X-Git-Url: https://git.donarmstrong.com/?a=blobdiff_plain;f=bam2bcf.c;h=b4f7fa6480a767d118f149ab1934e9c2ac781d2f;hb=125ac895854cf760a04192a257a4279f2c541164;hp=fe447e6aafb610fb984d3e07c9c507a5572630ca;hpb=4c8c9dfc1e3b3b066a62a703fd3ba04db6ad5a45;p=samtools.git diff --git a/bam2bcf.c b/bam2bcf.c index fe447e6..b4f7fa6 100644 --- a/bam2bcf.c +++ b/bam2bcf.c @@ -1,5 +1,6 @@ #include #include +#include #include "bam.h" #include "kstring.h" #include "bam2bcf.h" @@ -11,6 +12,7 @@ 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 @@ -22,21 +24,55 @@ bcf_callaux_t *bcf_call_init(double theta, int min_baseQ) bca->capQ = 60; bca->openQ = 40; bca->extQ = 20; bca->tandemQ = 100; bca->min_baseQ = min_baseQ; - bca->last_mnp_pos = -100; bca->e = errmod_init(1. - theta); + bca->min_frac = 0.002; + bca->min_support = 1; + bca->per_sample_flt = 0; return bca; } + +int dist_from_end(const bam_pileup1_t *p) +{ + int icig, n_tot_bases = 0, iread = 0, edist = p->qpos + 1; + for (icig=0; icigb->core.n_cigar; icig++) + { + // Conversion from uint32_t to MIDNSHP + // 0123456 + // MIDNSHP + int cig = bam1_cigar(p->b)[icig] & BAM_CIGAR_MASK; + int ncig = bam1_cigar(p->b)[icig] >> BAM_CIGAR_SHIFT; + if ( cig==0 ) + { + n_tot_bases += ncig; + iread += ncig; + } + else if ( cig==1 ) + { + n_tot_bases += ncig; + iread += ncig; + } + else if ( cig==4 ) + { + iread += ncig; + if ( iread<=p->qpos ) edist -= ncig; + } + } + // distance from either end + if ( edist > n_tot_bases/2. ) edist = n_tot_bases - edist + 1; + if ( edist<0 ) { fprintf(stderr,"uh: %d %d -> %d (%d)\n", p->qpos,n_tot_bases,edist,p->b->core.pos+1); exit(1); } + return edist; +} + void bcf_call_destroy(bcf_callaux_t *bca) { if (bca == 0) return; errmod_destroy(bca->e); + if (bca->npos) { free(bca->ref_pos); free(bca->alt_pos); bca->npos = 0; } free(bca->bases); free(bca->inscns); free(bca); } -/* Compute genotype likelihood by combining likelihood of each - * read. ref_base is the 4-bit representation of the reference base. It - * is negative if we are looking at an indel or an MNP. Indel and MNP - * are indistinguishable in this function. */ +/* 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, is_indel, ori_depth = 0; @@ -53,8 +89,8 @@ int bcf_call_glfgen(int _n, const bam_pileup1_t *pl, int ref_base, bcf_callaux_t 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) { + bca->read_len = 0; + 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, seqQ; // set base @@ -64,7 +100,8 @@ int bcf_call_glfgen(int _n, const bam_pileup1_t *pl, int ref_base, bcf_callaux_t seqQ = is_indel? (p->aux>>8&0xff) : 99; if (q < bca->min_baseQ) continue; if (q > seqQ) q = seqQ; - mapQ = p->b->core.qual < bca->capQ? p->b->core.qual : bca->capQ; + 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; @@ -76,41 +113,187 @@ int bcf_call_glfgen(int _n, const bam_pileup1_t *pl, int ref_base, bcf_callaux_t 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; + 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; + + // collect read positions for ReadPosBias + int epos = dist_from_end(p); + int npos = p->b->core.l_qseq; + if ( bca->npos < npos ) + { + bca->ref_pos = realloc(bca->ref_pos, sizeof(int)*npos); + bca->alt_pos = realloc(bca->alt_pos, sizeof(int)*npos); + int j; + for (j=bca->npos; jref_pos[j] = 0; + for (j=bca->npos; jalt_pos[j] = 0; + bca->npos = npos; + } + if ( bam1_seqi(bam1_seq(p->b),p->qpos) == ref_base ) + bca->ref_pos[epos]++; + else + bca->alt_pos[epos]++; + bca->read_len += npos; + //fprintf(stderr,"qpos=%d epos=%d fwd=%d var=%d len=%d %s\n", p->qpos, epos, p->b->core.flag&BAM_FREVERSE ? 