X-Git-Url: https://git.donarmstrong.com/?p=samtools.git;a=blobdiff_plain;f=bam2bcf.c;h=a51a406f249c05834b0d8e20f49403d5ed29a3b6;hp=ab5bfcd20d4a6233109821846842d9996aab93a9;hb=307c147168f7154e3755712797078c513e0b242a;hpb=df7cadac556f302bdd0bc48c235c2b61b699dfe1 diff --git a/bam2bcf.c b/bam2bcf.c index ab5bfcd..a51a406 100644 --- a/bam2bcf.c +++ b/bam2bcf.c @@ -3,6 +3,7 @@ #include "bam.h" #include "kstring.h" #include "bam2bcf.h" +#include "errmod.h" #include "bcftools/bcf.h" extern void ks_introsort_uint32_t(size_t n, uint32_t a[]); @@ -10,6 +11,7 @@ extern void ks_introsort_uint32_t(size_t n, uint32_t a[]); #define CALL_ETA 0.03f #define CALL_MAX 256 #define CALL_DEFTHETA 0.83f +#define DEF_MAPQ 20 #define CAP_DIST 25 @@ -19,11 +21,12 @@ bcf_callaux_t *bcf_call_init(double theta, int min_baseQ) if (theta <= 0.) theta = CALL_DEFTHETA; bca = calloc(1, sizeof(bcf_callaux_t)); bca->capQ = 60; - bca->openQ = 40; - bca->extQ = 20; - bca->tandemQ = 100; + bca->openQ = 40; bca->extQ = 20; bca->tandemQ = 100; bca->min_baseQ = min_baseQ; bca->e = errmod_init(1. - theta); + bca->min_frac = 0.002; + bca->min_support = 1; + bca->per_sample_flt = 0; return bca; } @@ -31,16 +34,20 @@ void bcf_call_destroy(bcf_callaux_t *bca) { if (bca == 0) return; errmod_destroy(bca->e); - free(bca->bases); free(bca); + free(bca->bases); free(bca->inscns); free(bca); } - -int bcf_call_glfgen(int _n, const bam_pileup1_t *pl, int ref_base /*4-bit*/, bcf_callaux_t *bca, bcf_callret1_t *r) +/* ref_base is the 4-bit representation of the reference base. It is + * negative if we are looking at an indel. */ +int bcf_call_glfgen(int _n, const bam_pileup1_t *pl, int ref_base, bcf_callaux_t *bca, bcf_callret1_t *r) { - int i, n, ref4; + static int *var_pos = NULL, nvar_pos = 0; + int i, n, ref4, is_indel, ori_depth = 0; memset(r, 0, sizeof(bcf_callret1_t)); - ref4 = bam_nt16_nt4_table[ref_base]; + if (ref_base >= 0) { + ref4 = bam_nt16_nt4_table[ref_base]; + is_indel = 0; + } else ref4 = 4, is_indel = 1; if (_n == 0) return -1; - // enlarge the bases array if necessary if (bca->max_bases < _n) { bca->max_bases = _n; @@ -48,166 +55,148 @@ int bcf_call_glfgen(int _n, const bam_pileup1_t *pl, int ref_base /*4-bit*/, bcf bca->bases = (uint16_t*)realloc(bca->bases, 2 * bca->max_bases); } // fill the bases array - memset(r, 0, sizeof(bcf_callret1_t)); - for (i = n = 0; i < _n; ++i) { + for (i = n = r->n_supp = 0; i < _n; ++i) { const bam_pileup1_t *p = pl + i; - int q, b, mapQ, baseQ, is_diff, min_dist; + int q, b, mapQ, baseQ, is_diff, min_dist, seqQ; // set base if (p->is_del || p->is_refskip || (p->b->core.flag&BAM_FUNMAP)) continue; - baseQ = q = (int)bam1_qual(p->b)[p->qpos]; // base quality + ++ori_depth; + baseQ = q = is_indel? p->aux&0xff : (int)bam1_qual(p->b)[p->qpos]; // base/indel quality + seqQ = is_indel? (p->aux>>8&0xff) : 99; if (q < bca->min_baseQ) continue; - mapQ = p->b->core.qual < bca->capQ? p->b->core.qual : bca->capQ; + if (q > seqQ) q = seqQ; + mapQ = p->b->core.qual < 255? p->b->core.qual : DEF_MAPQ; // special case for mapQ==255 + mapQ = mapQ < bca->capQ? mapQ : bca->capQ; if (q > mapQ) q = mapQ; if (q > 63) q = 63; if (q < 4) q = 4; - b = bam1_seqi(bam1_seq(p->b), p->qpos); // base - b = bam_nt16_nt4_table[b? b : ref_base]; // b is the 2-bit base + if (!is_indel) { + b = bam1_seqi(bam1_seq(p->b), p->qpos); // base + b = bam_nt16_nt4_table[b? b : ref_base]; // b is the 2-bit base + is_diff = (ref4 < 4 && b == ref4)? 0 : 1; + } else { + b = p->aux>>16&0x3f; + is_diff = (b != 0); + } + if (is_diff) ++r->n_supp; bca->bases[n++] = q<<5 | (int)bam1_strand(p->b)<<4 | b; // collect annotations - r->qsum[b] += q; - is_diff = (ref4 < 4 && b == ref4)? 0 : 1; + if (b < 4) r->qsum[b] += q; ++r->anno[0<<2|is_diff<<1|bam1_strand(p->b)]; min_dist = p->b->core.l_qseq - 1 - p->qpos; if (min_dist > p->qpos) min_dist = p->qpos; if (min_dist > CAP_DIST) min_dist = CAP_DIST; r->anno[1<<2|is_diff<<1|0] += baseQ; - r->anno[1<<2|is_diff<<1|1] += baseQ * baseQ; + r->anno[1<<2|is_diff<<1|1] += baseQ * baseQ; // FIXME: signed int is not enough for thousands of samples r->anno[2<<2|is_diff<<1|0] += mapQ; - r->anno[2<<2|is_diff<<1|1] += mapQ * mapQ; + r->anno[2<<2|is_diff<<1|1] += mapQ * mapQ; // FIXME: signed int is not enough for thousands of samples r->anno[3<<2|is_diff<<1|0] += min_dist; r->anno[3<<2|is_diff<<1|1] += min_dist * min_dist; } - r->depth = n; + r->depth = n; r->ori_depth = ori_depth; // glfgen errmod_cal(bca->e, n, 5, bca->bases, r->p); + + // Calculate the Variant Distance Bias (make it optional?) + if ( nvar_pos < _n ) { + nvar_pos = _n; + var_pos = realloc(var_pos,sizeof(int)*nvar_pos); + } + int alt_dp=0, read_len=0; + for (i=0; i<_n; i++) { + const bam_pileup1_t *p = pl + i; + if ( bam1_seqi(bam1_seq(p->b),p->qpos) == ref_base ) + continue; + + var_pos[alt_dp] = p->qpos; + if ( (bam1_cigar(p->b)[0]&BAM_CIGAR_MASK)==4 ) + var_pos[alt_dp] -= bam1_cigar(p->b)[0]>>BAM_CIGAR_SHIFT; + + alt_dp++; + read_len += p->b->core.l_qseq; + } + float mvd=0; + int j; + n=0; + for (i=0; imvd[0] = n ? mvd/n : 0; + r->mvd[1] = alt_dp; + r->mvd[2] = alt_dp ? read_len/alt_dp : 0; + return r->depth; } -int bcf_call_glfgen_gap(int pos, int _n, const bam_pileup1_t *pl, bcf_callaux_t *bca, bcf_callret1_t *r) + +void calc_vdb(int n, const bcf_callret1_t *calls, bcf_call_t *call) { - int i, n, n_ins, n_del; - memset(r, 0, sizeof(bcf_callret1_t)); - if (_n == 0) return -1; + // Variant distance bias. Samples merged by means of DP-weighted average. - // enlarge the bases array if necessary - if (bca->max_bases < _n) { - bca->max_bases = _n; - kroundup32(bca->max_bases); - bca->bases = (uint16_t*)realloc(bca->bases, 2 * bca->max_bases); - } - // fill the bases array - memset(r, 0, sizeof(bcf_callret1_t)); - r->indelreg = 10000; - n_ins = n_del = 0; - for (i = n = 0; i < _n; ++i) { - const bam_pileup1_t *p = pl + i; - int q, b, mapQ, indelQ, is_diff, min_dist; - if (p->is_del || p->is_refskip || (p->b->core.flag&BAM_FUNMAP)) continue; - { // compute indel (base) quality - // this can be made more efficient, but realignment is the bottleneck anyway - int j, k, x, y, op, len = 0, max_left, max_rght, seqQ, indelreg; - bam1_core_t *c = &p->b->core; - uint32_t *cigar = bam1_cigar(p->b); - uint8_t *qual = bam1_qual(p->b); - for (k = y = 0, x = c->pos; k < c->n_cigar && y <= p->qpos; ++k) { - op = cigar[k]&0xf; - len = cigar[k]>>4; - if (op == BAM_CMATCH) { - if (pos > x && pos < x + len) break; - x += len; y += len; - } else if (op == BAM_CINS || op == BAM_CSOFT_CLIP) y += len; - else if (op == BAM_CDEL || op == BAM_CREF_SKIP) x += len; - } - if (k == c->n_cigar) continue; // this actually should not happen - max_left = max_rght = 0; indelreg = 0; - if (pos == x + len - 1 && k+2 < c->n_cigar && ((cigar[k+1]&0xf) == BAM_CINS || (cigar[k+1]&0xf) == BAM_CDEL) - && (cigar[k+2]&0xf) == BAM_CMATCH) - { - for (j = y; j < y + len; ++j) - if (max_left < qual[j]) max_left = qual[j]; - if ((cigar[k+1]&0xf) == BAM_CINS) y += cigar[k+1]>>4; - else x += cigar[k+1]>>4; - op = cigar[k+2]&0xf; len = cigar[k+2]>>4; - for (j = y; j < y + len; ++j) { - if (max_rght < qual[j]) max_rght = qual[j]; - if (qual[j] > BAM2BCF_INDELREG_THRES && indelreg == 0) - indelreg = j - y + 1; - } - if (r->indelreg > indelreg) r->indelreg = indelreg; - } else { - for (j = y; j <= p->qpos; ++j) - if (max_left < qual[j]) max_left = qual[j]; - for (j = p->qpos + 1; j < y + len; ++j) - if (max_rght < qual[j]) max_rght = qual[j]; - - } - indelQ = max_left < max_rght? max_left : max_rght; - // estimate the sequencing error rate - seqQ = bca->openQ; - if (p->indel != 0) seqQ += bca->extQ * (abs(p->indel) - 1); // FIXME: better to model homopolymer - if (p->indel > 0) { - ++n_ins; r->ins_len += p->indel; - } else if (p->indel < 0) { - ++n_del; r->del_len += -p->indel; - } - if (p->indel != 0) { // a different model for tandem repeats - uint8_t *seq = bam1_seq(p->b); - int tandemQ, qb = bam1_seqi(seq, p->qpos), l; - for (j = p->qpos + 1; j < c->l_qseq; ++j) - if (qb != bam1_seqi(seq, j)) break; - l = j; - for (j = (int)p->qpos - 1; j >= 0; --j) - if (qb != bam1_seqi(seq, j)) break; - l = l - (j + 1); - tandemQ = (int)((double)(abs(p->indel)) / l * bca->tandemQ + .499); - if (seqQ > tandemQ) seqQ = tandemQ; - } -// fprintf(stderr, "%s\t%d\t%d\t%d\t%d\t%d\t%d\n", bam1_qname(p->b), pos+1, p->indel, indelQ, seqQ, max_left, max_rght); - if (indelQ > seqQ) indelQ = seqQ; - q = indelQ; - } - if (q < bca->min_baseQ) continue; - mapQ = p->b->core.qual < bca->capQ? p->b->core.qual : bca->capQ; - if (q > mapQ) q = mapQ; - if (q > 63) q = 63; - if (q < 4) q = 4; - b = p->indel? 