X-Git-Url: http://woldlab.caltech.edu/gitweb/?p=samtools.git;a=blobdiff_plain;f=bam_plcmd.c;h=cbf6ae8c13754be0c441b311eb14dfb9f04452a3;hp=ba787a98bd78726503f48af1fd16582d89a357dd;hb=d59366b97f0e6a0cbb0dfcdc5679f0ed88c8fe1e;hpb=317f5e8dd22cc9e1e5e05fbcaeb3b9aca7447351 diff --git a/bam_plcmd.c b/bam_plcmd.c index ba787a9..cbf6ae8 100644 --- a/bam_plcmd.c +++ b/bam_plcmd.c @@ -2,391 +2,545 @@ #include #include #include +#include +#include #include "sam.h" #include "faidx.h" -#include "bam_maqcns.h" -#include "khash.h" -#include "glf.h" #include "kstring.h" -typedef int *indel_list_t; -KHASH_MAP_INIT_INT64(64, indel_list_t) +static inline int printw(int c, FILE *fp) +{ + char buf[16]; + int l, x; + if (c == 0) return fputc('0', fp); + for (l = 0, x = c < 0? -c : c; x > 0; x /= 10) buf[l++] = x%10 + '0'; + if (c < 0) buf[l++] = '-'; + buf[l] = 0; + for (x = 0; x < l/2; ++x) { + int y = buf[x]; buf[x] = buf[l-1-x]; buf[l-1-x] = y; + } + fputs(buf, fp); + return 0; +} + +static inline void pileup_seq(const bam_pileup1_t *p, int pos, int ref_len, const char *ref) +{ + int j; + if (p->is_head) { + putchar('^'); + putchar(p->b->core.qual > 93? 126 : p->b->core.qual + 33); + } + if (!p->is_del) { + int c = bam_nt16_rev_table[bam1_seqi(bam1_seq(p->b), p->qpos)]; + if (ref) { + int rb = pos < ref_len? ref[pos] : 'N'; + if (c == '=' || bam_nt16_table[c] == bam_nt16_table[rb]) c = bam1_strand(p->b)? ',' : '.'; + else c = bam1_strand(p->b)? tolower(c) : toupper(c); + } else { + if (c == '=') c = bam1_strand(p->b)? ',' : '.'; + else c = bam1_strand(p->b)? tolower(c) : toupper(c); + } + putchar(c); + } else putchar(p->is_refskip? (bam1_strand(p->b)? '<' : '>') : '*'); + if (p->indel > 0) { + putchar('+'); printw(p->indel, stdout); + for (j = 1; j <= p->indel; ++j) { + int c = bam_nt16_rev_table[bam1_seqi(bam1_seq(p->b), p->qpos + j)]; + putchar(bam1_strand(p->b)? tolower(c) : toupper(c)); + } + } else if (p->indel < 0) { + printw(p->indel, stdout); + for (j = 1; j <= -p->indel; ++j) { + int c = (ref && (int)pos+j < ref_len)? ref[pos+j] : 'N'; + putchar(bam1_strand(p->b)? tolower(c) : toupper(c)); + } + } + if (p->is_tail) putchar('$'); +} -#define BAM_PLF_SIMPLE 0x01 -#define BAM_PLF_CNS 0x02 -#define BAM_PLF_INDEL_ONLY 0x04 -#define BAM_PLF_GLF 0x08 -#define BAM_PLF_VAR_ONLY 0x10 -#define BAM_PLF_2ND 0x20 +#include +#include "bam2bcf.h" +#include "sample.h" + +#define MPLP_GLF 0x10 +#define MPLP_NO_COMP 0x20 +#define MPLP_NO_ORPHAN 0x40 +#define MPLP_REALN 0x80 +#define MPLP_FMT_DP 0x100 +#define MPLP_FMT_SP 0x200 +#define MPLP_NO_INDEL 0x400 +#define MPLP_EXT_BAQ 0x800 +#define MPLP_ILLUMINA13 0x1000 +#define MPLP_IGNORE_RG 0x2000 +#define MPLP_PRINT_POS 0x4000 +#define MPLP_PRINT_MAPQ 0x8000 + +void *bed_read(const char *fn); +void bed_destroy(void *_h); +int bed_overlap(const void *_h, const char *chr, int beg, int end); typedef struct { - bam_header_t *h; - bam_maqcns_t *c; - bam_maqindel_opt_t *ido; + int max_mq, min_mq, flag, min_baseQ, capQ_thres, max_depth, max_indel_depth; + int openQ, extQ, tandemQ, min_support; // for indels + double min_frac; // for indels + char *reg, *pl_list; faidx_t *fai; - khash_t(64) *hash; - uint32_t format; - int tid, len, last_pos; - int mask; + void *bed, *rghash; +} mplp_conf_t; + +typedef struct { + bamFile fp; + bam_iter_t iter; + bam_header_t *h; + int ref_id; char *ref; - glfFile fp_glf; // for glf output only -} pu_data_t; + const mplp_conf_t *conf; +} mplp_aux_t; -char **__bam_get_lines(const char *fn, int *_n); -void bam_init_header_hash(bam_header_t *header); -int32_t bam_get_tid(const bam_header_t *header, const char *seq_name); +typedef struct { + int n; + int *n_plp, *m_plp; + bam_pileup1_t **plp; +} mplp_pileup_t; -static khash_t(64) *load_pos(const char *fn, bam_header_t *h) +static int mplp_func(void *data, bam1_t *b) { - char **list; - int i, j, n, *fields, max_fields; - khash_t(64) *hash; - bam_init_header_hash(h); - list = __bam_get_lines(fn, &n); - hash = kh_init(64); - max_fields = 0; fields = 0; - for (i = 0; i < n; ++i) { - char *str = list[i]; - int chr, n_fields, ret; - khint_t k; - uint64_t x; - n_fields = ksplit_core(str, 0, &max_fields, &fields); - if (n_fields < 2) continue; - chr = bam_get_tid(h, str + fields[0]); - if (chr < 0) { - fprintf(stderr, "[load_pos] unknown reference sequence name: %s\n", str + fields[0]); + extern int bam_realn(bam1_t *b, const char *ref); + extern int bam_prob_realn_core(bam1_t *b, const char *ref, int); + extern int bam_cap_mapQ(bam1_t *b, char *ref, int thres); + mplp_aux_t *ma = (mplp_aux_t*)data; + int ret, skip = 0; + do { + int has_ref; + ret = ma->iter? bam_iter_read(ma->fp, ma->iter, b) : bam_read1(ma->fp, b); + if (ret < 0) break; + if (b->core.tid < 0 || (b->core.flag&BAM_FUNMAP)) { // exclude unmapped reads + skip = 1; continue; } - x = (uint64_t)chr << 32 | (atoi(str + fields[1]) - 1); - k = kh_put(64, hash, x, &ret); - if (ret == 0) { - fprintf(stderr, "[load_pos] position %s:%s has been loaded.\n", str+fields[0], str+fields[1]); - continue; + if (ma->conf->bed) { // test overlap + skip = !bed_overlap(ma->conf->bed, ma->h->target_name[b->core.tid], b->core.pos, bam_calend(&b->core, bam1_cigar(b))); + if (skip) continue; } - kh_val(hash, k) = 0; - if (n_fields > 2) { - // count - for (j = 2; j < n_fields; ++j) { - char *s = str + fields[j]; - if ((*s != '+' && *s != '-') || !isdigit(s[1])) break; - } - if (j > 2) { // update kh_val() - int *q, y, z; - q = kh_val(hash, k) = (int*)calloc(j - 1, sizeof(int)); - q[0] = j - 2; z = j; y = 1; - for (j = 2; j < z; ++j) - q[y++] = atoi(str + fields[j]); - } + if (ma->conf->rghash) { // exclude read groups + uint8_t *rg = bam_aux_get(b, "RG"); + skip = (rg && bcf_str2id(ma->conf->rghash, (const char*)(rg+1)) >= 0); + if (skip) continue; } - free(str); - } - free(list); free(fields); - return hash; + if (ma->conf->flag & MPLP_ILLUMINA13) { + int i; + uint8_t *qual = bam1_qual(b); + for (i = 0; i < b->core.l_qseq; ++i) + qual[i] = qual[i] > 31? qual[i] - 31 : 0; + } + has_ref = (ma->ref && ma->ref_id == b->core.tid)? 1 : 0; + skip = 0; + if (has_ref && (ma->conf->flag&MPLP_REALN)) bam_prob_realn_core(b, ma->ref, (ma->conf->flag & MPLP_EXT_BAQ)? 3 : 1); + if (has_ref && ma->conf->capQ_thres > 10) { + int q = bam_cap_mapQ(b, ma->ref, ma->conf->capQ_thres); + if (q < 0) skip = 1; + else if (b->core.qual > q) b->core.qual = q; + } + else if (b->core.qual < ma->conf->min_mq) skip = 1; + else if ((ma->conf->flag&MPLP_NO_ORPHAN) && (b->core.flag&1) && !(b->core.flag&2)) skip = 1; + } while (skip); + return ret; } -// an analogy to pileup_func() below -static int glt3_func(uint32_t tid, uint32_t pos, int n, const bam_pileup1_t *pu, void *data) +static void group_smpl(mplp_pileup_t *m, bam_sample_t *sm, kstring_t *buf, + int n, char *const*fn, int *n_plp, const bam_pileup1_t **plp, int ignore_rg) { - pu_data_t *d = (pu_data_t*)data; - bam_maqindel_ret_t *r = 0; - int rb, *proposed_indels = 0; - glf1_t *g; - glf3_t *g3; - - if (d->fai == 0) { - fprintf(stderr, "[glt3_func] reference sequence is required for generating GLT. Abort!\n"); - exit(1); - } - if (d->hash) { // only output a list of sites - khint_t k = kh_get(64, d->hash, (uint64_t)tid<<32|pos); - if (k == kh_end(d->hash)) return 0; - proposed_indels = kh_val(d->hash, k); - } - g3 = glf3_init1(); - if (d->fai && (int)tid != d->tid) { - if (d->ref) { // then write the end mark - g3->rtype = GLF3_RTYPE_END; - glf3_write1(d->fp_glf, g3); + int i, j; + memset(m->n_plp, 0, m->n * sizeof(int)); + for (i = 0; i < n; ++i) { + for (j = 0; j < n_plp[i]; ++j) { + const bam_pileup1_t *p = plp[i] + j; + uint8_t *q; + int id = -1; + q = ignore_rg? 0 : bam_aux_get(p->b, "RG"); + if (q) id = bam_smpl_rg2smid(sm, fn[i], (char*)q+1, buf); + if (id < 0) id = bam_smpl_rg2smid(sm, fn[i], 0, buf); + if (id < 0 || id >= m->n) { + assert(q); // otherwise a bug + fprintf(stderr, "[%s] Read group %s used in file %s but absent from the header or an alignment missing read group.