0 : 1, bam1_seqi(bam1_seq(p->b),p->qpos) == ref_base ? 1 : 0, p->b->core.l_qseq, bam1_qname(p->b)); } + if ( n ) bca->read_len /= n; r->depth = n; r->ori_depth = ori_depth; // glfgen errmod_cal(bca->e, n, 5, bca->bases, r->p); return r->depth; } -/* Combine individual calls. ref_base is a 4-bit A/C/G/T for a SNP, or - * B2B_REF_MNP, or B2B_REF_INDEL. Indel and MNP are indistinguishable in - * this function. */ -int bcf_call_combine(int n, const bcf_callret1_t *calls, int ref_base /*4-bit*/, bcf_call_t *call) + +double mann_whitney_1947(int n, int m, int U) +{ + if (U<0) return 0; + if (n==0||m==0) return U==0 ? 1 : 0; + return (double)n/(n+m)*mann_whitney_1947(n-1,m,U-m) + (double)m/(n+m)*mann_whitney_1947(n,m-1,U); +} + +void calc_ReadPosBias(bcf_callaux_t *bca, bcf_call_t *call) +{ + int i, nref = 0, nalt = 0; + unsigned long int U = 0; + for (i=0; inpos; i++) + { + nref += bca->ref_pos[i]; + nalt += bca->alt_pos[i]; + U += nref*bca->alt_pos[i]; + bca->ref_pos[i] = 0; + bca->alt_pos[i] = 0; + } + if ( !nref || !nalt ) + { + call->read_pos_bias = -1; + return; + } + + if ( nref>=8 || nalt>=8 ) + { + // normal approximation + double mean = ((double)nref*nalt+1.0)/2.0; + double var2 = (double)nref*nalt*(nref+nalt+1.0)/12.0; + double z = (U-mean)/sqrt(var2); + call->read_pos_bias = z; + //fprintf(stderr,"nref=%d nalt=%d U=%ld mean=%e var=%e zval=%e\n", nref,nalt,U,mean,sqrt(var2),call->read_pos_bias); + } + else + { + double p = mann_whitney_1947(nalt,nref,U); + // biased form claimed by GATK to behave better empirically + // double var2 = (1.0+1.0/(nref+nalt+1.0))*(double)nref*nalt*(nref+nalt+1.0)/12.0; + double var2 = (double)nref*nalt*(nref+nalt+1.0)/12.0; + double z; + if ( p >= 1./sqrt(var2*2*M_PI) ) z = 0; // equal to mean + else + { + if ( U >= nref*nalt/2. ) z = sqrt(-2*log(sqrt(var2*2*M_PI)*p)); + else z = -sqrt(-2*log(sqrt(var2*2*M_PI)*p)); + } + call->read_pos_bias = z; + //fprintf(stderr,"nref=%d nalt=%d U=%ld p=%e var2=%e zval=%e\n", nref,nalt,U, p,var2,call->read_pos_bias); + } +} + +float mean_diff_to_prob(float mdiff, int dp, int readlen) +{ + if ( dp==2 ) + { + if ( mdiff==0 ) + return (2.0*readlen + 4.0*(readlen-1.0))/((float)readlen*readlen); + else + return 8.0*(readlen - 4.0*mdiff)/((float)readlen*readlen); + } + + // This is crude empirical approximation and is not very accurate for + // shorter read lengths (<100bp). There certainly is a room for + // improvement. + const float mv[24][2] = { {0,0}, {0,0}, {0,0}, + { 9.108, 4.934}, { 9.999, 3.991}, {10.273, 3.485}, {10.579, 3.160}, + {10.828, 2.889}, {11.014, 2.703}, {11.028, 2.546}, {11.244, 2.391}, + {11.231, 2.320}, {11.323, 2.138}, {11.403, 2.123}, {11.394, 1.994}, + {11.451, 1.928}, {11.445, 1.862}, {11.516, 1.815}, {11.560, 1.761}, + {11.544, 1.728}, {11.605, 1.674}, {11.592, 1.652}, {11.674, 1.613}, + {11.641, 1.570} }; + + float m, v; + if ( dp>=24 ) + { + m = (int)readlen/8.; + if (dp>100) dp = 100; + v = 1.476/(0.182*pow(dp,0.514)); + v = v*(readlen/100.); + } + else + { + m = mv[dp][0]; + v = mv[dp][1]; + m = (int)m*readlen/100.; + v = v*readlen/100.; + v *= 1.2; // allow more variability + } + return 1.0/(v*sqrt(2*M_PI)) * exp(-0.5*((mdiff-m)/v)*((mdiff-m)/v)); +} + +void calc_vdb(bcf_callaux_t *bca, bcf_call_t *call) +{ + int i, dp = 0; + float mean_pos = 0, mean_diff = 0; + for (i=0; inpos; i++) + { + if ( !bca->alt_pos[i] ) continue; + dp += bca->alt_pos[i]; + mean_pos += bca->alt_pos[i]*i; + } + if ( !dp ) + { + call->vdb = -1; + return; + } + mean_pos /= dp; + for (i=0; inpos; i++) + { + if ( !bca->alt_pos[i] ) continue; + mean_diff += bca->alt_pos[i] * fabs(i - mean_pos); + } + mean_diff /= dp; + call->vdb = mean_diff_to_prob(mean_diff, dp, bca->read_len); +} + +/** + * bcf_call_combine() - sets the PL array and VDB, RPB annotations, finds the top two alleles + * @n: number of samples + * @calls: each sample's calls + * @bca: auxiliary data structure for holding temporary values + * @ref_base: the reference base + * @call: filled with the annotations + */ +int bcf_call_combine(int n, const bcf_callret1_t *calls, bcf_callaux_t *bca, int ref_base /*4-bit*/, bcf_call_t *call) { int ref4, i, j, qsum[4]; int64_t tmp; if (ref_base >= 0) { call->ori_ref = ref4 = bam_nt16_nt4_table[ref_base]; if (ref4 > 4) ref4 = 4; - } else call->ori_ref = ref_base, ref4 = 0; // ref_base < 0 + } 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 @@ -122,15 +305,21 @@ 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 { // for indels and MNPs + } else { call->n_alleles = j; if (call->n_alleles == 1) return -1; // no reliable supporting read. stop doing anything } @@ -145,8 +334,8 @@ 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; @@ -171,11 +360,14 @@ int bcf_call_combine(int n, const bcf_callret1_t *calls, int ref_base /*4-bit*/, call->ori_depth += calls[i].ori_depth; for (j = 0; j < 16; ++j) call->anno[j] += calls[i].anno[j]; } + + calc_vdb(bca, call); + calc_ReadPosBias(bca, call); + return 0; } -/* Fill a bcf1_t record. The type of the record (SNP, MNP or INDEL) is - * determined by bca->ori_ref. */ -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); @@ -185,7 +377,7 @@ int bcf_call2bcf(int tid, int pos, bcf_call_t *bc, bcf1_t *b, bcf_callret1_t *bc 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->ori_ref == B2B_REF_INDEL) { // an indel + 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); @@ -208,16 +400,6 @@ int bcf_call2bcf(int tid, int pos, bcf_call_t *bc, bcf1_t *b, bcf_callret1_t *bc } } kputc('\0', &s); - } else if (bc->ori_ref == B2B_REF_MNP) { - for (j = 0; j < bca->indelreg; ++j) kputc(ref[pos+1+j], &s); - kputc('\0', &s); - for (i = 1; i < 4; ++i) { - if (bc->a[i] < 0) break; - if (i > 1) kputc(',', &s); - for (j = 0; j < bca->indelreg; ++j) - kputc("ACGT"[bca->indel_types[i]>>2*i&3], &s); - } - kputc('\0', &s); } else { // a SNP kputc("ACGTN"[bc->ori_ref], &s); kputc('\0', &s); for (i = 1; i < 5; ++i) { @@ -229,31 +411,38 @@ int bcf_call2bcf(int tid, int pos, bcf_call_t *bc, bcf1_t *b, bcf_callret1_t *bc } kputc('\0', &s); // INFO - if (bc->ori_ref == B2B_REF_INDEL) kputs("INDEL;", &s); - else if (bc->ori_ref == B2B_REF_MNP) kputs("MNP;", &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); + if (bc->read_pos_bias != -1 ) + ksprintf(&s, ";RPB=%.4f", bc->read_pos_bias); 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) {