < 4) bar_e = 4; // should not happen
198 if (bar_e > 63) bar_e = 63;
199 p[j<<2|j] = tmp1 + bm->coef[bar_e<<16|c<<8|tmp2];
200 } else p[j<<2|j] = 0.0; // all the bases are j
202 for (k = j + 1; k < 4; ++k) {
203 for (i = 0, tmp2 = 0, tmp1 = tmp3 = 0.0; i != 4; ++i) {
204 if (i == j || i == k) continue;
205 tmp1 += b->esum[i]; tmp2 += b->c[i]; tmp3 += b->fsum[i];
208 bar_e = (int)(tmp1 / tmp3 + 0.5);
209 if (bar_e < 4) bar_e = 4;
210 if (bar_e > 63) bar_e = 63;
211 p[j<<2|k] = p[k<<2|j] = -4.343 * bm->lhet[b->c[j]<<8|b->c[k]] + tmp1 + bm->coef[bar_e<<16|c<<8|tmp2];
212 } else p[j<<2|k] = p[k<<2|j] = -4.343 * bm->lhet[b->c[j]<<8|b->c[k]]; // all the bases are either j or k
215 for (k = 0; k != 4; ++k)
216 if (p[j<<2|k] < 0.0) p[j<<2|k] = 0.0;
220 float max1, max2, min1, min2;
223 max1 = max2 = -1.0; min1 = min2 = 1e30;
224 for (k = 0; k < 4; ++k) {
225 if (b->esum[k] > max1) {
226 max2 = max1; max1 = b->esum[k]; max_k = k;
227 } else if (b->esum[k] > max2) max2 = b->esum[k];
229 for (k = 0; k < 4; ++k) {
230 if (p[k<<2|k] < min1) {
231 min2 = min1; min1 = p[k<<2|k]; min_k = k;
232 } else if (p[k<<2|k] < min2) min2 = p[k<<2|k];
234 if (max1 > max2 && (min_k != max_k || min1 + 1.0 > min2))
235 p[max_k<<2|max_k] = min1 > 1.0? min1 - 1.0 : 0.0;
237 } else { // apply the SOAP model
238 // generate likelihood
239 for (j = 0; j != 4; ++j) {
242 for (k = 0, tmp = 0.0; k != 4; ++k)
243 if (j != k) tmp += b->esum[k];
246 for (k = j + 1; k < 4; ++k) {
247 for (i = 0, tmp = 0.0; i != 4; ++i)
248 if (i != j && i != k) tmp += b->esum[i];
249 p[j<<2|k] = p[k<<2|j] = -4.343 * bm->lhet[b->c[j]<<8|b->c[k]] + tmp;
254 // convert necessary information to glf1_t
255 g->ref_base = ref_base; g->max_mapQ = b->rms_mapQ;
256 g->depth = n > 16777215? 16777215 : n;
257 for (j = 0; j != 4; ++j)
258 for (k = j; k < 4; ++k)
259 if (p[j<<2|k] < min_p) min_p = p[j<<2|k];
260 g->min_lk = min_p > 255.0? 255 : (int)(min_p + 0.5);
261 for (j = c = 0; j != 4; ++j)
262 for (k = j; k < 4; ++k)
263 g->lk[c++] = p[j<<2|k]-min_p > 255.0? 255 : (int)(p[j<<2|k]-min_p + 0.5);
269 uint32_t glf2cns(const glf1_t *g, int q_r)
271 int i, j, k, tmp[16], min = 10000, min2 = 10000, min3 = 10000, min_g = -1, min_g2 = -1;
273 for (i = k = 0; i < 4; ++i)
274 for (j = i; j < 4; ++j) {
276 tmp[i<<2|j] = g->lk[k++] + (i == j? 0 : q_r);
278 for (i = 0; i < 16; ++i) {
279 if (tmp[i] < 0) continue;
281 min3 = min2; min2 = min; min = tmp[i]; min_g2 = min_g; min_g = i;
282 } else if (tmp[i] < min2) {
283 min3 = min2; min2 = tmp[i]; min_g2 = i;
284 } else if (tmp[i] < min3) min3 = tmp[i];
286 x = min_g >= 0? (1U<<(min_g>>2&3) | 1U<<(min_g&3)) << 28 : 0xf << 28;
287 x |= min_g2 >= 0? (1U<<(min_g2>>2&3) | 1U<<(min_g2&3)) << 24 : 0xf << 24;
288 x |= (uint32_t)g->max_mapQ << 16;
289 x |= min2 < 10000? (min2 - min < 256? min2 - min : 255) << 8 : 0xff << 8;
290 x |= min2 < 10000 && min3 < 10000? (min3 - min2 < 256? min3 - min2 : 255) : 0xff;
294 uint32_t bam_maqcns_call(int n, const bam_pileup1_t *pl, bam_maqcns_t *bm)
299 g = bam_maqcns_glfgen(n, pl, 0xf, bm);