\n", __func__, (char*)q+1, fn[i]); + exit(1); + } + if (m->n_plp[id] == m->m_plp[id]) { + m->m_plp[id] = m->m_plp[id]? m->m_plp[id]<<1 : 8; + m->plp[id] = realloc(m->plp[id], sizeof(bam_pileup1_t) * m->m_plp[id]); + } + m->plp[id][m->n_plp[id]++] = *p; } - glf3_ref_write(d->fp_glf, d->h->target_name[tid], d->h->target_len[tid]); // write reference - free(d->ref); - d->ref = fai_fetch(d->fai, d->h->target_name[tid], &d->len); - d->tid = tid; - d->last_pos = 0; - } - rb = (d->ref && (int)pos < d->len)? d->ref[pos] : 'N'; - g = bam_maqcns_glfgen(n, pu, bam_nt16_table[rb], d->c); - memcpy(g3, g, sizeof(glf1_t)); - g3->rtype = GLF3_RTYPE_SUB; - g3->offset = pos - d->last_pos; - d->last_pos = pos; - glf3_write1(d->fp_glf, g3); - if (pos < d->len) { - if (proposed_indels) - r = bam_maqindel(n, pos, d->ido, pu, d->ref, proposed_indels[0], proposed_indels+1); - else r = bam_maqindel(n, pos, d->ido, pu, d->ref, 0, 0); } - if (r) { // then write indel line - int het = 3 * n, min; - min = het; - if (min > r->gl[0]) min = r->gl[0]; - if (min > r->gl[1]) min = r->gl[1]; - g3->ref_base = 0; - g3->rtype = GLF3_RTYPE_INDEL; - memset(g3->lk, 0, 10); - g3->lk[0] = r->gl[0] - min < 255? r->gl[0] - min : 255; - g3->lk[1] = r->gl[1] - min < 255? r->gl[1] - min : 255; - g3->lk[2] = het - min < 255? het - min : 255; - g3->offset = 0; - g3->indel_len[0] = r->indel1; - g3->indel_len[1] = r->indel2; - g3->min_lk = min < 255? min : 255; - g3->max_len = (abs(r->indel1) > abs(r->indel2)? abs(r->indel1) : abs(r->indel2)) + 1; - g3->indel_seq[0] = strdup(r->s[0]+1); - g3->indel_seq[1] = strdup(r->s[1]+1); - glf3_write1(d->fp_glf, g3); - bam_maqindel_ret_destroy(r); - } - free(g); - glf3_destroy1(g3); - return 0; } -static int pileup_func(uint32_t tid, uint32_t pos, int n, const bam_pileup1_t *pu, void *data) +static int mpileup(mplp_conf_t *conf, int n, char **fn) { - pu_data_t *d = (pu_data_t*)data; - bam_maqindel_ret_t *r = 0; - int i, j, rb, rms_mapq = -1, *proposed_indels = 0; - uint64_t rms_aux; - uint32_t cns = 0; + extern void *bcf_call_add_rg(void *rghash, const char *hdtext, const char *list); + extern void bcf_call_del_rghash(void *rghash); + mplp_aux_t **data; + int i, tid, pos, *n_plp, tid0 = -1, beg0 = 0, end0 = 1u<<29, ref_len, ref_tid = -1, max_depth, max_indel_depth; + const bam_pileup1_t **plp; + bam_mplp_t iter; + bam_header_t *h = 0; + char *ref; + void *rghash = 0; - // if GLF is required, suppress -c completely - if (d->format & BAM_PLF_GLF) return glt3_func(tid, pos, n, pu, data); - // if d->hash is initialized, only output the sites in the hash table - if (d->hash) { - khint_t k = kh_get(64, d->hash, (uint64_t)tid<<32|pos); - if (k == kh_end(d->hash)) return 0; - proposed_indels = kh_val(d->hash, k); - } - // update d->ref if necessary - if (d->fai && (int)tid != d->tid) { - free(d->ref); - d->ref = fai_fetch(d->fai, d->h->target_name[tid], &d->len); - d->tid = tid; - } - rb = (d->ref && (int)pos < d->len)? d->ref[pos] : 'N'; - // when the indel-only mode is asked for, return if no reads mapped with indels - if (d->format & BAM_PLF_INDEL_ONLY) { - for (i = 0; i < n; ++i) - if (pu[i].indel != 0) break; - if (i == n) return 0; - } - // call the consensus and indel - if (d->format & BAM_PLF_CNS) // call consensus - cns = bam_maqcns_call(n, pu, d->c); - if ((d->format & (BAM_PLF_CNS|BAM_PLF_INDEL_ONLY)) && d->ref && pos < d->len) { // call indels - if (proposed_indels) // the first element gives the size of the array - r = bam_maqindel(n, pos, d->ido, pu, d->ref, proposed_indels[0], proposed_indels+1); - else r = bam_maqindel(n, pos, d->ido, pu, d->ref, 0, 0); - } - // when only variant sites are asked for, test if the site is a variant - if ((d->format & BAM_PLF_CNS) && (d->format & BAM_PLF_VAR_ONLY)) { - if (!(bam_nt16_table[rb] != 15 && cns>>28 != bam_nt16_table[rb])) { // not a SNP - if (!