#include #include #include #include #include #include #include "htslib/sam.h" #include "htslib/bgzf.h" #include "cram/cram.h" #include "htslib/hfile.h" #include "htslib/khash.h" KHASH_DECLARE(s2i, kh_cstr_t, int64_t) typedef khash_t(s2i) sdict_t; /********************** *** BAM header I/O *** **********************/ bam_hdr_t *bam_hdr_init() { return (bam_hdr_t*)calloc(1, sizeof(bam_hdr_t)); } void bam_hdr_destroy(bam_hdr_t *h) { int32_t i; if (h == NULL) return; if (h->target_name) { for (i = 0; i < h->n_targets; ++i) free(h->target_name[i]); free(h->target_name); free(h->target_len); } free(h->text); free(h->cigar_tab); if (h->sdict) kh_destroy(s2i, (sdict_t*)h->sdict); free(h); } bam_hdr_t *bam_hdr_dup(const bam_hdr_t *h0) { if (h0 == NULL) return NULL; bam_hdr_t *h; if ((h = bam_hdr_init()) == NULL) return NULL; // copy the simple data h->n_targets = h0->n_targets; h->ignore_sam_err = h0->ignore_sam_err; h->l_text = h0->l_text; // Then the pointery stuff h->cigar_tab = NULL; h->sdict = NULL; h->text = (char*)calloc(h->l_text + 1, 1); memcpy(h->text, h0->text, h->l_text); h->target_len = (uint32_t*)calloc(h->n_targets, 4); h->target_name = (char**)calloc(h->n_targets, sizeof(char*)); int i; for (i = 0; i < h->n_targets; ++i) { h->target_len[i] = h0->target_len[i]; h->target_name[i] = strdup(h0->target_name[i]); } return h; } static bam_hdr_t *hdr_from_dict(sdict_t *d) { bam_hdr_t *h; khint_t k; h = bam_hdr_init(); h->sdict = d; h->n_targets = kh_size(d); h->target_len = (uint32_t*)malloc(4 * h->n_targets); h->target_name = (char**)malloc(sizeof(char*) * h->n_targets); for (k = kh_begin(d); k != kh_end(d); ++k) { if (!kh_exist(d, k)) continue; h->target_name[kh_val(d, k)>>32] = (char*)kh_key(d, k); h->target_len[kh_val(d, k)>>32] = kh_val(d, k)<<32>>32; kh_val(d, k) >>= 32; } return h; } bam_hdr_t *bam_hdr_read(BGZF *fp) { bam_hdr_t *h; char buf[4]; int magic_len, has_EOF; int32_t i = 1, name_len; // check EOF has_EOF = bgzf_check_EOF(fp); if (has_EOF < 0) { perror("[W::sam_hdr_read] bgzf_check_EOF"); } else if (has_EOF == 0 && hts_verbose >= 2) fprintf(stderr, "[W::%s] EOF marker is absent. The input is probably truncated.\n", __func__); // read "BAM1" magic_len = bgzf_read(fp, buf, 4); if (magic_len != 4 || strncmp(buf, "BAM\1", 4)) { if (hts_verbose >= 1) fprintf(stderr, "[E::%s] invalid BAM binary header\n", __func__); return 0; } h = bam_hdr_init(); // read plain text and the number of reference sequences bgzf_read(fp, &h->l_text, 4); if (fp->is_be) ed_swap_4p(&h->l_text); h->text = (char*)malloc(h->l_text + 1); h->text[h->l_text] = 0; // make sure it is NULL terminated bgzf_read(fp, h->text, h->l_text); bgzf_read(fp, &h->n_targets, 4); if (fp->is_be) ed_swap_4p(&h->n_targets); // read reference sequence names and lengths h->target_name = (char**)calloc(h->n_targets, sizeof(char*)); h->target_len = (uint32_t*)calloc(h->n_targets, 4); for (i = 0; i != h->n_targets; ++i) { bgzf_read(fp, &name_len, 4); if (fp->is_be) ed_swap_4p(&name_len); h->target_name[i] = (char*)calloc(name_len, 1); bgzf_read(fp, h->target_name[i], name_len); bgzf_read(fp, &h->target_len[i], 4); if (fp->is_be) ed_swap_4p(&h->target_len[i]); } return h; } int bam_hdr_write(BGZF *fp, const bam_hdr_t *h) { char buf[4]; int32_t i, name_len, x; // write "BAM1" strncpy(buf, "BAM\1", 4); bgzf_write(fp, buf, 4); // write plain text and the number of reference sequences if (fp->is_be) { x = ed_swap_4(h->l_text); bgzf_write(fp, &x, 4); if (h->l_text) bgzf_write(fp, h->text, h->l_text); x = ed_swap_4(h->n_targets); bgzf_write(fp, &x, 4); } else { bgzf_write(fp, &h->l_text, 4); if (h->l_text) bgzf_write(fp, h->text, h->l_text); bgzf_write(fp, &h->n_targets, 4); } // write sequence names and lengths for (i = 0; i != h->n_targets; ++i) { char *p = h->target_name[i]; name_len = strlen(p) + 1; if (fp->is_be) { x = ed_swap_4(name_len); bgzf_write(fp, &x, 4); } else bgzf_write(fp, &name_len, 4); bgzf_write(fp, p, name_len); if (fp->is_be) { x = ed_swap_4(h->target_len[i]); bgzf_write(fp, &x, 4); } else bgzf_write(fp, &h->target_len[i], 4); } bgzf_flush(fp); return 0; } int bam_name2id(bam_hdr_t *h, const char *ref) { sdict_t *d = (sdict_t*)h->sdict; khint_t k; if (h->sdict == 0) { int i, absent; d = kh_init(s2i); for (i = 0; i < h->n_targets; ++i) { k = kh_put(s2i, d, h->target_name[i], &absent); kh_val(d, k) = i; } h->sdict = d; } k = kh_get(s2i, d, ref); return k == kh_end(d)? -1 : kh_val(d, k); } /************************* *** BAM alignment I/O *** *************************/ bam1_t *bam_init1() { return (bam1_t*)calloc(1, sizeof(bam1_t)); } void bam_destroy1(bam1_t *b) { if (b == 0) return; free(b->data); free(b); } bam1_t *bam_copy1(bam1_t *bdst, const bam1_t *bsrc) { uint8_t *data = bdst->data; int m_data = bdst->m_data; // backup data and m_data if (m_data < bsrc->l_data) { // double the capacity m_data = bsrc->l_data; kroundup32(m_data); data = (uint8_t*)realloc(data, m_data); } memcpy(data, bsrc->data, bsrc->l_data); // copy var-len data *bdst = *bsrc; // copy the rest // restore the backup bdst->m_data = m_data; bdst->data = data; return bdst; } bam1_t *bam_dup1(const bam1_t *bsrc) { if (bsrc == NULL) return NULL; bam1_t *bdst = bam_init1(); if (bdst == NULL) return NULL; return bam_copy1(bdst, bsrc); } int bam_cigar2qlen(int n_cigar, const uint32_t *cigar) { int k, l; for (k = l = 0; k < n_cigar; ++k) if (bam_cigar_type(bam_cigar_op(cigar[k]))&1) l += bam_cigar_oplen(cigar[k]); return l; } int bam_cigar2rlen(int n_cigar, const uint32_t *cigar) { int k, l; for (k = l = 0; k < n_cigar; ++k) if (bam_cigar_type(bam_cigar_op(cigar[k]))&2) l += bam_cigar_oplen(cigar[k]); return l; } int32_t bam_endpos(const bam1_t *b) { if (!