-
-#define END_SKIP 4 // must be larger than B2B_MAX_MNP
-#define MIN_MNP_FLANK_BAQ 20
-
-// the following routine is modified from bcf_call_gap_prep()
-int bcf_call_mnp_prep(int n, int *n_plp, bam_pileup1_t **plp, int pos, bcf_callaux_t *bca, const char *ref)
-{
- extern void ks_introsort_uint32_t(int, uint32_t*);
- int i, s, j, k, t, n_types, *types, max_rd_len, left, right, *score1;
- int N, K, ref_seq, ref_type, n_alt;
- char *ref2, *query;
- uint8_t *_seqQ;
- if (ref == 0 || bca == 0) return -1;
- if (pos < bca->last_mnp_pos + B2B_MNP_WIN) return -2; // to avoid calling a TNP multiple times
- if (pos < END_SKIP || ref[pos] == 0 || ref[pos+1] == 0) return -2; // end of the reference
- { // determine if there is an MNP
- int r[2];
- uint8_t *tt;
- r[0] = bam_nt16_table[(int)ref[pos]];
- r[1] = bam_nt16_table[(int)ref[pos+1]];
- for (s = 0; s < n; ++s) {
- for (i = 0; i < n_plp[s]; ++i) {
- bam_pileup1_t *p = plp[s] + i;
- int left, rght;
- if (p->qpos < END_SKIP || p->qpos >= p->b->core.l_qseq - END_SKIP) continue;
- tt = bam1_seq(p->b);
- if (bam1_seqi(tt, p->qpos) == r[0] || bam1_seqi(tt, p->qpos+1) == r[1]) continue; // no MNP
- tt = bam1_qual(p->b);
- for (left = 0, k = p->qpos - END_SKIP; k <= p->qpos; ++k)
- left = left > tt[k]? left : tt[k];
- for (rght = 0, k = p->qpos; k < p->b->core.l_qseq - END_SKIP; ++k)
- rght = rght > tt[k]? left : tt[k];
- if (left >= MIN_MNP_FLANK_BAQ && rght >= MIN_MNP_FLANK_BAQ) break; // bracketed by good bases
- }
- if (i != n_plp[s]) break;
- }
- if (s == n) return -1; // there is no MNP at this position.
- }
- for (s = N = 0; s < n; ++s) N += n_plp[s]; // N is the total number of reads
- { // construct the MNP consensus
- uint8_t *tt;
- uint32_t *aux, x, *cnt;
- int m;
- m = max_rd_len = 0;
- aux = calloc(N + 1, 4);
- for (i = 0, x = 0; i < B2B_MAX_MNP; ++i)
- x |= bam_nt16_nt4_table[bam_nt16_table[(int)ref[pos+i]]] << 2*i;
- ref_seq = x;
- bca->indelreg = 0;
- for (s = 0; s < n; ++s) {
- for (i = 0; i < n_plp[s]; ++i) {
- bam_pileup1_t *p = plp[s] + i;
- int stop;
- j = bam_cigar2qlen(&p->b->core, bam1_cigar(p->b));
- if (j > max_rd_len) max_rd_len = j;
- if (p->qpos < END_SKIP || p->qpos >= p->b->core.l_qseq - END_SKIP) continue;
- tt = bam1_seq(p->b);
- for (k = j = stop = 0, x = 0; k < B2B_MAX_MNP; ++k) {
- int c = bam_nt16_nt4_table[bam1_seqi(tt, p->qpos + k)];
- if (c > 3) break;
- if (c != (ref_seq>>k*2&3) && !stop) ++j;
- else stop = 1;
- x |= c << 2*k;
- }
-// if (j >= 2 && k == B2B_MAX_MNP) fprintf(stderr, "* %c%c%c%c, %d\n", "ACGT"[x&3], "ACGT"[x>>2&3], "ACGT"[x>>4&3], "ACGT"[x>>6&3], j);
- if (k == B2B_MAX_MNP && j >= 2) aux[m++] = x;
- }
- }
- ks_introsort(uint32_t, m, aux);
- // squeeze out indentical types
- for (i = 1, n_types = 1; i < m; ++i)
- if (aux[i] != aux[i-1]) ++n_types;
- if (n_types == 1) { // no indels
- free(aux); return -1;
- }
- // count reads for each type
- cnt = alloca(n_types * 4);
- cnt[0] = 1<<8 | aux[0];
- for (i = 1, t = 0; i < m; ++i) {
- if (aux[i] != aux[i-1]) {
- ++t;
- cnt[t] = 1<<8 | aux[i];
- } else cnt[t] += 1<<8;