(r && (r->gt == 2 || strcmp(r->s[r->gt], "*")))) { // not an indel - if (r) bam_maqindel_ret_destroy(r); - return 0; + bcf_callaux_t *bca = 0; + bcf_callret1_t *bcr = 0; + bcf_call_t bc; + bcf_t *bp = 0; + bcf_hdr_t *bh = 0; + + bam_sample_t *sm = 0; + kstring_t buf; + mplp_pileup_t gplp; + + memset(&gplp, 0, sizeof(mplp_pileup_t)); + memset(&buf, 0, sizeof(kstring_t)); + memset(&bc, 0, sizeof(bcf_call_t)); + data = calloc(n, sizeof(void*)); + plp = calloc(n, sizeof(void*)); + n_plp = calloc(n, sizeof(int*)); + sm = bam_smpl_init(); + + // read the header and initialize data + for (i = 0; i < n; ++i) { + bam_header_t *h_tmp; + data[i] = calloc(1, sizeof(mplp_aux_t)); + data[i]->fp = strcmp(fn[i], "-") == 0? bam_dopen(fileno(stdin), "r") : bam_open(fn[i], "r"); + data[i]->conf = conf; + h_tmp = bam_header_read(data[i]->fp); + data[i]->h = i? h : h_tmp; // for i==0, "h" has not been set yet + bam_smpl_add(sm, fn[i], (conf->flag&MPLP_IGNORE_RG)? 0 : h_tmp->text); + rghash = bcf_call_add_rg(rghash, h_tmp->text, conf->pl_list); + if (conf->reg) { + int beg, end; + bam_index_t *idx; + idx = bam_index_load(fn[i]); + if (idx == 0) { + fprintf(stderr, "[%s] fail to load index for %d-th input.\n", __func__, i+1); + exit(1); + } + if (bam_parse_region(h_tmp, conf->reg, &tid, &beg, &end) < 0) { + fprintf(stderr, "[%s] malformatted region or wrong seqname for %d-th input.\n", __func__, i+1); + exit(1); } + if (i == 0) tid0 = tid, beg0 = beg, end0 = end; + data[i]->iter = bam_iter_query(idx, tid, beg, end); + bam_index_destroy(idx); + } + if (i == 0) h = h_tmp; + else { + // FIXME: to check consistency + bam_header_destroy(h_tmp); } } - // print the first 3 columns - printf("%s\t%d\t%c\t", d->h->target_name[tid], pos + 1, rb); - // print consensus information if required - if (d->format & BAM_PLF_CNS) { - int ref_q, rb4 = bam_nt16_table[rb]; - ref_q = 0; - if (rb4 != 15 && cns>>28 != 15 && cns>>28 != rb4) { // a SNP - ref_q = ((cns>>24&0xf) == rb4)? cns>>8&0xff : (cns>>8&0xff) + (cns&0xff); - if (ref_q > 255) ref_q = 255; + gplp.n = sm->n; + gplp.n_plp = calloc(sm->n, sizeof(int)); + gplp.m_plp = calloc(sm->n, sizeof(int)); + gplp.plp = calloc(sm->n, sizeof(void*)); + + fprintf(stderr, "[%s] %d samples in %d input files\n", __func__, sm->n, n); + // write the VCF header + if (conf->flag & MPLP_GLF) { + kstring_t s; + bh = calloc(1, sizeof(bcf_hdr_t)); + s.l = s.m = 0; s.s = 0; + bp = bcf_open("-", (conf->flag&MPLP_NO_COMP)? "wu" : "w"); + for (i = 0; i < h->n_targets; ++i) { + kputs(h->target_name[i], &s); + kputc('\0', &s); + } + bh->l_nm = s.l; + bh->name = malloc(s.l); + memcpy(bh->name, s.s, s.l); + s.l = 0; + for (i = 0; i < sm->n; ++i) { + kputs(sm->smpl[i], &s); kputc('\0', &s); } - rms_mapq = cns>>16&0xff; - printf("%c\t%d\t%d\t%d\t", bam_nt16_rev_table[cns>>28], cns>>8&0xff, ref_q, rms_mapq); + bh->l_smpl = s.l; + bh->sname = malloc(s.l); + memcpy(bh->sname, s.s, s.l); + bh->txt = malloc(strlen(BAM_VERSION) + 64); + bh->l_txt = 1 + sprintf(bh->txt, "##samtoolsVersion=%s\n", BAM_VERSION); + free(s.s); + bcf_hdr_sync(bh); + bcf_hdr_write(bp, bh); + bca = bcf_call_init(-1., conf->min_baseQ); + bcr = calloc(sm->n, sizeof(bcf_callret1_t)); + bca->rghash = rghash; + bca->openQ = conf->openQ, bca->extQ = conf->extQ, bca->tandemQ = conf->tandemQ; + bca->min_frac = conf->min_frac; + bca->min_support = conf->min_support; } - // print pileup sequences - printf("%d\t", n); - rms_aux = 0; // we need to recalculate rms_mapq when -c is not flagged on the command line - for (i = 0; i < n; ++i) { - const bam_pileup1_t *p = pu + i; - int tmp = p->b->core.qual < d->c->cap_mapQ? p->b->core.qual : d->c->cap_mapQ; - rms_aux += tmp * tmp; - if (p->is_head) printf("^%c", p->b->core.qual > 93? 126 : p->b->core.qual + 33); - if (!p->is_del) { - int c = bam_nt16_rev_table[bam1_seqi(bam1_seq(p->b), p->qpos)]; - if (c == '=' || toupper(c) == toupper(rb)) c = bam1_strand(p->b)? ',' : '.'; - else c = bam1_strand(p->b)? tolower(c) : toupper(c); - putchar(c); - if (p->indel > 0) { - printf("+%d", p->indel); - for (j = 1; j <= p->indel; ++j) { - c = bam_nt16_rev_table[bam1_seqi(bam1_seq(p->b), p->qpos + j)]; - putchar(bam1_strand(p->b)? tolower(c) : toupper(c)); + if (tid0 >= 0 && conf->fai) { // region is set + ref = faidx_fetch_seq(conf->fai, h->target_name[tid0], 0, 0x7fffffff, &ref_len); + ref_tid = tid0; + for (i = 0; i < n; ++i) data[i]->ref = ref, data[i]->ref_id = tid0; + } else ref_tid = -1, ref = 0; + iter = bam_mplp_init(n, mplp_func, (void**)data); + max_depth = conf->max_depth; + if (max_depth * sm->n > 1<<20) + fprintf(stderr, "(%s) Max depth is above 1M. Potential memory hog!