#include #include #include #include "kstring.h" #include "sam_header.h" #include "sam.h" #include "bam.h" #include "faidx.h" bam_header_t *bam_header_dup(const bam_header_t *h0); /*in sam.c*/ static void replace_cigar(bam1_t *b, int n, uint32_t *cigar) { if (n != b->core.n_cigar) { int o = b->core.l_qname + b->core.n_cigar * 4; if (b->data_len + (n - b->core.n_cigar) * 4 > b->m_data) { b->m_data = b->data_len + (n - b->core.n_cigar) * 4; kroundup32(b->m_data); b->data = (uint8_t*)realloc(b->data, b->m_data); } memmove(b->data + b->core.l_qname + n * 4, b->data + o, b->data_len - o); memcpy(b->data + b->core.l_qname, cigar, n * 4); b->data_len += (n - b->core.n_cigar) * 4; b->core.n_cigar = n; } else memcpy(b->data + b->core.l_qname, cigar, n * 4); } #define write_cigar(_c, _n, _m, _v) do { \ if (_n == _m) { \ _m = _m? _m<<1 : 4; \ _c = (uint32_t*)realloc(_c, _m * 4); \ } \ _c[_n++] = (_v); \ } while (0) static void unpad_seq(bam1_t *b, kstring_t *s) { int k, j, i; int length; uint32_t *cigar = bam1_cigar(b); uint8_t *seq = bam1_seq(b); // b->core.l_qseq gives length of the SEQ entry (including soft clips, S) // We need the padded length after alignment from the CIGAR (excluding // soft clips S, but including pads from CIGAR D operations) length = 0; for (k = 0; k < b->core.n_cigar; ++k) { int op, ol; op= bam_cigar_op(cigar[k]); ol = bam_cigar_oplen(cigar[k]); if (op == BAM_CMATCH || op == BAM_CEQUAL || op == BAM_CDIFF || op == BAM_CDEL) length += ol; } ks_resize(s, length); for (k = 0, s->l = 0, j = 0; k < b->core.n_cigar; ++k) { int op, ol; op = bam_cigar_op(cigar[k]); ol = bam_cigar_oplen(cigar[k]); if (op == BAM_CMATCH || op == BAM_CEQUAL || op == BAM_CDIFF) { for (i = 0; i < ol; ++i, ++j) s->s[s->l++] = bam1_seqi(seq, j); } else if (op == BAM_CSOFT_CLIP) { j += ol; } else if (op == BAM_CHARD_CLIP) { /* do nothing */ } else if (op == BAM_CDEL) { for (i = 0; i < ol; ++i) s->s[s->l++] = 0; } else { fprintf(stderr, "[depad] ERROR: Didn't expect CIGAR op %c in read %s\n", BAM_CIGAR_STR[op], bam1_qname(b)); assert(-1); } } assert(length == s->l); } int load_unpadded_ref(faidx_t *fai, char *ref_name, int ref_len, kstring_t *seq) { char base; char *fai_ref = 0; int fai_ref_len = 0, k; fai_ref = fai_fetch(fai, ref_name, &fai_ref_len); if (fai_ref_len != ref_len) { fprintf(stderr, "[depad] ERROR: FASTA sequence %s length %i, expected %i\n", ref_name, fai_ref_len, ref_len); free(fai_ref); return -1; } ks_resize(seq, ref_len); seq->l = 0; for (k = 0; k < ref_len; ++k) { base = fai_ref[k]; if (base == '-' || base == '*') { // Map gaps to null to match unpad_seq function seq->s[seq->l++] = 0; } else { int i = bam_nt16_table[(int)base]; if (i == 0 || i==16) { // Equals maps to 0, anything unexpected to 16 fprintf(stderr, "[depad] ERROR: Invalid character %c (ASCII %i) in FASTA sequence %s\n", base, (int)base, ref_name); free(fai_ref); return -1; } seq->s[seq->l++] = i; } } assert(ref_len == seq->l); free(fai_ref); return 0; } int get_unpadded_len(faidx_t *fai, char *ref_name, int padded_len) { char base; char *fai_ref = 0; int fai_ref_len = 0, k; int bases=0, gaps=0; fai_ref = fai_fetch(fai, ref_name, &fai_ref_len); if (fai_ref_len != padded_len) { fprintf(stderr, "[depad] ERROR: FASTA sequence '%s' length %i, expected %i\n", ref_name, fai_ref_len, padded_len); free(fai_ref); return -1; } for (k = 0; k < padded_len; ++k) { //fprintf(stderr, "[depad] checking base %i of %i or %i\n", k+1, ref_len, strlen(fai_ref)); base = fai_ref[k]; if (base == '-' || base == '*') { gaps += 1; } else { int i = bam_nt16_table[(int)base]; if (i == 0 || i==16) { // Equals maps to 0, anything unexpected to 16 fprintf(stderr, "[depad] ERROR: Invalid character %c (ASCII %i) in FASTA sequence '%s'\n", base, (int)base, ref_name); free(fai_ref); return -1; } bases += 1; } } free(fai_ref); assert (padded_len == bases + gaps); return bases; } inline int * update_posmap(int *posmap, kstring_t ref) { int i, k; posmap = realloc(posmap, ref.m * sizeof(int)); for (i = k = 0; i < ref.l; ++i) { posmap[i] = k; if (ref.s[i]) ++k; } return posmap; } int bam_pad2unpad(samfile_t *in, samfile_t *out, faidx_t *fai) { bam_header_t *h = 0; bam1_t *b = 0; kstring_t r, q; int