(b->core.flag & BAM_FUNMAP) && b->core.n_cigar > 0) return b->core.pos + bam_cigar2rlen(b->core.n_cigar, bam_get_cigar(b)); else return b->core.pos + 1; } static inline int aux_type2size(uint8_t type) { switch (type) { case 'A': case 'c': case 'C': return 1; case 's': case 'S': return 2; case 'i': case 'I': case 'f': return 4; case 'd': return 8; case 'Z': case 'H': case 'B': return type; default: return 0; } } static void swap_data(const bam1_core_t *c, int l_data, uint8_t *data, int is_host) { uint8_t *s; uint32_t *cigar = (uint32_t*)(data + c->l_qname); uint32_t i, n; s = data + c->n_cigar*4 + c->l_qname + c->l_qseq + (c->l_qseq + 1)/2; for (i = 0; i < c->n_cigar; ++i) ed_swap_4p(&cigar[i]); while (s < data + l_data) { int size; s += 2; // skip key size = aux_type2size(*s); ++s; // skip type switch (size) { case 1: ++s; break; case 2: ed_swap_2p(s); s += 2; break; case 4: ed_swap_4p(s); s += 4; break; case 8: ed_swap_8p(s); s += 8; break; case 'Z': case 'H': while (*s) ++s; ++s; break; case 'B': size = aux_type2size(*s); ++s; if (is_host) memcpy(&n, s, 4), ed_swap_4p(s); else ed_swap_4p(s), memcpy(&n, s, 4); s += 4; switch (size) { case 1: s += n; break; case 2: for (i = 0; i < n; ++i, s += 2) ed_swap_2p(s); break; case 4: for (i = 0; i < n; ++i, s += 4) ed_swap_4p(s); break; case 8: for (i = 0; i < n; ++i, s += 8) ed_swap_8p(s); break; } break; } } } int bam_read1(BGZF *fp, bam1_t *b) { bam1_core_t *c = &b->core; int32_t block_len, ret, i; uint32_t x[8]; if ((ret = bgzf_read(fp, &block_len, 4)) != 4) { if (ret == 0) return -1; // normal end-of-file else return -2; // truncated } if (bgzf_read(fp, x, 32) != 32) return -3; if (fp->is_be) { ed_swap_4p(&block_len); for (i = 0; i < 8; ++i) ed_swap_4p(x + i); } c->tid = x[0]; c->pos = x[1]; c->bin = x[2]>>16; c->qual = x[2]>>8&0xff; c->l_qname = x[2]&0xff; c->flag = x[3]>>16; c->n_cigar = x[3]&0xffff; c->l_qseq = x[4]; c->mtid = x[5]; c->mpos = x[6]; c->isize = x[7]; b->l_data = block_len - 32; if (b->l_data < 0 || c->l_qseq < 0) return -4; if ((char *)bam_get_aux(b) - (char *)b->data > b->l_data) return -4; if (b->m_data < b->l_data) { b->m_data = b->l_data; kroundup32(b->m_data); b->data = (uint8_t*)realloc(b->data, b->m_data); if (!b->data) return -4; } if (bgzf_read(fp, b->data, b->l_data) != b->l_data) return -4; //b->l_aux = b->l_data - c->n_cigar * 4 - c->l_qname - c->l_qseq - (c->l_qseq+1)/2; if (fp->is_be) swap_data(c, b->l_data, b->data, 0); return 4 + block_len; } int bam_write1(BGZF *fp, const bam1_t *b) { const bam1_core_t *c = &b->core; uint32_t x[8], block_len = b->l_data + 32, y; int i, ok; x[0] = c->tid; x[1] = c->pos; x[2] = (uint32_t)c->bin<<16 | c->qual<<8 | c->l_qname; x[3] = (uint32_t)c->flag<<16 | c->n_cigar; x[4] = c->l_qseq; x[5] = c->mtid; x[6] = c->mpos; x[7] = c->isize; ok = (bgzf_flush_try(fp, 4 + block_len) >= 0); if (fp->is_be) { for (i = 0; i < 8; ++i) ed_swap_4p(x + i); y = block_len; if (ok) ok = (bgzf_write(fp, ed_swap_4p(&y), 4) >= 0); swap_data(c, b->l_data, b->data, 1); } else { if (ok) ok = (bgzf_write(fp, &block_len, 4) >= 0); } if (ok) ok = (bgzf_write(fp, x, 32) >= 0); if (ok) ok = (bgzf_write(fp, b->data, b->l_data) >= 0); if (fp->is_be) swap_data(c, b->l_data, b->data, 0); return ok? 4 + block_len : -1; } /******************** *** BAM indexing *** ********************/ static hts_idx_t *bam_index(BGZF *fp, int min_shift) { int n_lvls, i, fmt; bam1_t *b; hts_idx_t *idx; bam_hdr_t *h; h = bam_hdr_read(fp); if (min_shift > 0) { int64_t max_len = 0, s; for (i = 0; i < h->n_targets; ++i) if (max_len < h->target_len[i]) max_len = h->target_len[i]; max_len += 256; for (n_lvls = 0, s = 1< s; ++n_lvls, s <<= 3); fmt = HTS_FMT_CSI; } else min_shift = 14, n_lvls = 5, fmt = HTS_FMT_BAI; idx = hts_idx_init(h->n_targets, fmt, bgzf_tell(fp), min_shift, n_lvls); bam_hdr_destroy(h); b = bam_init1(); while (bam_read1(fp, b) >= 0) { int l, ret; l = bam_cigar2rlen(b->core.n_cigar, bam_get_cigar(b)); if (l == 0) l = 1; // no zero-length records ret = hts_idx_push(idx, b->core.tid, b->core.pos, b->core.pos + l, bgzf_tell(fp), !(b->core.flag&BAM_FUNMAP)); if (ret < 0) { // unsorted bam_destroy1(b); hts_idx_destroy(idx); return NULL; } } hts_idx_finish(idx, bgzf_tell(fp)); bam_destroy1(b); return idx; } int bam_index_build(const char *fn, int min_shift) { hts_idx_t *idx; htsFile *fp; int ret = 0; if ((fp = hts_open(fn, "r")) == 0) return -1; if (fp->is_cram) { ret = cram_index_build(fp->fp.cram, fn); } else { idx = bam_index(fp->fp.bgzf, min_shift); if ( !idx ) { hts_close(fp); return -1; } hts_idx_save(idx, fn, min_shift > 0 ? HTS_FMT_CSI : HTS_FMT_BAI); hts_idx_destroy(idx); } hts_close(fp); return ret; } static int bam_readrec(BGZF *fp, void *ignored, void *bv, int *tid, int *beg, int *end) { bam1_t *b = bv; int ret; if ((ret = bam_read1(fp, b)) >= 0) { *tid = b->core.tid; *beg = b->core.pos; *end = b->core.pos + (b->core.n_cigar? bam_cigar2rlen(b->core.n_cigar, bam_get_cigar(b)) : 1); } return ret; } // This is used only with read_rest=1 iterators, so need not set tid/beg/end. static int cram_readrec(BGZF *ignored, void *fpv, void *bv, int *tid, int *beg, int *end) { htsFile *fp = fpv; bam1_t *b = bv; return cram_get_bam_seq(fp->fp.cram, &b); } // This is used only with read_rest=1 iterators, so need not set tid/beg/end. static int sam_bam_cram_readrec(BGZF *bgzfp, void *fpv, void *bv, int *tid, int *beg, int *end) { htsFile *fp = fpv; bam1_t *b = bv; if (fp->is_bin) return bam_read1(bgzfp, b); else if (fp->is_cram) return cram_get_bam_seq(fp->fp.cram, &b); else { // TODO Need headers available to implement this for SAM files fprintf(stderr, "[sam_bam_cram_readrec] Not implemented for SAM files -- Exiting\n"); abort(); } } // The CRAM implementation stores the loaded index within the cram_fd rather // than separately as is done elsewhere in htslib. So if p is a pointer to // an hts_idx_t with p->fmt == HTS_FMT_CRAI, then it actually points to an // hts_cram_idx_t and should be cast accordingly. typedef struct hts_cram_idx_t { int fmt; cram_fd *cram; } hts_cram_idx_t; hts_idx_t *sam_index_load(samFile *fp, const char *fn) { if (fp->is_bin) return bam_index_load(fn); else if (fp->is_cram) { if (cram_index_load(fp->fp.cram, fn) < 0) return NULL; // Cons up a fake "index" just pointing at the associated cram_fd: hts_cram_idx_t *idx = malloc(sizeof (hts_cram_idx_t)); if (idx == NULL) return NULL; idx->fmt = HTS_FMT_CRAI; idx->cram = fp->fp.cram; return (hts_idx_t *) idx; } else return NULL; // TODO Would use tbx_index_load if it returned hts_idx_t } static hts_itr_t *cram_itr_query(const hts_idx_t *idx, int tid, int beg, int end, hts_readrec_func *readrec) { const hts_cram_idx_t *cidx = (const hts_cram_idx_t *) idx; hts_itr_t *iter = (hts_itr_t *) calloc(1, sizeof(hts_itr_t)); if (iter == NULL) return NULL; // Cons up a dummy iterator for which hts_itr_next() will simply invoke // the readrec function: iter->read_rest = 1; iter->off = NULL; iter->bins.a = NULL; iter->readrec = readrec; if (tid >= 0) { cram_range r = { tid, beg+1, end }; if (cram_set_option(cidx->cram, CRAM_OPT_RANGE, &r) != 0) { free(iter); return NULL; } iter->curr_off = 0; // The following fields are not required by hts_itr_next(), but are // filled in in case user code wants to look at them. iter->tid = tid; iter->beg = beg; iter->end = end; } else switch (tid) { case HTS_IDX_REST: iter->curr_off = 0; break; case HTS_IDX_NONE: iter->curr_off = 0; iter->finished = 1; break; default: fprintf(stderr, "[cram_itr_query] tid=%d not implemented for CRAM files -- Exiting\n", tid); abort(); break; } return iter; } hts_itr_t *sam_itr_queryi(const hts_idx_t *idx, int tid, int beg, int end) { const hts_cram_idx_t *cidx = (const hts_cram_idx_t *) idx; if (idx == NULL) return hts_itr_query(NULL, tid, beg, end, sam_bam_cram_readrec); else if (cidx->fmt == HTS_FMT_CRAI) return cram_itr_query(idx, tid, beg, end, cram_readrec); else return hts_itr_query(idx, tid, beg, end, bam_readrec); } static int cram_name2id(void *fdv, const char *ref) { cram_fd *fd = (cram_fd *) fdv; return sam_hdr_name2ref(fd->header, ref); } hts_itr_t *sam_itr_querys(const hts_idx_t *idx, bam_hdr_t *hdr, const char *region) { const hts_cram_idx_t *cidx = (const hts_cram_idx_t *) idx; if (cidx->fmt == HTS_FMT_CRAI) return hts_itr_querys(idx, region, cram_name2id, cidx->cram, cram_itr_query, cram_readrec); else return hts_itr_querys(idx, region, (hts_name2id_f)(bam_name2id), hdr, hts_itr_query, bam_readrec); } /********************** *** SAM header I/O *** **********************/ #include "htslib/kseq.h" #include "htslib/kstring.h" bam_hdr_t *sam_hdr_parse(int l_text, const char *text) { const char *q, *r, *p; khash_t(s2i) *d; d = kh_init(s2i); for (p = text; *p; ++p) { if (strncmp(p, "@SQ", 3) == 0) { char *sn = 0; int ln = -1; for (q = p + 4;; ++q) { if (strncmp(q, "SN:", 3) == 0) { q += 3; for (r = q; *r != '\t' && *r != '\n'; ++r); sn = (char*)calloc(r - q + 1, 1); strncpy(sn, q, r - q); q = r; } else if (strncmp(q, "LN:", 3) == 0) ln = strtol(q + 3, (char**)&q, 10); while (*q != '\t' && *q != '\n') ++q; if (*q == '\n') break; } p = q; if (sn && ln >= 0) { khint_t k; int absent; k = kh_put(s2i, d, sn, &absent); if (!absent) { if (hts_verbose >= 2) fprintf(stderr, "[W::%s] duplicated sequence '%s'\n", __func__, sn); free(sn); } else kh_val(d, k) = (int64_t)(kh_size(d) - 1)<<32 | ln; } } while (*p != '\n') ++p; } return hdr_from_dict(d); } bam_hdr_t *sam_hdr_read(htsFile *fp) { if (fp->is_bin) { return bam_hdr_read(fp->fp.bgzf); } else if (fp->is_cram) { return cram_header_to_bam(fp->fp.cram->header); } else { kstring_t str; bam_hdr_t *h; int has_SQ = 0; str.l = str.m = 0; str.s = 0; while (hts_getline(fp, KS_SEP_LINE, &fp->line) >= 0) { if (fp->line.s[0] != '@') break; if (fp->line.l > 3 && strncmp(fp->line.s,"@SQ",3) == 0) has_SQ = 1; kputsn(fp->line.s, fp->line.l, &str); kputc('\n', &str); } if (! has_SQ && fp->fn_aux) { char line[2048]; FILE *f = fopen(fp->fn_aux, "r"); if (f == NULL) return NULL; while (fgets(line, sizeof line, f)) { const char *name = strtok(line, "\t"); const char *length = strtok(NULL, "\t"); ksprintf(&str, "@SQ\tSN:%s\tLN:%s\n", name, length); } fclose(f); } if (str.l == 0) kputsn("", 0, &str); h = sam_hdr_parse(str.l, str.s); h->l_text = str.l; h->text = str.s; return h; } } int sam_hdr_write(htsFile *fp, const bam_hdr_t *h) { if (fp->is_bin) { bam_hdr_write(fp->fp.bgzf, h); } else if (fp->is_cram) { cram_fd *fd = fp->fp.cram; if (cram_set_header(fd, bam_header_to_cram((bam_hdr_t *)h)) < 0) return -1; if (fp->fn_aux) cram_load_reference(fd, fp->fn_aux); if (cram_write_SAM_hdr(fd, fd->header) < 0) return -1; } else { char *p; hputs(h->text, fp->fp.hfile); p = strstr(h->text, "@SQ\t"); // FIXME: we need a loop to make sure "@SQ\t" does not match something unwanted!!! if (p == 0) { int i; for (i = 0; i < h->n_targets; ++i) { fp->line.l = 0; kputsn("@SQ\tSN:", 7, &fp->line); kputs(h->target_name[i], &fp->line); kputsn("\tLN:", 4, &fp->line); kputw(h->target_len[i], &fp->line); kputc('\n', &fp->line); if ( hwrite(fp->fp.hfile, fp->line.s, fp->line.l) != fp->line.l ) return -1; } } if ( hflush(fp->fp.hfile) != 0 ) return -1; } return 0; } /********************** *** SAM record I/O *** **********************/ int sam_parse1(kstring_t *s, bam_hdr_t *h, bam1_t *b) { #define _read_token(_p) (_p); for (; *(_p) && *(_p) != '\t'; ++(_p)); if (*(_p) != '\t') goto err_ret; *(_p)++ = 0 #define _read_token_aux(_p) (_p); for (; *(_p) && *(_p) != '\t'; ++(_p)); *(_p)++ = 0 // this is different in that it does not test *(_p)=='\t' #define _get_mem(type_t, _x, _s, _l) ks_resize((_s), (_s)->l + (_l)); *(_x) = (type_t*)((_s)->s + (_s)->l); (_s)->l += (_l) #define _parse_err(cond, msg) do { if ((cond) && hts_verbose >= 1) { fprintf(stderr, "[E::%s] " msg "\n", __func__); goto err_ret; } } while (0) #define _parse_warn(cond, msg) if ((cond) && hts_verbose >= 2) fprintf(stderr, "[W::%s] " msg "\n", __func__) uint8_t *t; char *p = s->s, *q; int i; kstring_t str; bam1_core_t *c = &b->core; str.l = b->l_data = 0; str.s = (char*)b->data; str.m = b->m_data; memset(c, 0, 32); if (h->cigar_tab == 0) { h->cigar_tab = (int8_t*) malloc(128); for (i = 0; i < 128; ++i) h->cigar_tab[i] = -1; for (i = 0; BAM_CIGAR_STR[i]; ++i) h->cigar_tab[(int)BAM_CIGAR_STR[i]] = i; } // qname q = _read_token(p); kputsn_(q, p - q, &str); c->l_qname = p - q; // flag c->flag = strtol(p, &p, 0); if (*p++ != '\t') goto err_ret; // malformated flag // chr q = _read_token(p); if (strcmp(q, "*")) { _parse_err(h->n_targets == 0, "missing SAM header"); c->tid = bam_name2id(h, q); _parse_warn(c->tid < 0, "urecognized reference name; treated as unmapped"); } else c->tid = -1; // pos c->pos = strtol(p, &p, 10) - 1; if (*p++ != '\t') goto err_ret; if (c->pos < 0 && c->tid >= 0) { _parse_warn(1, "mapped query cannot have zero coordinate; treated as unmapped"); c->tid = -1; } if (c->tid < 0) c->flag |= BAM_FUNMAP; // mapq c->qual = strtol(p, &p, 10); if (*p++ != '\t') goto err_ret; // cigar if (*p != '*') { uint32_t *cigar; size_t n_cigar = 0; for (q = p; *p && *p != '\t'; ++p) if (!isdigit(*p)) ++n_cigar; if (*p++ != '\t') goto err_ret; _parse_err(n_cigar >= 65536, "too many CIGAR operations"); c->n_cigar = n_cigar; _get_mem(uint32_t, &cigar, &str, c->n_cigar<<2); for (i = 0; i < c->n_cigar; ++i, ++q) { int op; cigar[i] = strtol(q, &q, 10)<= 128? -1 : h->cigar_tab[(int)*q]; _parse_err(op < 0, "unrecognized CIGAR operator"); cigar[i] |= op; } i = bam_cigar2rlen(c->n_cigar, cigar); } else { _parse_warn(!