1 : 0; - bca->bases[n++] = q<<5 | (int)bam1_strand(p->b)<<4 | b; - // collect annotations - r->qsum[b] += q; - is_diff = b; - ++r->anno[0<<2|is_diff<<1|bam1_strand(p->b)]; - min_dist = p->b->core.l_qseq - 1 - p->qpos; - if (min_dist > p->qpos) min_dist = p->qpos; - if (min_dist > CAP_DIST) min_dist = CAP_DIST; - r->anno[1<<2|is_diff<<1|0] += indelQ; - r->anno[1<<2|is_diff<<1|1] += indelQ * indelQ; - r->anno[2<<2|is_diff<<1|0] += mapQ; - r->anno[2<<2|is_diff<<1|1] += mapQ * mapQ; - r->anno[3<<2|is_diff<<1|0] += min_dist; - r->anno[3<<2|is_diff<<1|1] += min_dist * min_dist; - } - r->depth = n; - r->ins_len = n_ins? r->ins_len / n_ins : 0; - r->del_len = n_del? r->del_len / n_del : 0; - if (r->indelreg >= 10000) r->indelreg = 0; - // glfgen - errmod_cal(bca->e, n, 2, bca->bases, r->p); - return r->depth; + float weight=0, tot_prob=0; + + int i; + for (i=0; i2*mu ? 0 : sin(mvd*3.14/2/mu) / (4*mu/3.14); + } + else + { + // Scaled gaussian curve, crude approximation, but behaves well. Using fixed depth for bigger depths. + if ( dp>5 ) + dp = 5; + float sigma2 = (read_len/1.9/(dp+1)) * (read_len/1.9/(dp+1)); + float norm = 1.125*sqrt(2*3.14*sigma2); + float mu = read_len/2.9; + if ( mvd < mu ) + prob = exp(-(mvd-mu)*(mvd-mu)/2/sigma2)/norm; + else + prob = exp(-(mvd-mu)*(mvd-mu)/3.125/sigma2)/norm; + } + + //fprintf(stderr,"dp=%d mvd=%d read_len=%d -> prob=%f\n", dp,mvd,read_len,prob); + tot_prob += prob*dp; + weight += dp; + } + tot_prob = weight ? tot_prob/weight : 1; + //fprintf(stderr,"prob=%f\n", tot_prob); + call->vdb = tot_prob; } int bcf_call_combine(int n, const bcf_callret1_t *calls, int ref_base /*4-bit*/, bcf_call_t *call) { int ref4, i, j, qsum[4]; int64_t tmp; - call->ori_ref = ref4 = bam_nt16_nt4_table[ref_base]; - call->ins_len = call->del_len = 0; call->indelreg = 0; - if (ref4 > 4) ref4 = 4; + if (ref_base >= 0) { + call->ori_ref = ref4 = bam_nt16_nt4_table[ref_base]; + if (ref4 > 4) ref4 = 4; + } else call->ori_ref = -1, ref4 = 0; // calculate qsum memset(qsum, 0, 4 * sizeof(int)); for (i = 0; i < n; ++i) for (j = 0; j < 4; ++j) qsum[j] += calls[i].qsum[j]; + int qsum_tot=0; + for (j=0; j<4; j++) { qsum_tot += qsum[j]; call->qsum[j] = 0; } for (j = 0; j < 4; ++j) qsum[j] = qsum[j] << 2 | j; // find the top 2 alleles for (i = 1; i < 4; ++i) // insertion sort @@ -219,13 +208,24 @@ int bcf_call_combine(int n, const bcf_callret1_t *calls, int ref_base /*4-bit*/, call->a[0] = ref4; for (i = 3, j = 1; i >= 0; --i) { if ((qsum[i]&3) != ref4) { - if (qsum[i]>>2 != 0) call->a[j++] = qsum[i]&3; + if (qsum[i]>>2 != 0) + { + if ( j<4 ) call->qsum[j] = (float)(qsum[i]>>2)/qsum_tot; // ref N can make j>=4 + call->a[j++] = qsum[i]&3; + } else break; } + else + call->qsum[0] = (float)(qsum[i]>>2)/qsum_tot; + } + if (ref_base >= 0) { // for SNPs, find the "unseen" base + if (((ref4 < 4 && j < 4) || (ref4 == 4 && j < 5)) && i >= 0) + call->unseen = j, call->a[j++] = qsum[i]&3; + call->n_alleles = j; + } else { + call->n_alleles = j; + if (call->n_alleles == 1) return -1; // no reliable supporting read. stop doing anything } - if (((ref4 < 4 && j < 4) || (ref4 == 4 && j < 5)) && i >= 0) - call->unseen = j, call->a[j++] = qsum[i]&3; - call->n_alleles = j; // set the PL array if (call->n < n) { call->n = n; @@ -237,8 +237,8 @@ int bcf_call_combine(int n, const bcf_callret1_t *calls, int ref_base /*4-bit*/, x = call->n_alleles * (call->n_alleles + 1) / 2; // get the possible genotypes for (i = z = 0; i < call->n_alleles; ++i) - for (j = i; j < call->n_alleles; ++j) - g[z++] = call->a[i] * 5 + call->a[j]; + for (j = 0; j <= i; ++j) + g[z++] = call->a[j] * 5 + call->a[i]; for (i = 0; i < n; ++i) { uint8_t *PL = call->PL + x * i; const bcf_callret1_t *r = calls + i; @@ -253,126 +253,96 @@ int bcf_call_combine(int n, const bcf_callret1_t *calls, int ref_base /*4-bit*/, PL[j] = y; } } +// if (ref_base < 0) fprintf(stderr, "%d,%d,%f,%d\n", call->n_alleles, x, sum_min, call->unseen); call->shift = (int)(sum_min + .499); } // combine annotations memset(call->anno, 0, 16 * sizeof(int)); - for (i = call->depth = 0, tmp = 0; i < n; ++i) { + for (i = call->depth = call->ori_depth = 0, tmp = 0; i < n; ++i) { call->depth += calls[i].depth; + call->ori_depth += calls[i].ori_depth; for (j = 0; j < 16; ++j) call->anno[j] += calls[i].anno[j]; } - return 0; -} -int bcf_call_combine_gap(int n, const bcf_callret1_t *calls, bcf_call_t *call) -{ - int i, j, n_ins, n_del; - // combine annotations - call->ori_ref = 4; - memset(call->anno, 0, 16 * sizeof(int)); - call->ins_len = call->del_len = 0; call->indelreg = 10000; - for (i = call->depth = 0, n_ins = n_del = 0; i < n; ++i) { - const bcf_callret1_t *r = calls + i; - if (r->depth > 0) { - call->depth += r->depth; - if (r->ins_len > 0) { - call->ins_len += r->ins_len; - ++n_ins; - } - if (r->del_len > 0) { - call->del_len += r->del_len; - ++n_del; - } - if (r->indelreg > 0 && call->indelreg > r->indelreg) - call->indelreg = r->indelreg; - for (j = 0; j < 16; ++j) call->anno[j] += r->anno[j]; - } - } - if (call->depth == 0) return 0; // no indels - call->ins_len = n_ins? call->ins_len / n_ins : 0; - call->del_len = n_del? call->del_len / n_del : 0; - // - for (i = 0; i < 5; ++i) call->a[i] = -1; - call->a[0] = 0; call->a[1] = 1; - call->unseen = -1; - call->n_alleles = 2; - // set the PL array - if (call->n < n) { - call->n = n; - call->PL = realloc(call->PL, 15 * n); - } - { - int g[3]; - double sum_min = 0.; - g[0] = 0; g[1] = 1; g[2] = 3; - for (i = 0; i < n; ++i) { - uint8_t *PL = call->PL + 3 * i; - const bcf_callret1_t *r = calls + i; - float min = 1e37; - for (j = 0; j < 3; ++j) - if (min > r->p[g[j]]) min = r->p[g[j]]; - sum_min += min; - for (j = 0; j < 3; ++j) { - int y; - y = (int)(r->p[g[j]] - min + .499); - if (y > 255) y = 255; - PL[j] = y; - } - } - call->shift = (int)(sum_min + .499); - } + calc_vdb(n, calls, call); + return 0; } -int bcf_call2bcf(int tid, int pos, bcf_call_t *bc, bcf1_t *b, bcf_callret1_t *bcr, int is_SP) +int bcf_call2bcf(int tid, int pos, bcf_call_t *bc, bcf1_t *b, bcf_callret1_t *bcr, int fmt_flag, + const bcf_callaux_t *bca, const char *ref) { extern double kt_fisher_exact(int n11, int n12, int n21, int n22, double *_left, double *_right, double *two); kstring_t s; - int i; + int