300 x = glf2cns(g, (int)(bm->q_r + 0.5));
302 } else x = 0xfU<<28 | 0xfU<<24;
306 /************** *****************/
308 bam_maqindel_opt_t *bam_maqindel_opt_init()
310 bam_maqindel_opt_t *mi = (bam_maqindel_opt_t*)calloc(1, sizeof(bam_maqindel_opt_t));
312 mi->r_indel = 0.00015;
320 void bam_maqindel_ret_destroy(bam_maqindel_ret_t *mir)
322 if (mir == 0) return;
323 free(mir->s[0]); free(mir->s[1]); free(mir);
326 int bam_tpos2qpos(const bam1_core_t *c, const uint32_t *cigar, int32_t tpos, int is_left, int32_t *_tpos)
328 int k, x = c->pos, y = 0, last_y = 0;
330 for (k = 0; k < c->n_cigar; ++k) {
331 int op = cigar[k] & BAM_CIGAR_MASK;
332 int l = cigar[k] >> BAM_CIGAR_SHIFT;
333 if (op == BAM_CMATCH) {
334 if (c->pos > tpos) return y;
337 return y + (tpos - x);
341 } else if (op == BAM_CINS || op == BAM_CSOFT_CLIP) y += l;
342 else if (op == BAM_CDEL || op == BAM_CREF_SKIP) {
344 *_tpos = is_left? x : x + l;
354 #define MINUS_CONST 0x10000000
356 bam_maqindel_ret_t *bam_maqindel(int n, int pos, const bam_maqindel_opt_t *mi, const bam_pileup1_t *pl, const char *ref,
357 int _n_types, int *_types)
359 int i, j, n_types, *types, left, right, max_rd_len = 0;
360 bam_maqindel_ret_t *ret = 0;
361 // if there is no proposed indel, check if there is an indel from the alignment
363 for (i = 0; i < n; ++i) {
364 const bam_pileup1_t *p = pl + i;
365 if (!(p->b->core.flag&BAM_FUNMAP) && p->indel != 0) break;
367 if (i == n) return 0; // no indel
369 { // calculate how many types of indels are available (set n_types and types)
372 aux = (uint32_t*)calloc(n + _n_types + 1, 4);
374 aux[m++] = MINUS_CONST; // zero indel is always a type
375 for (i = 0; i < n; ++i) {
376 const bam_pileup1_t *p = pl + i;
377 if (!(p->b->core.flag&BAM_FUNMAP) && p->indel != 0)
378 aux[m++] = MINUS_CONST + p->indel;
379 j = bam_cigar2qlen(&p->b->core, bam1_cigar(p->b));
380 if (j > max_rd_len) max_rd_len = j;
382 if (_n_types) // then also add this to aux[]
383 for (i = 0; i < _n_types; ++i)
384 if (_types[i]) aux[m++] = MINUS_CONST + _types[i];
385 ks_introsort(uint32_t, m, aux);
386 // squeeze out identical types
387 for (i = 1, n_types = 1; i < m; ++i)
388 if (aux[i] != aux[i-1]) ++n_types;
389 types = (int*)calloc(n_types, sizeof(int));
391 types[j++] = aux[0] - MINUS_CONST;
392 for (i = 1; i < m; ++i) {
393 if (aux[i] != aux[i-1])
394 types[j++] = aux[i] - MINUS_CONST;
398 { // calculate left and right boundary
399 left = pos > INDEL_WINDOW_SIZE? pos - INDEL_WINDOW_SIZE : 0;
400 right = pos + INDEL_WINDOW_SIZE;
401 if (types[0] < 0) right -= types[0];
402 // in case the alignments stand out the reference
403 for (i = pos; i < right; ++i)
404 if (ref[i] == 0) break;
408 char *ref2, *rs, *inscns = 0;
409 int k, l, *score, *pscore, max_ins = types[n_types-1];
410 if (max_ins > 0) { // get the consensus of inserted sequences
411 int *inscns_aux = (int*)calloc(4 * n_types * max_ins, sizeof(int));
413 for (i = 0; i < n_types; ++i) {
414 if (types[i] <= 0) continue; // not insertion
415 for (j = 0; j < n; ++j) {
416 const bam_pileup1_t *p = pl + j;