(c->flag&BAM_FUNMAP), "mapped query must have a CIGAR; treated as unmapped"); c->flag |= BAM_FUNMAP; q = _read_token(p); i = 1; } c->bin = hts_reg2bin(c->pos, c->pos + i, 14, 5); // mate chr q = _read_token(p); if (strcmp(q, "=") == 0) c->mtid = c->tid; else if (strcmp(q, "*") == 0) c->mtid = -1; else c->mtid = bam_name2id(h, q); // mpos c->mpos = strtol(p, &p, 10) - 1; if (*p++ != '\t') goto err_ret; if (c->mpos < 0 && c->mtid >= 0) { _parse_warn(1, "mapped mate cannot have zero coordinate; treated as unmapped"); c->mtid = -1; } // tlen c->isize = strtol(p, &p, 10); if (*p++ != '\t') goto err_ret; // seq q = _read_token(p); if (strcmp(q, "*")) { c->l_qseq = p - q - 1; i = bam_cigar2qlen(c->n_cigar, (uint32_t*)(str.s + c->l_qname)); _parse_err(c->n_cigar && i != c->l_qseq, "CIGAR and query sequence are of different length"); i = (c->l_qseq + 1) >> 1; _get_mem(uint8_t, &t, &str, i); memset(t, 0, i); for (i = 0; i < c->l_qseq; ++i) t[i>>1] |= seq_nt16_table[(int)q[i]] << ((~i&1)<<2); } else c->l_qseq = 0; // qual q = _read_token_aux(p); _get_mem(uint8_t, &t, &str, c->l_qseq); if (strcmp(q, "*")) { _parse_err(p - q - 1 != c->l_qseq, "SEQ and QUAL are of different length"); for (i = 0; i < c->l_qseq; ++i) t[i] = q[i] - 33; } else memset(t, 0xff, c->l_qseq); // aux // Note that (like the bam1_core_t fields) this aux data in b->data is // stored in host endianness; so there is no byte swapping needed here. while (p < s->s + s->l) { uint8_t type; q = _read_token_aux(p); // FIXME: can be accelerated for long 'B' arrays _parse_err(p - q - 1 < 6, "incomplete aux field"); kputsn_(q, 2, &str); q += 3; type = *q++; ++q; // q points to value if (type == 'A' || type == 'a' || type == 'c' || type == 'C') { kputc_('A', &str); kputc_(*q, &str); } else if (type == 'i' || type == 'I') { long x; x = strtol(q, &q, 10); if (x < 0) { if (x >= INT8_MIN) { kputc_('c', &str); kputc_(x, &str); } else if (x >= INT16_MIN) { int16_t y = x; kputc_('s', &str); kputsn_((char*)&y, 2, &str); } else { int32_t y = x; kputc_('i', &str); kputsn_(&y, 4, &str); } } else { if (x <= UINT8_MAX) { kputc_('C', &str); kputc_(x, &str); } else if (x <= UINT16_MAX) { uint16_t y = x; kputc_('S', &str); kputsn_(&y, 2, &str); } else { uint32_t y = x; kputc_('I', &str); kputsn_(&y, 4, &str); } } } else if (type == 'f') { float x; x = strtod(q, &q); kputc_('f', &str); kputsn_(&x, 4, &str); } else if (type == 'd') { double x; x = strtod(q, &q); kputc_('d', &str); kputsn_(&x, 8, &str); } else if (type == 'Z' || type == 'H') { kputc_(type, &str);kputsn_(q, p - q, &str); // note that this include the trailing NULL } else if (type == 'B') { int32_t n; char *r; _parse_err(p - q - 1 < 3, "incomplete B-typed aux field"); type = *q++; // q points to the first ',' following the typing byte for (r = q, n = 0; *r; ++r) if (*r == ',') ++n; kputc_('B', &str); kputc_(type, &str); kputsn_(&n, 4, &str); // FIXME: to evaluate which is faster: a) aligned array and then memmove(); b) unaligned array; c) kputsn_() if (type == 'c') while (q + 1 < p) { int8_t x = strtol(q + 1, &q, 0); kputc_(x, &str); } else if (type == 'C') while (q + 1 < p) { uint8_t x = strtoul(q + 1, &q, 0); kputc_(x, &str); } else if (type == 's') while (q + 1 < p) { int16_t x = strtol(q + 1, &q, 0); kputsn_(&x, 2, &str); } else if (type == 'S') while (q + 1 < p) { uint16_t x = strtoul(q + 1, &q, 0); kputsn_(&x, 2, &str); } else if (type == 'i') while (q + 1 < p) { int32_t x = strtol(q + 1, &q, 0); kputsn_(&x, 4, &str); } else if (type == 'I') while (q + 1 < p) { uint32_t x = strtoul(q + 1, &q, 0); kputsn_(&x, 4, &str); } else if (type == 'f') while (q + 1 < p) { float x = strtod(q + 1, &q); kputsn_(&x, 4, &str); } else _parse_err(1, "unrecognized type"); } else _parse_err(1, "unrecognized type"); } b->data = (uint8_t*)str.s; b->l_data = str.l; b->m_data = str.m; return 0; #undef _parse_warn #undef _parse_err #undef _get_mem #undef _read_token_aux #undef _read_token err_ret: b->data = (uint8_t*)str.s; b->l_data = str.l; b->m_data = str.m; return -2; } int sam_read1(htsFile *fp, bam_hdr_t *h, bam1_t *b) { if (fp->is_bin) { int r = bam_read1(fp->fp.bgzf, b); if (r >= 0) { if (b->core.tid >= h->n_targets || b->core.tid < -1 || b->core.mtid >= h->n_targets || b->core.mtid < -1) return -3; } return r; } else if (fp->is_cram) { return cram_get_bam_seq(fp->fp.cram, &b); } else { int ret; err_recover: if (fp->line.l == 0) { ret = hts_getline(fp, KS_SEP_LINE, &fp->line); if (ret < 0) return -1; } ret = sam_parse1(&fp->line, h, b); fp->line.l = 0; if (ret < 0) { if (hts_verbose >= 1) fprintf(stderr, "[W::%s] parse error at line %lld\n", __func__, (long long)fp->lineno); if (h->ignore_sam_err) goto err_recover; } return ret; } } int sam_format1(const bam_hdr_t *h, const bam1_t *b, kstring_t *str) { int i; uint8_t *s; const bam1_core_t *c = &b->core; str->l = 0; kputsn(bam_get_qname(b), c->l_qname-1, str); kputc('\t', str); // query name kputw(c->flag, str); kputc('\t', str); // flag if (c->tid >= 0) { // chr kputs(h->target_name[c->tid] , str); kputc('\t', str); } else kputsn("*\t", 2, str); kputw(c->pos + 1, str); kputc('\t', str); // pos kputw(c->qual, str); kputc('\t', str); // qual if (c->n_cigar) { // cigar uint32_t *cigar = bam_get_cigar(b); for (i = 0; i < c->n_cigar; ++i) { kputw(bam_cigar_oplen(cigar[i]), str); kputc(bam_cigar_opchr(cigar[i]), str); } } else kputc('*', str); kputc('\t', str); if (c->mtid < 0) kputsn("*\t", 2, str); // mate chr else if (c->mtid == c->tid) kputsn("=\t", 2, str); else { kputs(h->target_name[c->mtid], str); kputc('\t', str); } kputw(c->mpos + 1, str); kputc('\t', str); // mate pos kputw(c->isize, str); kputc('\t', str); // template len if (c->l_qseq) { // seq and qual uint8_t *s = bam_get_seq(b); for (i = 0; i < c->l_qseq; ++i) kputc("=ACMGRSVTWYHKDBN"[bam_seqi(s, i)], str); kputc('\t', str); s = bam_get_qual(b); if (s[0] == 0xff) kputc('*', str); else for (i = 0; i < c->l_qseq; ++i) kputc(s[i] + 33, str); } else kputsn("*\t*", 3, str); s = bam_get_aux(b); // aux while (s+4 <= b->data + b->l_data) { uint8_t type, key[2]; key[0] = s[0]; key[1] = s[1]; s += 2; type = *s++; kputc('\t', str); kputsn((char*)key, 2, str); kputc(':', str); if (type == 'A') { kputsn("A:", 2, str); kputc(*s, str); ++s; } else if (type == 'C') { kputsn("i:", 2, str); kputw(*s, str); ++s; } else