i, j; b->n_smpl = bc->n; b->tid = tid; b->pos = pos; b->qual = 0; s.s = b->str; s.m = b->m_str; s.l = 0; kputc('\0', &s); - if (bc->ins_len > 0 || bc->del_len > 0) { // an indel - for (i = 0; i < bc->indelreg; ++i) kputc('N', &s); + if (bc->ori_ref < 0) { // an indel + // write REF + kputc(ref[pos], &s); + for (j = 0; j < bca->indelreg; ++j) kputc(ref[pos+1+j], &s); + kputc('\0', &s); + // write ALT + kputc(ref[pos], &s); + for (i = 1; i < 4; ++i) { + if (bc->a[i] < 0) break; + if (i > 1) { + kputc(',', &s); kputc(ref[pos], &s); + } + if (bca->indel_types[bc->a[i]] < 0) { // deletion + for (j = -bca->indel_types[bc->a[i]]; j < bca->indelreg; ++j) + kputc(ref[pos+1+j], &s); + } else { // insertion; cannot be a reference unless a bug + char *inscns = &bca->inscns[bc->a[i] * bca->maxins]; + for (j = 0; j < bca->indel_types[bc->a[i]]; ++j) + kputc("ACGTN"[(int)inscns[j]], &s); + for (j = 0; j < bca->indelreg; ++j) kputc(ref[pos+1+j], &s); + } + } kputc('\0', &s); - if (bc->ins_len > 0 && bc->del_len > 0) kputs("", &s); - else if (bc->ins_len > 0) kputs("", &s); - else if (bc->del_len > 0) kputs("", &s); - } else { // SNP + } else { // a SNP kputc("ACGTN"[bc->ori_ref], &s); kputc('\0', &s); for (i = 1; i < 5; ++i) { if (bc->a[i] < 0) break; if (i > 1) kputc(',', &s); kputc(bc->unseen == i? 'X' : "ACGT"[bc->a[i]], &s); } + kputc('\0', &s); } kputc('\0', &s); - kputc('\0', &s); // INFO - kputs("I16=", &s); + if (bc->ori_ref < 0) ksprintf(&s,"INDEL;IS=%d,%f;", bca->max_support, bca->max_frac); + kputs("DP=", &s); kputw(bc->ori_depth, &s); kputs(";I16=", &s); for (i = 0; i < 16; ++i) { if (i) kputc(',', &s); kputw(bc->anno[i], &s); } + ksprintf(&s,";QS=%f,%f,%f,%f", bc->qsum[0],bc->qsum[1],bc->qsum[2],bc->qsum[3]); + if (bc->vdb != 1) + ksprintf(&s, ";VDB=%.4f", bc->vdb); kputc('\0', &s); // FMT kputs("PL", &s); - if (bcr) { - kputs(":DP", &s); - if (is_SP) kputs(":SP", &s); + if (bcr && fmt_flag) { + if (fmt_flag & B2B_FMT_DP) kputs(":DP", &s); + if (fmt_flag & B2B_FMT_DV) kputs(":DV", &s); + if (fmt_flag & B2B_FMT_SP) kputs(":SP", &s); } kputc('\0', &s); b->m_str = s.m; b->str = s.s; b->l_str = s.l; bcf_sync(b); memcpy(b->gi[0].data, bc->PL, b->gi[0].len * bc->n); - if (bcr) { - uint16_t *dp = (uint16_t*)b->gi[1].data; - uint8_t *sp = is_SP? b->gi[2].data : 0; + if (bcr && fmt_flag) { + uint16_t *dp = (fmt_flag & B2B_FMT_DP)? b->gi[1].data : 0; + uint16_t *dv = (fmt_flag & B2B_FMT_DV)? b->gi[1 + ((fmt_flag & B2B_FMT_DP) != 0)].data : 0; + int32_t *sp = (fmt_flag & B2B_FMT_SP)? b->gi[1 + ((fmt_flag & B2B_FMT_DP) != 0) + ((fmt_flag & B2B_FMT_DV) != 0)].data : 0; for (i = 0; i < bc->n; ++i) { bcf_callret1_t *p = bcr + i; - dp[i] = p->depth < 0xffff? p->depth : 0xffff; - if (is_SP) { + if (dp) dp[i] = p->depth < 0xffff? p->depth : 0xffff; + if (dv) dv[i] = p->n_supp < 0xffff? p->n_supp : 0xffff; + if (sp) { if (p->anno[0] + p->anno[1] < 2 || p->anno[2] + p->anno[3] < 2 || p->anno[0] + p->anno[2] < 2 || p->anno[1] + p->anno[3] < 2) {