\n", __func__); + if (max_depth * sm->n < 8000) { + max_depth = 8000 / sm->n; + fprintf(stderr, "<%s> Set max per-file depth to %d\n", __func__, max_depth); + } + max_indel_depth = conf->max_indel_depth * sm->n; + bam_mplp_set_maxcnt(iter, max_depth); + while (bam_mplp_auto(iter, &tid, &pos, n_plp, plp) > 0) { + if (conf->reg && (pos < beg0 || pos >= end0)) continue; // out of the region requested + if (conf->bed && tid >= 0 && !bed_overlap(conf->bed, h->target_name[tid], pos, pos+1)) continue; + if (tid != ref_tid) { + free(ref); ref = 0; + if (conf->fai) ref = faidx_fetch_seq(conf->fai, h->target_name[tid], 0, 0x7fffffff, &ref_len); + for (i = 0; i < n; ++i) data[i]->ref = ref, data[i]->ref_id = tid; + ref_tid = tid; + } + if (conf->flag & MPLP_GLF) { + int total_depth, _ref0, ref16; + bcf1_t *b = calloc(1, sizeof(bcf1_t)); + for (i = total_depth = 0; i < n; ++i) total_depth += n_plp[i]; + group_smpl(&gplp, sm, &buf, n, fn, n_plp, plp, conf->flag & MPLP_IGNORE_RG); + _ref0 = (ref && pos < ref_len)? ref[pos] : 'N'; + ref16 = bam_nt16_table[_ref0]; + for (i = 0; i < gplp.n; ++i) + bcf_call_glfgen(gplp.n_plp[i], gplp.plp[i], ref16, bca, bcr + i); + bcf_call_combine(gplp.n, bcr, ref16, &bc); + bcf_call2bcf(tid, pos, &bc, b, (conf->flag&(MPLP_FMT_DP|MPLP_FMT_SP))? bcr : 0, + (conf->flag&MPLP_FMT_SP), 0, 0); + bcf_write(bp, bh, b); + bcf_destroy(b); + // call indels + if (!(conf->flag&MPLP_NO_INDEL) && total_depth < max_indel_depth && bcf_call_gap_prep(gplp.n, gplp.n_plp, gplp.plp, pos, bca, ref, rghash) >= 0) { + for (i = 0; i < gplp.n; ++i) + bcf_call_glfgen(gplp.n_plp[i], gplp.plp[i], -1, bca, bcr + i); + if (bcf_call_combine(gplp.n, bcr, -1, &bc) >= 0) { + b = calloc(1, sizeof(bcf1_t)); + bcf_call2bcf(tid, pos, &bc, b, (conf->flag&(MPLP_FMT_DP|MPLP_FMT_SP))? bcr : 0, + (conf->flag&MPLP_FMT_SP), bca, ref); + bcf_write(bp, bh, b); + bcf_destroy(b); } - } else if (p->indel < 0) { - printf("%d", p->indel); - for (j = 1; j <= -p->indel; ++j) { - c = (d->ref && (int)pos+j < d->len)? d->ref[pos+j] : 'N'; - putchar(bam1_strand(p->b)? tolower(c) : toupper(c)); + } + } else { + printf("%s\t%d\t%c", h->target_name[tid], pos + 1, (ref && pos < ref_len)? ref[pos] : 'N'); + for (i = 0; i < n; ++i) { + int j; + printf("\t%d\t", n_plp[i]); + if (n_plp[i] == 0) { + printf("*\t*"); // FIXME: printf() is very slow... + if (conf->flag & MPLP_PRINT_POS) printf("\t*"); + } else { + for (j = 0; j < n_plp[i]; ++j) + pileup_seq(plp[i] + j, pos, ref_len, ref); + putchar('\t'); + for (j = 0; j < n_plp[i]; ++j) { + const bam_pileup1_t *p = plp[i] + j; + int c = bam1_qual(p->b)[p->qpos] + 33; + if (c > 126) c = 126; + putchar(c); + } + if (conf->flag & MPLP_PRINT_MAPQ) { + putchar('\t'); + for (j = 0; j < n_plp[i]; ++j) { + int c = plp[i][j].b->core.qual + 33; + if (c > 126) c = 126; + putchar(c); + } + } + if (conf->flag & MPLP_PRINT_POS) { + putchar('\t'); + for (j = 0; j < n_plp[i]; ++j) { + if (j > 0) putchar(','); + printf("%d", plp[i][j].qpos + 1); // FIXME: printf() is very slow... + } + } } } - } else putchar('*'); - if (p->is_tail) putchar('$'); - } - // finalize rms_mapq - rms_aux = (uint64_t)(sqrt((double)rms_aux / n) + .499); - if (rms_mapq < 0) rms_mapq = rms_aux; - putchar('\t'); - // print quality - for (i = 0; i < n; ++i) { - const bam_pileup1_t *p = pu + i; - int c = bam1_qual(p->b)[p->qpos] + 33; - if (c > 126) c = 126; - putchar(c); - } - if (d->format & BAM_PLF_2ND) { // print 2nd calls and qualities - const unsigned char *q; - putchar('\t'); - for (i = 0; i < n; ++i) { - const bam_pileup1_t *p = pu + i; - q = bam_aux_get(p->b, "E2"); - putchar(q? q[p->qpos + 1] : 'N'); - } - putchar('\t'); - for (i = 0; i < n; ++i) { - const bam_pileup1_t *p = pu + i; - q = bam_aux_get(p->b, "U2"); - putchar(q? q[p->qpos + 1] : '!'); + putchar('\n'); } } - // print mapping quality if -s is flagged on the command line - if (d->format & BAM_PLF_SIMPLE) { - putchar('\t'); - for (i = 0; i < n; ++i) { - int c = pu[i].b->core.qual + 33; - if (c > 126) c = 126; - putchar(c); - } - } - putchar('\n'); - // print the indel line if r has been calculated. This only happens if: - // a) -c or -i are flagged, AND b) the reference sequence is available - if (r) { - printf("%s\t%d\t*\t", d->h->target_name[tid], pos + 1); - if (r->gt < 2) printf("%s/%s\t", r->s[r->gt], r->s[r->gt]); - else printf("%s/%s\t", r->s[0], r->s[1]); - printf("%d\t%d\t", r->q_cns, r->q_ref); - printf("%d\t%d\t", rms_mapq, n); - printf("%s\t%s\t", r->s[0], r->s[1]); - //printf("%d\t%d\t", r->gl[0], r->gl[1]); - printf("%d\t%d\t%d\t", r->cnt1, r->cnt2, r->cnt_anti); - printf("%d\t%d\n", r->cnt_ref, r->cnt_ambi); - bam_maqindel_ret_destroy(r); + + bcf_close(bp); + bam_smpl_destroy(sm); free(buf.s); + for (i = 0; i < gplp.n; ++i) free(gplp.plp[i]); + free(gplp.plp); free(gplp.n_plp); free(gplp.m_plp); + bcf_call_del_rghash(rghash); + bcf_hdr_destroy(bh); bcf_call_destroy(bca); free(bc.PL); free(bcr); + bam_mplp_destroy(iter); + bam_header_destroy(h); + for (i = 0; i < n; ++i) { + bam_close(data[i]->fp); + if (data[i]->iter) bam_iter_destroy(data[i]->iter); + free(data[i]); } + free(data); free(plp); free(ref); free(n_plp); return 0; } -int bam_pileup(int argc, char *argv[]) +#define MAX_PATH_LEN 1024 +static int read_file_list(const char *file_list,int *n,char **argv[]) { - int c, is_SAM = 0; - char *fn_list = 0, *fn_fa = 0, *fn_pos = 0; - pu_data_t *d = (pu_data_t*)calloc(1, sizeof(pu_data_t)); - d->tid = -1; d->mask = BAM_DEF_MASK; - d->c = bam_maqcns_init(); - d->ido = bam_maqindel_opt_init(); - while ((c = getopt(argc, argv, "st:f:cT:N:r:l:im:gI:G:vM:S2a")) >= 0) { + char buf[MAX_PATH_LEN]; + int len, nfiles; + char **files; + + FILE *fh = fopen(file_list,"r"); + if ( !fh ) + { + fprintf(stderr,"%s: %s\n", file_list,strerror(errno)); + return 1; + } + + // Speed is not an issue here, determine the number of files by reading the file twice + nfiles = 0; + while ( fgets(buf,MAX_PATH_LEN,fh) ) nfiles++; + + if ( fseek(fh, 0L, SEEK_SET) ) + { + fprintf(stderr,"%s: %s\n", file_list,strerror(errno)); + return 1; + } + + files = calloc(nfiles,sizeof(char*)); + nfiles = 0; + while ( fgets(buf,MAX_PATH_LEN,fh) ) + { + len = strlen(buf); + while ( len>0 && isspace(buf[len-1]) ) len--; + if ( !len ) continue; + + files[nfiles] = malloc(sizeof(char)*(len+1)); + strncpy(files[nfiles],buf,len); + files[nfiles][len] = 0; + nfiles++; + } + fclose(fh); + if ( !nfiles ) + { + fprintf(stderr,"No files read from %s\n", file_list); + return 1; + } + *argv = files; + *n = nfiles; + return 0; +} +#undef MAX_PATH_LEN + +int bam_mpileup(int argc, char *argv[]) +{ + int c; + const char *file_list = NULL; + char **fn = NULL; + int nfiles = 0, use_orphan = 0; + mplp_conf_t mplp; + memset(&mplp, 0, sizeof(mplp_conf_t)); + #define MPLP_PRINT_POS 0x4000 + mplp.max_mq = 60; + mplp.min_baseQ = 13; + mplp.capQ_thres = 0; + mplp.max_depth = 250; mplp.max_indel_depth = 250; + mplp.openQ = 40; mplp.extQ = 20; mplp.tandemQ = 100; + mplp.min_frac = 0.002; mplp.min_support = 1; + mplp.flag = MPLP_NO_ORPHAN | MPLP_REALN; + while ((c = getopt(argc, argv, "Agf:r:l:M:q:Q:uaRC:BDSd:L:b:P:o:e:h:Im:F:EG:6Os")) >= 0) { switch (c) { - case 'a': d->c->is_soap = 1; break; - case 's': d->format |= BAM_PLF_SIMPLE; break; - case 't': fn_list = strdup(optarg); break; - case 'l': fn_pos = strdup(optarg); break; - case 'f': fn_fa = strdup(optarg); break; - case 'T': d->c->theta = atof(optarg); break; - case 'N': d->c->n_hap = atoi(optarg); break; - case 'r': d->c->het_rate = atof(optarg); break; - case 'M': d->c->cap_mapQ = atoi(optarg); break; - case 'c': d->format |= BAM_PLF_CNS; break; - case 'i': d->format |= BAM_PLF_INDEL_ONLY; break; - case 'v': d->format |= BAM_PLF_VAR_ONLY; break; - case 'm': d->mask = strtol(optarg, 0, 0); break; - case 'g': d->format |= BAM_PLF_GLF; break; - case '2': d->format |= BAM_PLF_2ND; break; - case 'I': d->ido->q_indel = atoi(optarg); break; - case 'G': d->ido->r_indel = atof(optarg); break; - case 'S': is_SAM = 1; break; - default: fprintf(stderr, "Unrecognizd option '-%c'.