if (type == 'c') { kputsn("i:", 2, str); kputw(*(int8_t*)s, str); ++s; } else if (type == 'S') { if (s+2 <= b->data + b->l_data) { kputsn("i:", 2, str); kputw(*(uint16_t*)s, str); s += 2; } else return -1; } else if (type == 's') { if (s+2 <= b->data + b->l_data) { kputsn("i:", 2, str); kputw(*(int16_t*)s, str); s += 2; } else return -1; } else if (type == 'I') { if (s+4 <= b->data + b->l_data) { kputsn("i:", 2, str); kputuw(*(uint32_t*)s, str); s += 4; } else return -1; } else if (type == 'i') { if (s+4 <= b->data + b->l_data) { kputsn("i:", 2, str); kputw(*(int32_t*)s, str); s += 4; } else return -1; } else if (type == 'f') { if (s+4 <= b->data + b->l_data) { ksprintf(str, "f:%g", *(float*)s); s += 4; } else return -1; } else if (type == 'd') { if (s+8 <= b->data + b->l_data) { ksprintf(str, "d:%g", *(double*)s); s += 8; } else return -1; } else if (type == 'Z' || type == 'H') { kputc(type, str); kputc(':', str); while (s < b->data + b->l_data && *s) kputc(*s++, str); if (s >= b->data + b->l_data) return -1; ++s; } else if (type == 'B') { uint8_t sub_type = *(s++); int32_t n; memcpy(&n, s, 4); s += 4; // no point to the start of the array if (s + n >= b->data + b->l_data) return -1; kputsn("B:", 2, str); kputc(sub_type, str); // write the typing for (i = 0; i < n; ++i) { // FIXME: for better performance, put the loop after "if" kputc(',', str); if ('c' == sub_type) { kputw(*(int8_t*)s, str); ++s; } else if ('C' == sub_type) { kputw(*(uint8_t*)s, str); ++s; } else if ('s' == sub_type) { kputw(*(int16_t*)s, str); s += 2; } else if ('S' == sub_type) { kputw(*(uint16_t*)s, str); s += 2; } else if ('i' == sub_type) { kputw(*(int32_t*)s, str); s += 4; } else if ('I' == sub_type) { kputuw(*(uint32_t*)s, str); s += 4; } else if ('f' == sub_type) { ksprintf(str, "%g", *(float*)s); s += 4; } } } } return str->l; } int sam_write1(htsFile *fp, const bam_hdr_t *h, const bam1_t *b) { if (fp->is_bin) { return bam_write1(fp->fp.bgzf, b); } else if (fp->is_cram) { return cram_put_bam_seq(fp->fp.cram, (bam1_t *)b); } else { if (sam_format1(h, b, &fp->line) < 0) return -1; kputc('\n', &fp->line); if ( hwrite(fp->fp.hfile, fp->line.s, fp->line.l) != fp->line.l ) return -1; return fp->line.l; } } /************************ *** Auxiliary fields *** ************************/ void bam_aux_append(bam1_t *b, const char tag[2], char type, int len, uint8_t *data) { int ori_len = b->l_data; b->l_data += 3 + len; if (b->m_data < b->l_data) { b->m_data = b->l_data; kroundup32(b->m_data); b->data = (uint8_t*)realloc(b->data, b->m_data); } b->data[ori_len] = tag[0]; b->data[ori_len + 1] = tag[1]; b->data[ori_len + 2] = type; memcpy(b->data + ori_len + 3, data, len); } static inline uint8_t *skip_aux(uint8_t *s) { int size = aux_type2size(*s); ++s; // skip type uint32_t n; switch (size) { case 'Z': case 'H': while (*s) ++s; return s + 1; case 'B': size = aux_type2size(*s); ++s; memcpy(&n, s, 4); s += 4; return s + size * n; case 0: abort(); break; default: return s + size; } } uint8_t *bam_aux_get(const bam1_t *b, const char tag[2]) { uint8_t *s; int y = tag[0]<<8 | tag[1]; s = bam_get_aux(b); while (s < b->data + b->l_data) { int x = (int)s[0]<<8 | s[1]; s += 2; if (x == y) return s; s = skip_aux(s); } return 0; } // s MUST BE returned by bam_aux_get() int bam_aux_del(bam1_t *b, uint8_t *s) { uint8_t *p, *aux; int l_aux = bam_get_l_aux(b); aux = bam_get_aux(b); p = s - 2; s = skip_aux(s); memmove(p, s, l_aux - (s - aux)); b->l_data -= s - p; return 0; } int32_t bam_aux2i(const uint8_t *s) { int type; type = *s++; if (type == 'c') return (int32_t)*(int8_t*)s; else if (type == 'C') return (int32_t)*(uint8_t*)s; else if (type == 's') return (int32_t)*(int16_t*)s; else if (type == 'S') return (int32_t)*(uint16_t*)s; else if (type == 'i' || type == 'I') return *(int32_t*)s; else return 0; } double bam_aux2f(const uint8_t *s) { int type; type = *s++; if (type == 'd') return *(double*)s; else if (type == 'f') return *(float*)s; else return 0.0; } char bam_aux2A(const uint8_t *s) { int type; type = *s++; if (type == 'A') return *(char*)s; else return 0; } char *bam_aux2Z(const uint8_t *s) { int type; type = *s++; if (type == 'Z' || type == 'H') return (char*)s; else return 0; } int sam_open_mode(char *mode, const char *fn, const char *format) { // TODO Parse "bam5" etc for compression level if (format == NULL) { // Try to pick a format based on the filename extension const char *ext = fn? strrchr(fn, '.') : NULL; if (ext == NULL || strchr(ext, '/')) return -1; return sam_open_mode(mode, fn, ext+1); } else if (strcmp(format, "bam") == 0) strcpy(mode, "b"); else if (strcmp(format, "cram") == 0) strcpy(mode, "c"); else if (strcmp(format, "sam") == 0) strcpy(mode, ""); else return -1; return 0; } #define STRNCMP(a,b,n) (strncasecmp((a),(b),(n)) || strlen(a)!=(n)) int bam_str2flag(const char *str) { char *end, *beg = (char*) str; long int flag = strtol(str, &end, 0); if ( end!=str ) return flag; // the conversion was successful flag = 0; while ( *str ) { end = beg; while ( *end && *end!=',' ) end++; if ( !STRNCMP("PAIRED",beg,end-beg) ) flag |= BAM_FPAIRED; else if ( !STRNCMP("PROPER_PAIR",beg,end-beg) ) flag |= BAM_FPROPER_PAIR; else if ( !STRNCMP("UNMAP",beg,end-beg) ) flag |= BAM_FUNMAP; else if ( !STRNCMP("MUNMAP",beg,end-beg) ) flag |= BAM_FMUNMAP; else if ( !STRNCMP("REVERSE",beg,end-beg) ) flag |= BAM_FREVERSE; else if ( !STRNCMP("MREVERSE",beg,end-beg) ) flag |= BAM_FMREVERSE; else if ( !STRNCMP("READ1",beg,end-beg) ) flag |= BAM_FREAD1; else if ( !STRNCMP("READ2",beg,end-beg) ) flag |= BAM_FREAD2; else if ( !STRNCMP("SECONDARY",beg,end-beg) ) flag |= BAM_FSECONDARY; else if ( !STRNCMP("QCFAIL",beg,end-beg) ) flag |= BAM_FQCFAIL; else if ( !STRNCMP("DUP",beg,end-beg) ) flag |= BAM_FDUP; else if ( !STRNCMP("SUPPLEMENTARY",beg,end-beg) ) flag |= BAM_FSUPPLEMENTARY; else return -1; if ( !