417 if (!(p->b->core.flag&BAM_FUNMAP) && p->indel == types[i]) {
418 for (k = 1; k <= p->indel; ++k) {
419 int c = bam_nt16_nt4_table[bam1_seqi(bam1_seq(p->b), p->qpos + k)];
420 if (c < 4) ++inscns_aux[i*max_ins*4 + (k-1)*4 + c];
425 // construct the consensus of inserted sequence
426 inscns = (char*)calloc(n_types * max_ins, sizeof(char));
427 for (i = 0; i < n_types; ++i) {
428 for (j = 0; j < types[i]; ++j) {
429 int max = 0, max_k = -1, *ia = inscns_aux + i*max_ins*4 + j*4;
430 for (k = 0; k < 4; ++k) {
436 inscns[i*max_ins + j] = max? 1<<max_k : 15;
442 ref2 = (char*)calloc(right - left + types[n_types-1] + 2, 1);
443 rs = (char*)calloc(right - left + max_rd_len + types[n_types-1] + 2, 1);
444 score = (int*)calloc(n_types * n, sizeof(int));
445 pscore = (int*)calloc(n_types * n, sizeof(int));
446 for (i = 0; i < n_types; ++i) {
447 ka_param_t ap = ka_param_blast;
448 ap.band_width = 2 * types[n_types - 1] + 2;
450 for (k = 0, j = left; j <= pos; ++j)
451 ref2[k++] = bam_nt16_nt4_table[bam_nt16_table[(int)ref[j]]];
452 if (types[i] <= 0) j += -types[i];
453 else for (l = 0; l < types[i]; ++l)
454 ref2[k++] = bam_nt16_nt4_table[(int)inscns[i*max_ins + l]];
455 for (; j < right && ref[j]; ++j)
456 ref2[k++] = bam_nt16_nt4_table[bam_nt16_table[(int)ref[j]]];
457 if (j < right) right = j;
458 // calculate score for each read
459 for (j = 0; j < n; ++j) {
460 const bam_pileup1_t *p = pl + j;
461 int qbeg, qend, tbeg, tend;
462 if (p->b->core.flag & BAM_FUNMAP) continue;
463 qbeg = bam_tpos2qpos(&p->b->core, bam1_cigar(p->b), left, 0, &tbeg);
464 qend = bam_tpos2qpos(&p->b->core, bam1_cigar(p->b), right, 1, &tend);
465 assert(tbeg >= left);
466 for (l = qbeg; l < qend; ++l)
467 rs[l - qbeg] = bam_nt16_nt4_table[bam1_seqi(bam1_seq(p->b), l)];
469 int x, y, n_acigar, ps;
472 if (tend - tbeg + types[i] <= 0) {
473 score[i*n+j] = -(1<<20);
474 pscore[i*n+j] = 1<<20;
477 acigar = ka_global_core((uint8_t*)ref2 + tbeg - left, tend - tbeg + types[i], (uint8_t*)rs, qend - qbeg, &ap, &score[i*n+j], &n_acigar);
478 x = tbeg - left; y = 0;
479 for (l = 0; l < n_acigar; ++l) {
480 int op = acigar[l]&0xf;
481 int len = acigar[l]>>4;
482 if (op == BAM_CMATCH) {
484 for (k = 0; k < len; ++k)
485 if (ref2[x+k] != rs[y+k]) ps += bam1_qual(p->b)[y+k];
487 } else if (op == BAM_CINS || op == BAM_CSOFT_CLIP) {
488 if (op == BAM_CINS) ps += mi->q_indel * len;
490 } else if (op == BAM_CDEL) {
491 ps += mi->q_indel * len;
496 /*if (pos == 2618517) { // for debugging only
497 fprintf(stderr, "pos=%d, type=%d, j=%d, score=%d, psore=%d, %d, %d, %d, %d, ", pos+1, types[i], j, score[i*n+j], pscore[i*n+j], tbeg, tend, qbeg, qend);
498 for (l = 0; l < n_acigar; ++l) fprintf(stderr, "%d%c", acigar[l]>>4, "MIDS"[acigar[l]&0xf]); fprintf(stderr, "\n");
499 for (l = 0; l < tend - tbeg + types[i]; ++l) fputc("ACGTN"[ref2[l]], stderr); fputc('\n', stderr);
500 for (l = 0; l < qend - qbeg; ++l) fputc("ACGTN"[rs[l]], stderr); fputc('\n', stderr);
506 { // get final result
507 int *sum, max1, max2, max1_i, max2_i;
508 // pick up the best two score