\n", c); return 1; + case 'f': + mplp.fai = fai_load(optarg); + if (mplp.fai == 0) return 1; + break; + case 'd': mplp.max_depth = atoi(optarg); break; + case 'r': mplp.reg = strdup(optarg); break; + case 'l': mplp.bed = bed_read(optarg); break; + case 'P': mplp.pl_list = strdup(optarg); break; + case 'g': mplp.flag |= MPLP_GLF; break; + case 'u': mplp.flag |= MPLP_NO_COMP | MPLP_GLF; break; + case 'a': mplp.flag |= MPLP_NO_ORPHAN | MPLP_REALN; break; + case 'B': mplp.flag &= ~MPLP_REALN; break; + case 'D': mplp.flag |= MPLP_FMT_DP; break; + case 'S': mplp.flag |= MPLP_FMT_SP; break; + case 'I': mplp.flag |= MPLP_NO_INDEL; break; + case 'E': mplp.flag |= MPLP_EXT_BAQ; break; + case '6': mplp.flag |= MPLP_ILLUMINA13; break; + case 'R': mplp.flag |= MPLP_IGNORE_RG; break; + case 's': mplp.flag |= MPLP_PRINT_MAPQ; break; + case 'O': mplp.flag |= MPLP_PRINT_POS; break; + case 'C': mplp.capQ_thres = atoi(optarg); break; + case 'M': mplp.max_mq = atoi(optarg); break; + case 'q': mplp.min_mq = atoi(optarg); break; + case 'Q': mplp.min_baseQ = atoi(optarg); break; + case 'b': file_list = optarg; break; + case 'o': mplp.openQ = atoi(optarg); break; + case 'e': mplp.extQ = atoi(optarg); break; + case 'h': mplp.tandemQ = atoi(optarg); break; + case 'A': use_orphan = 1; break; + case 'F': mplp.min_frac = atof(optarg); break; + case 'm': mplp.min_support = atoi(optarg); break; + case 'L': mplp.max_indel_depth = atoi(optarg); break; + case 'G': { + FILE *fp_rg; + char buf[1024]; + mplp.rghash = bcf_str2id_init(); + if ((fp_rg = fopen(optarg, "r")) == 0) + fprintf(stderr, "(%s) Fail to open file %s. Continue anyway.\n", __func__, optarg); + while (!feof(fp_rg) && fscanf(fp_rg, "%s", buf) > 0) // this is not a good style, but forgive me... + bcf_str2id_add(mplp.rghash, strdup(buf)); + fclose(fp_rg); + } + break; } } - if (fn_list) is_SAM = 1; - if (optind == argc) { + if (use_orphan) mplp.flag &= ~MPLP_NO_ORPHAN; + if (argc == 1) { fprintf(stderr, "\n"); - fprintf(stderr, "Usage: samtools pileup [options] |\n\n"); - fprintf(stderr, "Option: -s simple (yet incomplete) pileup format\n"); - fprintf(stderr, " -S the input is in SAM\n"); - fprintf(stderr, " -a use the SOAPsnp model for SNP calling\n"); - fprintf(stderr, " -2 output the 2nd best call and quality\n"); - fprintf(stderr, " -i only show lines/consensus with indels\n"); - fprintf(stderr, " -m INT filtering reads with bits in INT [%d]\n", d->mask); - fprintf(stderr, " -M INT cap mapping quality at INT [%d]\n", d->c->cap_mapQ); - fprintf(stderr, " -t FILE list of reference sequences (force -S)\n"); - fprintf(stderr, " -l FILE list of sites at which pileup is output\n"); - fprintf(stderr, " -f FILE reference sequence in the FASTA format\n\n"); - fprintf(stderr, " -c output the maq consensus sequence\n"); - fprintf(stderr, " -v print variants only (for -c)\n"); - fprintf(stderr, " -g output in the GLFv3 format (suppressing -c/-i/-s)\n"); - fprintf(stderr, " -T FLOAT theta in maq consensus calling model (for -c/-g) [%f]\n", d->c->theta); - fprintf(stderr, " -N INT number of haplotypes in the sample (for -c/-g) [%d]\n", d->c->n_hap); - fprintf(stderr, " -r FLOAT prior of a difference between two haplotypes (for -c/-g) [%f]\n", d->c->het_rate); - fprintf(stderr, " -G FLOAT prior of an indel between two haplotypes (for -c/-g) [%f]\n", d->ido->r_indel); - fprintf(stderr, " -I INT phred prob. of an indel in sequencing/prep. (for -c/-g) [%d]\n", d->ido->q_indel); + fprintf(stderr, "Usage: samtools mpileup [options] in1.bam [in2.bam [...]]