*end ) break; beg = end + 1; } return flag; } char *bam_flag2str(int flag) { kstring_t str = {0,0,0}; if ( flag&BAM_FPAIRED ) ksprintf(&str,"%s%s", str.l?",":"","PAIRED"); if ( flag&BAM_FPROPER_PAIR ) ksprintf(&str,"%s%s", str.l?",":"","PROPER_PAIR"); if ( flag&BAM_FUNMAP ) ksprintf(&str,"%s%s", str.l?",":"","UNMAP"); if ( flag&BAM_FMUNMAP ) ksprintf(&str,"%s%s", str.l?",":"","MUNMAP"); if ( flag&BAM_FREVERSE ) ksprintf(&str,"%s%s", str.l?",":"","REVERSE"); if ( flag&BAM_FMREVERSE ) ksprintf(&str,"%s%s", str.l?",":"","MREVERSE"); if ( flag&BAM_FREAD1 ) ksprintf(&str,"%s%s", str.l?",":"","READ1"); if ( flag&BAM_FREAD2 ) ksprintf(&str,"%s%s", str.l?",":"","READ2"); if ( flag&BAM_FSECONDARY ) ksprintf(&str,"%s%s", str.l?",":"","SECONDARY"); if ( flag&BAM_FQCFAIL ) ksprintf(&str,"%s%s", str.l?",":"","QCFAIL"); if ( flag&BAM_FDUP ) ksprintf(&str,"%s%s", str.l?",":"","DUP"); if ( flag&BAM_FSUPPLEMENTARY ) ksprintf(&str,"%s%s", str.l?",":"","SUPPLEMENTARY"); if ( str.l == 0 ) kputsn("", 0, &str); return str.s; } /************************** *** Pileup and Mpileup *** **************************/ #if !defined(BAM_NO_PILEUP) #include /******************* *** Memory pool *** *******************/ typedef struct { int k, x, y, end; } cstate_t; static cstate_t g_cstate_null = { -1, 0, 0, 0 }; typedef struct __linkbuf_t { bam1_t b; int32_t beg, end; cstate_t s; struct __linkbuf_t *next; } lbnode_t; typedef struct { int cnt, n, max; lbnode_t **buf; } mempool_t; static mempool_t *mp_init(void) { mempool_t *mp; mp = (mempool_t*)calloc(1, sizeof(mempool_t)); return mp; } static void mp_destroy(mempool_t *mp) { int k; for (k = 0; k < mp->n; ++k) { free(mp->buf[k]->b.data); free(mp->buf[k]); } free(mp->buf); free(mp); } static inline lbnode_t *mp_alloc(mempool_t *mp) { ++mp->cnt; if (mp->n == 0) return (lbnode_t*)calloc(1, sizeof(lbnode_t)); else return mp->buf[--mp->n]; } static inline void mp_free(mempool_t *mp, lbnode_t *p) { --mp->cnt; p->next = 0; // clear lbnode_t::next here if (mp->n == mp->max) { mp->max = mp->max? mp->max<<1 : 256; mp->buf = (lbnode_t**)realloc(mp->buf, sizeof(lbnode_t*) * mp->max); } mp->buf[mp->n++] = p; } /********************** *** CIGAR resolver *** **********************/ /* s->k: the index of the CIGAR operator that has just been processed. s->x: the reference coordinate of the start of s->k s->y: the query coordiante of the start of s->k */ static inline int resolve_cigar2(bam_pileup1_t *p, int32_t pos, cstate_t *s) { #define _cop(c) ((c)&BAM_CIGAR_MASK) #define _cln(c) ((c)>>BAM_CIGAR_SHIFT) bam1_t *b = p->b; bam1_core_t *c = &b->core; uint32_t *cigar = bam_get_cigar(b); int k; // determine the current CIGAR operation // fprintf(stderr, "%s\tpos=%d\tend=%d\t(%d,%d,%d)\n", bam_get_qname(b), pos, s->end, s->k, s->x, s->y); if (s->k == -1) { // never processed if (c->n_cigar == 1) { // just one operation, save a loop if (_cop(cigar[0]) == BAM_CMATCH || _cop(cigar[0]) == BAM_CEQUAL || _cop(cigar[0]) == BAM_CDIFF) s->k = 0, s->x = c->pos, s->y = 0; } else { // find the first match or deletion for (k = 0, s->x = c->pos, s->y = 0; k < c->n_cigar; ++k) { int op = _cop(cigar[k]); int l = _cln(cigar[k]); if (op == BAM_CMATCH || op == BAM_CDEL || op == BAM_CEQUAL || op == BAM_CDIFF) break; else if (op == BAM_CREF_SKIP) s->x += l; else if (op == BAM_CINS || op == BAM_CSOFT_CLIP) s->y += l; } assert(k < c->n_cigar); s->k = k; } } else { // the read has been processed before int op, l = _cln(cigar[s->k]); if (pos - s->x >= l) { // jump to the next operation assert(s->k < c->n_cigar); // otherwise a bug: this function should not be called in this case op = _cop(cigar[s->k+1]); if (op == BAM_CMATCH || op == BAM_CDEL || op == BAM_CREF_SKIP || op == BAM_CEQUAL || op == BAM_CDIFF) { // jump to the next without a loop if (_cop(cigar[s->k]) == BAM_CMATCH|| _cop(cigar[s->k]) == BAM_CEQUAL || _cop(cigar[s->k]) == BAM_CDIFF) s->y += l; s->x += l; ++s->k; } else { // find the next M/D/N/=/X if (_cop(cigar[s->k]) == BAM_CMATCH|| _cop(cigar[s->k]) == BAM_CEQUAL || _cop(cigar[s->k]) == BAM_CDIFF) s->y += l; s->x += l; for (k = s->k + 1; k < c->n_cigar; ++k) { op = _cop(cigar[k]), l = _cln(cigar[k]); if (op == BAM_CMATCH || op == BAM_CDEL || op == BAM_CREF_SKIP || op == BAM_CEQUAL || op == BAM_CDIFF) break; else if (op == BAM_CINS || op == BAM_CSOFT_CLIP) s->y += l; } s->k = k; } assert(s->k < c->n_cigar); // otherwise a bug } // else, do nothing } { // collect pileup information int op, l; op = _cop(cigar[s->k]); l = _cln(cigar[s->k]); p->is_del = p->indel = p->is_refskip = 0; if (s->x + l - 1 == pos && s->k + 1 < c->n_cigar) { // peek the next operation int op2 = _cop(cigar[s->k+1]); int l2 = _cln(cigar[s->k+1]); if (op2 == BAM_CDEL) p->indel = -(int)l2; else if (op2 == BAM_CINS) p->indel = l2; else if (op2 == BAM_CPAD && s->k + 2 < c->n_cigar) { // no working for adjacent padding int l3 = 0; for (k = s->k + 2; k < c->n_cigar; ++k) { op2 = _cop(cigar[k]); l2 = _cln(cigar[k]); if (op2 == BAM_CINS) l3 += l2; else if (op2 == BAM_CDEL || op2 == BAM_CMATCH || op2 == BAM_CREF_SKIP || op2 == BAM_CEQUAL || op2 == BAM_CDIFF) break; } if (l3 > 0) p->indel = l3; } } if (op == BAM_CMATCH || op == BAM_CEQUAL || op == BAM_CDIFF) { p->qpos = s->y + (pos - s->x); } else if (op == BAM_CDEL || op == BAM_CREF_SKIP) { p->is_del = 1; p->qpos = s->y; // FIXME: distinguish D and N!!!!! p->is_refskip = (op == BAM_CREF_SKIP); } // cannot be other operations; otherwise a bug p->is_head = (pos == c->pos); p->is_tail = (pos == s->end); } return 1; } /*********************** *** Pileup iterator *** ***********************/ // Dictionary of overlapping reads KHASH_MAP_INIT_STR(olap_hash, lbnode_t *) typedef khash_t(olap_hash) olap_hash_t; struct __bam_plp_t { mempool_t *mp; lbnode_t *head, *tail, *dummy; int32_t tid, pos, max_tid, max_pos; int is_eof, max_plp, error, maxcnt; uint64_t id; bam_pileup1_t *plp; // for the "auto" interface only bam1_t *b; bam_plp_auto_f func; void *data; olap_hash_t *overlaps; }; bam_plp_t bam_plp_init(bam_plp_auto_f func, void *data) { bam_plp_t iter; iter = (bam_plp_t)calloc(1, sizeof(struct __bam_plp_t)); iter->mp = mp_init(); iter->head = iter->tail = mp_alloc(iter->mp); iter->dummy = mp_alloc(iter->mp); iter->max_tid = iter->max_pos = -1; iter->maxcnt = 8000; if (func) { iter->func = func; iter->data = data; iter->b = bam_init1(); } return iter; } void bam_plp_init_overlaps(bam_plp_t iter) { iter->overlaps = kh_init(olap_hash); // hash for tweaking quality of bases in overlapping reads } void bam_plp_destroy(bam_plp_t iter) { if ( iter->overlaps ) kh_destroy(olap_hash, iter->overlaps); mp_free(iter->mp, iter->dummy); mp_free(iter->mp, iter->head); if (iter->mp->cnt != 0) fprintf(stderr, "[bam_plp_destroy] memory leak: %d. Continue anyway.