- }
- free(aux);
- // collect types
- ks_introsort(uint32_t, n_types, cnt);
- types = (int*)calloc(2, sizeof(int));
- types[0] = ref_seq; types[1] = cnt[n_types-1]&0xff;
- ref_type = 0; n_types = 2;
- // calculate MNP length
- for (i = B2B_MAX_MNP - 1, k = 0; i >= 0; --i) {
- int c = types[0] >> 2*i & 3;
- for (t = 1; t < n_types; ++t)
- if ((types[t] >> 2*i & 3) != c) break;
- if (t == n_types) ++k;
- else break;
- }
- bca->indelreg = B2B_MAX_MNP - k;
- x = (1<<2*bca->indelreg) - 1;
- for (t = 0; t < n_types; ++t) types[t] &= x;
- ref_seq &= x;
-// x = types[1]; fprintf(stderr, "%d, %d, %d, %c%c%c%c\n", pos, n_types, bca->indelreg, "ACGT"[x&3], "ACGT"[x>>2&3], "ACGT"[x>>4&3], "ACGT"[x>>6&3]);
- }
- { // calculate left and right boundary
- left = pos > INDEL_WINDOW_SIZE? pos - INDEL_WINDOW_SIZE : 0;
- right = pos + INDEL_WINDOW_SIZE;
- // in case the alignments stand out the reference
- for (i = pos; i < right; ++i)
- if (ref[i] == 0) break;
- right = i;
- }
- { // find the highest base quality
- _seqQ = calloc(N, 1);
- for (s = K = 0; s < n; ++s) {
- for (i = 0; i < n_plp[s]; ++i, ++K) {
- bam_pileup1_t *p = plp[s] + i;
- uint8_t *qq, *bq;
- int max = 0;
- qq = bam1_qual(p->b);
- bq = (uint8_t*)bam_aux_get(p->b, "ZQ");
- if (bq) ++bq;
- for (j = p->qpos; j < p->b->core.l_qseq && j < p->qpos + bca->indelreg; ++j) {
- int q = bq? qq[j] + (bq[j] - 64) : qq[j];
- max = max > q? max : q;
- }
- _seqQ[K] = max;
- }
- }
- }
- // compute the likelihood given each type of indel for each read
- ref2 = calloc(right - left + 2, 1);
- query = calloc(right - left + max_rd_len + 2, 1);
- score1 = calloc(N * n_types, sizeof(int));
- for (t = 0; t < n_types; ++t) {
- int l;
- kpa_par_t apf1 = { 1e-4, 1e-2, 10 };
- apf1.bw = abs(types[t]) + 3;
- // write ref2
- for (k = 0, j = left; j < pos; ++j)
- ref2[k++] = bam_nt16_nt4_table[bam_nt16_table[(int)ref[j]]];
- for (i = 0; i < bca->indelreg; ++i, ++j)
- ref2[k++] = types[t]>>2*i&3;
- 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);
- // 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);
- // 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,
- (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] = sc<<8 | l;
- free(qq);
- }
-/*
- for (l = 0; l < tend - tbeg; ++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], 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;
- if ((sc[0]&0x3f) == ref_type) {
- indelQ = (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;
- indelQ = (sc[t]>>14) - (sc[0]>>14);
- }
- seqQ = _seqQ[K];
- tmp = sc[0]>>6 & 0xff;
- indelQ = tmp > 111? 0 : (int)((1. - tmp/111.) * indelQ + .499); // reduce indelQ
- if (indelQ > seqQ) indelQ = seqQ; // this is different from indel calling!
- p->aux = (sc[0]&0x3f)<<16 | seqQ<<8 | indelQ;
- sumq[sc[0]&0x3f] += indelQ < seqQ? indelQ : seqQ;
- }
- }
- // determine bca->indel_types[] and bca->inscns
- 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];
- // 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
- free(types); free(_seqQ);
- return n_alt > 0? 0 : -1;
-}