\n\n"); + fprintf(stderr, "Input options:\n\n"); + fprintf(stderr, " -6 assume the quality is in the Illumina-1.3+ encoding\n"); + fprintf(stderr, " -A count anomalous read pairs\n"); + fprintf(stderr, " -B disable BAQ computation\n"); + fprintf(stderr, " -b FILE list of input BAM files [null]\n"); + fprintf(stderr, " -C INT parameter for adjusting mapQ; 0 to disable [0]\n"); + fprintf(stderr, " -d INT max per-BAM depth to avoid excessive memory usage [%d]\n", mplp.max_depth); + fprintf(stderr, " -E extended BAQ for higher sensitivity but lower specificity\n"); + fprintf(stderr, " -f FILE faidx indexed reference sequence file [null]\n"); + fprintf(stderr, " -G FILE exclude read groups listed in FILE [null]\n"); + fprintf(stderr, " -l FILE list of positions (chr pos) or regions (BED) [null]\n"); + fprintf(stderr, " -M INT cap mapping quality at INT [%d]\n", mplp.max_mq); + fprintf(stderr, " -r STR region in which pileup is generated [null]\n"); + fprintf(stderr, " -R ignore RG tags\n"); + fprintf(stderr, " -q INT skip alignments with mapQ smaller than INT [%d]\n", mplp.min_mq); + fprintf(stderr, " -Q INT skip bases with baseQ/BAQ smaller than INT [%d]\n", mplp.min_baseQ); + fprintf(stderr, "\nOutput options:\n\n"); + fprintf(stderr, " -D output per-sample DP in BCF (require -g/-u)\n"); + fprintf(stderr, " -g generate BCF output (genotype likelihoods)\n"); + fprintf(stderr, " -O output base positions on reads (disabled by -g/-u)\n"); + fprintf(stderr, " -s output mapping quality (disabled by -g/-u)\n"); + fprintf(stderr, " -S output per-sample strand bias P-value in BCF (require -g/-u)\n"); + fprintf(stderr, " -u generate uncompress BCF output\n"); + fprintf(stderr, "\nSNP/INDEL genotype likelihoods options (effective with `-g' or `-u'):\n\n"); + fprintf(stderr, " -e INT Phred-scaled gap extension seq error probability [%d]\n", mplp.extQ); + fprintf(stderr, " -F FLOAT minimum fraction of gapped reads for candidates [%g]\n", mplp.min_frac); + fprintf(stderr, " -h INT coefficient for homopolymer errors [%d]\n", mplp.tandemQ); + fprintf(stderr, " -I do not perform indel calling\n"); + fprintf(stderr, " -L INT max per-sample depth for INDEL calling [%d]\n", mplp.max_indel_depth); + fprintf(stderr, " -m INT minimum gapped reads for indel candidates [%d]\n", mplp.min_support); + fprintf(stderr, " -o INT Phred-scaled gap open sequencing error probability [%d]\n", mplp.openQ); + fprintf(stderr, " -P STR comma separated list of platforms for indels [all]\n"); fprintf(stderr, "\n"); - free(fn_list); free(fn_fa); free(d); + fprintf(stderr, "Notes: Assuming diploid individuals.\n\n"); return 1; } - if (fn_fa) d->fai = fai_load(fn_fa); - if (d->format & (BAM_PLF_CNS|BAM_PLF_GLF)) bam_maqcns_prepare(d->c); // consensus calling - if (d->format & BAM_PLF_GLF) { // for glf output - glf3_header_t *h; - h = glf3_header_init(); - d->fp_glf = bgzf_fdopen(fileno(stdout), "w"); - glf3_header_write(d->fp_glf, h); - glf3_header_destroy(h); - } - if (d->fai == 0 && (d->format & (BAM_PLF_CNS|BAM_PLF_INDEL_ONLY))) - fprintf(stderr, "[bam_pileup] indels will not be called when -f is absent.\n"); - if (fn_fa && is_SAM && fn_list == 0) fn_list = samfaipath(fn_fa); - - { - samfile_t *fp; - fp = is_SAM? samopen(argv[optind], "r", fn_list) : samopen(argv[optind], "rb", 0); - if (fp == 0 || fp->header == 0) { - fprintf(stderr, "[bam_pileup] fail to read the header: non-exisiting file or wrong format.\n"); - return 1; - } - d->h = fp->header; - if (fn_pos) d->hash = load_pos(fn_pos, d->h); - sampileup(fp, d->mask, pileup_func, d); - samclose(fp); // d->h will be destroyed here - } - - // free - if (d->format & BAM_PLF_GLF) bgzf_close(d->fp_glf); - if (fn_pos) { // free the hash table - khint_t k; - for (k = kh_begin(d->hash); k < kh_end(d->hash); ++k) - if (kh_exist(d->hash, k)) free(kh_val(d->hash, k)); - kh_destroy(64, d->hash); - } - free(fn_pos); free(fn_list); free(fn_fa); - if (d->fai) fai_destroy(d->fai); - bam_maqcns_destroy(d->c); - free(d->ido); free(d->ref); free(d); + if (file_list) { + if ( read_file_list(file_list,&nfiles,&fn) ) return 1; + mpileup(&mplp,nfiles,fn); + for (c=0; c