\n", iter->mp->cnt); mp_destroy(iter->mp); if (iter->b) bam_destroy1(iter->b); free(iter->plp); free(iter); } //--------------------------------- //--- Tweak overlapping reads //--------------------------------- /** * cigar_iref2iseq_set() - find the first CMATCH setting the ref and the read index * cigar_iref2iseq_next() - get the next CMATCH base * @cigar: pointer to current cigar block (rw) * @cigar_max: pointer just beyond the last cigar block * @icig: position within the current cigar block (rw) * @iseq: position in the sequence (rw) * @iref: position with respect to the beginning of the read (iref_pos - b->core.pos) (rw) * * Returns BAM_CMATCH or -1 when there is no more cigar to process or the requested position is not covered. */ static inline int cigar_iref2iseq_set(uint32_t **cigar, uint32_t *cigar_max, int *icig, int *iseq, int *iref) { int pos = *iref; if ( pos < 0 ) return -1; *icig = 0; *iseq = 0; *iref = 0; while ( *cigar> BAM_CIGAR_SHIFT; if ( cig==BAM_CSOFT_CLIP ) { (*cigar)++; *iseq += ncig; *icig = 0; continue; } if ( cig==BAM_CHARD_CLIP ) { (*cigar)++; *icig = 0; continue; } if ( cig==BAM_CMATCH || cig==BAM_CEQUAL || cig==BAM_CDIFF ) { pos -= ncig; if ( pos < 0 ) { *icig = ncig + pos; *iseq += *icig; *iref += *icig; return BAM_CMATCH; } (*cigar)++; *iseq += ncig; *icig = 0; *iref += ncig; continue; } if ( cig==BAM_CINS ) { (*cigar)++; *iseq += ncig; *icig = 0; continue; } if ( cig==BAM_CDEL ) { pos -= ncig; if ( pos<0 ) pos = 0; (*cigar)++; *icig = 0; *iref += ncig; continue; } fprintf(stderr,"todo: cigar %d\n", cig); assert(0); } *iseq = -1; return -1; } static inline int cigar_iref2iseq_next(uint32_t **cigar, uint32_t *cigar_max, int *icig, int *iseq, int *iref) { while ( *cigar < cigar_max ) { int cig = (**cigar) & BAM_CIGAR_MASK; int ncig = (**cigar) >> BAM_CIGAR_SHIFT; if ( cig==BAM_CMATCH || cig==BAM_CEQUAL || cig==BAM_CDIFF ) { if ( *icig >= ncig - 1 ) { *icig = 0; (*cigar)++; continue; } (*iseq)++; (*icig)++; (*iref)++; return BAM_CMATCH; } if ( cig==BAM_CDEL ) { (*cigar)++; (*iref) += ncig; *icig = 0; continue; } if ( cig==BAM_CINS ) { (*cigar)++; *iseq += ncig; *icig = 0; continue; } if ( cig==BAM_CSOFT_CLIP ) { (*cigar)++; *iseq += ncig; *icig = 0; continue; } if ( cig==BAM_CHARD_CLIP ) { (*cigar)++; *icig = 0; continue; } fprintf(stderr,"todo: cigar %d\n", cig); assert(0); } *iseq = -1; *iref = -1; return -1; } static void tweak_overlap_quality(bam1_t *a, bam1_t *b) { uint32_t *a_cigar = bam_get_cigar(a), *a_cigar_max = a_cigar + a->core.n_cigar; uint32_t *b_cigar = bam_get_cigar(b), *b_cigar_max = b_cigar + b->core.n_cigar; int a_icig = 0, a_iseq = 0; int b_icig = 0, b_iseq = 0; uint8_t *a_qual = bam_get_qual(a), *b_qual = bam_get_qual(b); uint8_t *a_seq = bam_get_seq(a), *b_seq = bam_get_seq(b); int iref = b->core.pos; int a_iref = iref - a->core.pos; int b_iref = iref - b->core.pos; int a_ret = cigar_iref2iseq_set(&a_cigar, a_cigar_max, &a_icig, &a_iseq, &a_iref); if ( a_ret<0 ) return; // no overlap int b_ret = cigar_iref2iseq_set(&b_cigar, b_cigar_max, &b_icig, &b_iseq, &b_iref); if ( b_ret<0 ) return; // no overlap #if DBG fprintf(stderr,"tweak %s n_cigar=%d %d .. %d-%d vs %d-%d\n", bam_get_qname(a), a->core.n_cigar, b->core.n_cigar, a->core.pos+1,a->core.pos+bam_cigar2rlen(a->core.n_cigar,bam_get_cigar(a)), b->core.pos+1, b->core.pos+bam_cigar2rlen(b->core.n_cigar,bam_get_cigar(b))); #endif while ( 1 ) { // Increment reference position while ( a_iref>=0 && a_iref < iref - a->core.pos ) a_ret = cigar_iref2iseq_next(&a_cigar, a_cigar_max, &a_icig, &a_iseq, &a_iref); if ( a_ret<0 ) break; // done if ( iref < a_iref + a->core.pos ) iref = a_iref + a->core.pos; while ( b_iref>=0 && b_iref < iref - b->core.pos ) b_ret = cigar_iref2iseq_next(&b_cigar, b_cigar_max, &b_icig, &b_iseq, &b_iref); if ( b_ret<0 ) break; // done if ( iref < b_iref + b->core.pos ) iref = b_iref + b->core.pos; iref++; if ( a_iref+a->core.pos != b_iref+b->core.pos ) continue; // only CMATCH positions, don't know what to do with indels if ( bam_seqi(a_seq,a_iseq) == bam_seqi(b_seq,b_iseq) ) { #if DBG fprintf(stderr,"%c",seq_nt16_str[bam_seqi(a_seq,a_iseq)]); #endif // we are very confident about this base int qual = a_qual[a_iseq] + b_qual[b_iseq]; a_qual[a_iseq] = qual>200 ? 200 : qual; b_qual[b_iseq] = 0; } else { if ( a_qual[a_iseq] >= b_qual[b_iseq] ) { #if DBG fprintf(stderr,"[%c/%c]",seq_nt16_str[bam_seqi(a_seq,a_iseq)],tolower(seq_nt16_str[bam_seqi(b_seq,b_iseq)])); #endif a_qual[a_iseq] = 0.8 * a_qual[a_iseq]; // not so confident about a_qual anymore given the mismatch b_qual[b_iseq] = 0; } else { #if DBG fprintf(stderr,"[%c/%c]",tolower(seq_nt16_str[bam_seqi(a_seq,a_iseq)]),seq_nt16_str[bam_seqi(b_seq,b_iseq)]); #endif b_qual[b_iseq] = 0.8 * b_qual[b_iseq]; a_qual[a_iseq] = 0; } } } #if DBG fprintf(stderr,"\n"); #endif } // Fix overlapping reads. Simple soft-clipping did not give good results. // Lowering qualities of unwanted bases is more selective and works better. // static void overlap_push(bam_plp_t iter, lbnode_t *node) { if ( !iter->overlaps ) return; // mapped mates and paired reads only if ( node->b.core.flag&BAM_FMUNMAP || !(node->b.core.flag&BAM_FPROPER_PAIR) ) return; // no overlap possible, unless some wild cigar if ( abs(node->b.core.isize) >= 2*node->b.core.l_qseq ) return; khiter_t kitr = kh_get(olap_hash, iter->overlaps, bam_get_qname(&node->b)); if ( kitr==kh_end(iter->overlaps) ) { int ret; kitr = kh_put(olap_hash, iter->overlaps, bam_get_qname(&node->b), &ret); kh_value(iter->overlaps, kitr) = node; } else { lbnode_t *a = kh_value(iter->overlaps, kitr); tweak_overlap_quality(&a->b, &node->b); kh_del(olap_hash, iter->overlaps, kitr); assert(a->end-1 == a->s.end); a->end = a->b.core.pos + bam_cigar2rlen(a->b.core.n_cigar, bam_get_cigar(&a->b)); a->s.end = a->end - 1; } } static void overlap_remove(bam_plp_t iter, const bam1_t *b) { if ( !iter->overlaps ) return; khiter_t kitr; if ( b ) { kitr = kh_get(olap_hash, iter->overlaps, bam_get_qname(b)); if ( kitr!