509 sum = (int*)calloc(n_types, sizeof(int));
510 for (i = 0; i < n_types; ++i)
511 for (j = 0; j < n; ++j)
512 sum[i] += -pscore[i*n+j];
513 max1 = max2 = -0x7fffffff; max1_i = max2_i = -1;
514 for (i = 0; i < n_types; ++i) {
516 max2 = max1; max2_i = max1_i; max1 = sum[i]; max1_i = i;
517 } else if (sum[i] > max2) {
518 max2 = sum[i]; max2_i = i;
523 ret = (bam_maqindel_ret_t*)calloc(1, sizeof(bam_maqindel_ret_t));
524 ret->indel1 = types[max1_i]; ret->indel2 = types[max2_i];
525 ret->s[0] = (char*)calloc(abs(ret->indel1) + 2, 1);
526 ret->s[1] = (char*)calloc(abs(ret->indel2) + 2, 1);
527 // write indel sequence
528 if (ret->indel1 > 0) {
530 for (k = 0; k < ret->indel1; ++k)
531 ret->s[0][k+1] = bam_nt16_rev_table[(int)inscns[max1_i*max_ins + k]];
532 } else if (ret->indel1 < 0) {
534 for (k = 0; k < -ret->indel1 && ref[pos + k + 1]; ++k)
535 ret->s[0][k+1] = ref[pos + k + 1];
536 } else ret->s[0][0] = '*';
537 if (ret->indel2 > 0) {
539 for (k = 0; k < ret->indel2; ++k)
540 ret->s[1][k+1] = bam_nt16_rev_table[(int)inscns[max2_i*max_ins + k]];
541 } else if (ret->indel2 < 0) {
543 for (k = 0; k < -ret->indel2 && ref[pos + k + 1]; ++k)
544 ret->s[1][k+1] = ref[pos + k + 1];
545 } else ret->s[1][0] = '*';
547 for (i = 0; i < n; ++i) {
548 const bam_pileup1_t *p = pl + i;
549 if (p->indel == ret->indel1) ++ret->cnt1;
550 else if (p->indel == ret->indel2) ++ret->cnt2;
551 else ++ret->cnt_anti;
554 int tmp, seq_err = 0;
556 tmp = max1_i - max2_i;
557 if (tmp < 0) tmp = -tmp;
558 for (j = 0; j < tmp + 1; ++j) x *= INDEL_EXT_DEP;
559 seq_err = mi->q_indel * (1.0 - x) / (1.0 - INDEL_EXT_DEP);
560 ret->gl[0] = ret->gl[1] = 0;
561 for (j = 0; j < n; ++j) {
562 int s1 = pscore[max1_i*n + j], s2 = pscore[max2_i*n + j];
563 //printf("%d, %d, %d, %d, %d\n", pl[j].b->core.pos+1, max1_i, max2_i, s1, s2);
564 if (s1 > s2) ret->gl[0] += s1 - s2 < seq_err? s1 - s2 : seq_err;
565 else ret->gl[1] += s2 - s1 < seq_err? s2 - s1 : seq_err;
568 // write cnt_ref and cnt_ambi
569 if (max1_i != 0 && max2_i != 0) {
570 for (j = 0; j < n; ++j) {
571 int diff1 = score[j] - score[max1_i * n + j];
572 int diff2 = score[j] - score[max2_i * n + j];
573 if (diff1 > 0 && diff2 > 0) ++ret->cnt_ref;
574 else if (diff1 == 0 || diff2 == 0) ++ret->cnt_ambi;
578 free(score); free(pscore); free(ref2); free(rs); free(inscns);
581 int q[3], qr_indel = (int)(-4.343 * log(mi->r_indel) + 0.5);
582 int min1, min2, min1_i;
583 q[0] = ret->gl[0] + (ret->s[0][0] != '*'? 0 : 0) * qr_indel;
584 q[1] = ret->gl[1] + (ret->s[1][0] != '*'? 0 : 0) * qr_indel;
585 q[2] = n * 3 + (ret->s[0][0] == '*' || ret->s[1][0] == '*'? 1 : 1) * qr_indel;
586 min1 = min2 = 0x7fffffff; min1_i = -1;
587 for (i = 0; i < 3; ++i) {
589 min2 = min1; min1 = q[i]; min1_i = i;
590 } else if (q[i] < min2) min2 = q[i];
593 ret->q_cns = min2 - min1;
595 if (ret->gt < 2) ret->q_ref = (ret->s[ret->gt][0] == '*')? 0 : q[1-ret->gt] - q[ret->gt] - qr_indel - 3;
596 else ret->q_ref = (ret->s[0][0] == '*')? q[0] - q[2] : q[1] - q[2];
597 if (ret->q_ref < 0) ret->q_ref = 0;