=kh_end(iter->overlaps) ) kh_del(olap_hash, iter->overlaps, kitr); } else { // remove all for (kitr = kh_begin(iter->overlaps); kitroverlaps); kitr++) if ( kh_exist(iter->overlaps, kitr) ) kh_del(olap_hash, iter->overlaps, kitr); } } // Prepares next pileup position in bam records collected by bam_plp_auto -> user func -> bam_plp_push. Returns // pointer to the piled records if next position is ready or NULL if there is not enough records in the // buffer yet (the current position is still the maximum position across all buffered reads). const bam_pileup1_t *bam_plp_next(bam_plp_t iter, int *_tid, int *_pos, int *_n_plp) { if (iter->error) { *_n_plp = -1; return 0; } *_n_plp = 0; if (iter->is_eof && iter->head->next == 0) return 0; while (iter->is_eof || iter->max_tid > iter->tid || (iter->max_tid == iter->tid && iter->max_pos > iter->pos)) { int n_plp = 0; lbnode_t *p, *q; // write iter->plp at iter->pos iter->dummy->next = iter->head; for (p = iter->head, q = iter->dummy; p->next; q = p, p = p->next) { if (p->b.core.tid < iter->tid || (p->b.core.tid == iter->tid && p->end <= iter->pos)) { // then remove overlap_remove(iter, &p->b); q->next = p->next; mp_free(iter->mp, p); p = q; } else if (p->b.core.tid == iter->tid && p->beg <= iter->pos) { // here: p->end > pos; then add to pileup if (n_plp == iter->max_plp) { // then double the capacity iter->max_plp = iter->max_plp? iter->max_plp<<1 : 256; iter->plp = (bam_pileup1_t*)realloc(iter->plp, sizeof(bam_pileup1_t) * iter->max_plp); } iter->plp[n_plp].b = &p->b; if (resolve_cigar2(iter->plp + n_plp, iter->pos, &p->s)) ++n_plp; // actually always true... } } iter->head = iter->dummy->next; // dummy->next may be changed *_n_plp = n_plp; *_tid = iter->tid; *_pos = iter->pos; // update iter->tid and iter->pos if (iter->head->next) { if (iter->tid > iter->head->b.core.tid) { fprintf(stderr, "[%s] unsorted input. Pileup aborts.\n", __func__); iter->error = 1; *_n_plp = -1; return 0; } } if (iter->tid < iter->head->b.core.tid) { // come to a new reference sequence iter->tid = iter->head->b.core.tid; iter->pos = iter->head->beg; // jump to the next reference } else if (iter->pos < iter->head->beg) { // here: tid == head->b.core.tid iter->pos = iter->head->beg; // jump to the next position } else ++iter->pos; // scan contiguously // return if (n_plp) return iter->plp; if (iter->is_eof && iter->head->next == 0) break; } return 0; } int bam_plp_push(bam_plp_t iter, const bam1_t *b) { if (iter->error) return -1; if (b) { if (b->core.tid < 0) { overlap_remove(iter, b); return 0; } // Skip only unmapped reads here, any additional filtering must be done in iter->func if (b->core.flag & BAM_FUNMAP) { overlap_remove(iter, b); return 0; } if (iter->tid == b->core.tid && iter->pos == b->core.pos && iter->mp->cnt > iter->maxcnt) { overlap_remove(iter, b); return 0; } bam_copy1(&iter->tail->b, b); overlap_push(iter, iter->tail); #ifndef BAM_NO_ID iter->tail->b.id = iter->id++; #endif iter->tail->beg = b->core.pos; iter->tail->end = b->core.pos + bam_cigar2rlen(b->core.n_cigar, bam_get_cigar(b)); iter->tail->s = g_cstate_null; iter->tail->s.end = iter->tail->end - 1; // initialize cstate_t if (b->core.tid < iter->max_tid) { fprintf(stderr, "[bam_pileup_core] the input is not sorted (chromosomes out of order)\n"); iter->error = 1; return -1; } if ((b->core.tid == iter->max_tid) && (iter->tail->beg < iter->max_pos)) { fprintf(stderr, "[bam_pileup_core] the input is not sorted (reads out of order)\n"); iter->error = 1; return -1; } iter->max_tid = b->core.tid; iter->max_pos = iter->tail->beg; if (iter->tail->end > iter->pos || iter->tail->b.core.tid > iter->tid) { iter->tail->next = mp_alloc(iter->mp); iter->tail = iter->tail->next; } } else iter->is_eof = 1; return 0; } const bam_pileup1_t *bam_plp_auto(bam_plp_t iter, int *_tid, int *_pos, int *_n_plp) { const bam_pileup1_t *plp; if (iter->func == 0 || iter->error) { *_n_plp = -1; return 0; } if ((plp = bam_plp_next(iter, _tid, _pos, _n_plp)) != 0) return plp; else { // no pileup line can be obtained; read alignments *_n_plp = 0; if (iter->is_eof) return 0; int ret; while ( (ret=iter->func(iter->data, iter->b)) >= 0) { if (bam_plp_push(iter, iter->b) < 0) { *_n_plp = -1; return 0; } if ((plp = bam_plp_next(iter, _tid, _pos, _n_plp)) != 0) return plp; // otherwise no pileup line can be returned; read the next alignment. } if ( ret < -1 ) { iter->error = ret; *_n_plp = -1; return 0; } bam_plp_push(iter, 0); if ((plp = bam_plp_next(iter, _tid, _pos, _n_plp)) != 0) return plp; return 0; } } void bam_plp_reset(bam_plp_t iter) { lbnode_t *p, *q; iter->max_tid = iter->max_pos = -1; iter->tid = iter->pos = 0; iter->is_eof = 0; for (p = iter->head; p->next;) { overlap_remove(iter, NULL); q = p->next; mp_free(iter->mp, p); p = q; } iter->head = iter->tail; } void bam_plp_set_maxcnt(bam_plp_t iter, int maxcnt) { iter->maxcnt = maxcnt; } /************************ *** Mpileup iterator *** ************************/ struct __bam_mplp_t { int n; uint64_t min, *pos; bam_plp_t *iter; int *n_plp; const bam_pileup1_t **plp; }; bam_mplp_t bam_mplp_init(int n, bam_plp_auto_f func, void **data) { int i; bam_mplp_t iter; iter = (bam_mplp_t)calloc(1, sizeof(struct __bam_mplp_t)); iter->pos = (uint64_t*)calloc(n, 8); iter->n_plp = (int*)calloc(n, sizeof(int)); iter->plp = (const bam_pileup1_t**)calloc(n, sizeof(bam_pileup1_t*)); iter->iter = (bam_plp_t*)calloc(n, sizeof(bam_plp_t)); iter->n = n; iter->min = (uint64_t)-1; for (i = 0; i < n; ++i) { iter->iter[i] = bam_plp_init(func, data[i]); iter->pos[i] = iter->min; } return iter; } void bam_mplp_init_overlaps(bam_mplp_t iter) { int i; for (i = 0; i < iter->n; ++i) bam_plp_init_overlaps(iter->iter[i]); } void bam_mplp_set_maxcnt(bam_mplp_t iter, int maxcnt) { int i; for (i = 0; i < iter->n; ++i) iter->iter[i]->maxcnt = maxcnt; } void bam_mplp_destroy(bam_mplp_t iter) { int i; for (i = 0; i < iter->n; ++i) bam_plp_destroy(iter->iter[i]); free(iter->iter); free(iter->pos); free(iter->n_plp); free(iter->plp); free(iter); } int bam_mplp_auto(bam_mplp_t iter, int *_tid, int *_pos, int *n_plp, const bam_pileup1_t **plp) { int i, ret = 0; uint64_t new_min = (uint64_t)-1; for (i = 0; i < iter->n; ++i) { if (iter->pos[i] == iter->min) { int tid, pos; iter->plp[i] = bam_plp_auto(iter->iter[i], &tid, &pos, &iter->n_plp[i]); if ( iter->iter[i]->error ) return -1; iter->pos[i] = iter->plp[i] ? (uint64_t)tid<<32 | pos : 0; } if (iter->plp[i] && iter->pos[i] < new_min) new_min = iter->pos[i]; } iter->min = new_min; if (new_min == (uint64_t)-1) return 0; *_tid = new_min>>32; *_pos = (uint32_t)new_min; for (i = 0; i < iter->n; ++i) { if (iter->pos[i] == iter->min) { // FIXME: valgrind reports "uninitialised value(s) at this line" n_plp[i] = iter->n_plp[i], plp[i] = iter->plp[i]; ++ret; } else n_plp[i] = 0, plp[i] = 0; } return ret; } #endif // ~!defined(BAM_NO_PILEUP)