r_tid = -1; uint32_t *cigar2 = 0; int ret = 0, n2 = 0, m2 = 0, *posmap = 0; b = bam_init1(); r.l = r.m = q.l = q.m = 0; r.s = q.s = 0; int read_ret; h = in->header; while ((read_ret = samread(in, b)) >= 0) { // read one alignment from `in' uint32_t *cigar = bam1_cigar(b); n2 = 0; if (b->core.pos == 0 && b->core.tid >= 0 && strcmp(bam1_qname(b), h->target_name[b->core.tid]) == 0) { // fprintf(stderr, "[depad] Found embedded reference '%s'\n", bam1_qname(b)); r_tid = b->core.tid; unpad_seq(b, &r); if (h->target_len[r_tid] != r.l) { fprintf(stderr, "[depad] ERROR: (Padded) length of '%s' is %d in BAM header, but %ld in embedded reference\n", bam1_qname(b), h->target_len[r_tid], r.l); return -1; } if (fai) { // Check the embedded reference matches the FASTA file if (load_unpadded_ref(fai, h->target_name[b->core.tid], h->target_len[b->core.tid], &q)) { fprintf(stderr, "[depad] ERROR: Failed to load embedded reference '%s' from FASTA\n", h->target_name[b->core.tid]); return -1; } assert(r.l == q.l); int i; for (i = 0; i < r.l; ++i) { if (r.s[i] != q.s[i]) { // Show gaps as ASCII 45 fprintf(stderr, "[depad] ERROR: Embedded sequence and reference FASTA don't match for %s base %i, '%c' vs '%c'\n", h->target_name[b->core.tid], i+1, r.s[i] ? bam_nt16_rev_table[(int)r.s[i]] : 45, q.s[i] ? bam_nt16_rev_table[(int)q.s[i]] : 45); return -1; } } } write_cigar(cigar2, n2, m2, bam_cigar_gen(b->core.l_qseq, BAM_CMATCH)); replace_cigar(b, n2, cigar2); posmap = update_posmap(posmap, r); } else if (b->core.n_cigar > 0) { int i, k, op; if (b->core.tid < 0) { fprintf(stderr, "[depad] ERROR: Read '%s' has CIGAR but no RNAME\n", bam1_qname(b)); return -1; } else if (b->core.tid == r_tid) { ; // good case, reference available //fprintf(stderr, "[depad] Have ref '%s' for read '%s'\n", h->target_name[b->core.tid], bam1_qname(b)); } else if (fai) { if (load_unpadded_ref(fai, h->target_name[b->core.tid], h->target_len[b->core.tid], &r)) { fprintf(stderr, "[depad] ERROR: Failed to load '%s' from reference FASTA\n", h->target_name[b->core.tid]); return -1; } posmap = update_posmap(posmap, r); r_tid = b->core.tid; // fprintf(stderr, "[depad] Loaded %s from FASTA file\n", h->target_name[b->core.tid]); } else { fprintf(stderr, "[depad] ERROR: Missing %s embedded reference sequence (and no FASTA file)\n", h->target_name[b->core.tid]); return -1; } unpad_seq(b, &q); if (bam_cigar_op(cigar[0]) == BAM_CSOFT_CLIP) { write_cigar(cigar2, n2, m2, cigar[0]); } else if (bam_cigar_op(cigar[0]) == BAM_CHARD_CLIP) { write_cigar(cigar2, n2, m2, cigar[0]); if (b->core.n_cigar > 2 && bam_cigar_op(cigar[1]) == BAM_CSOFT_CLIP) { write_cigar(cigar2, n2, m2, cigar[1]); } } /* Determine CIGAR operator for each base in the aligned read */ for (i = 0, k = b->core.pos; i < q.l; ++i, ++k) q.s[i] = q.s[i]? (r.s[k]? BAM_CMATCH : BAM_CINS) : (r.s[k]? BAM_CDEL : BAM_CPAD); /* Include any pads if starts with an insert */ if (q.s[0] == BAM_CINS) { for (k = 0; k+1 < b->core.pos && !r.s[b->core.pos - k - 1]; ++k); if (k) write_cigar(cigar2, n2, m2, bam_cigar_gen(k, BAM_CPAD)); } /* Count consecutive CIGAR operators to turn into a CIGAR string */ for (i = k = 1, op = q.s[0]; i < q.l; ++i) { if (op != q.s[i]) { write_cigar(cigar2, n2, m2, bam_cigar_gen(k, op)); op = q.s[i]; k = 1; } else ++k; } write_cigar(cigar2, n2, m2, bam_cigar_gen(k, op)); if (bam_cigar_op(cigar[b->core.n_cigar-1]) == BAM_CSOFT_CLIP) { write_cigar(cigar2, n2, m2, cigar[b->core.n_cigar-1]); } else if (bam_cigar_op(cigar[b->core.n_cigar-1]) == BAM_CHARD_CLIP) { if (b->core.n_cigar > 2 && bam_cigar_op(cigar[b->core.n_cigar-2]) == BAM_CSOFT_CLIP) { write_cigar(cigar2, n2, m2, cigar[b->core.n_cigar-2]); } write_cigar(cigar2, n2, m2, cigar[b->core.n_cigar-1]); } /* Remove redundant P operators between M/X/=/D operators, e.g. 5M2P10M -> 15M */ int pre_op, post_op; for (i = 2; i < n2; ++i) if (bam_cigar_op(cigar2[i-1]) == BAM_CPAD) { pre_op = bam_cigar_op(cigar2[i-2]); post_op = bam_cigar_op(cigar2[i]); /* Note don't need to check for X/= as code above will use M only */ if ((pre_op == BAM_CMATCH || pre_op == BAM_CDEL) && (post_op == BAM_CMATCH || post_op == BAM_CDEL)) { /* This is a redundant P operator */ cigar2[i-1] = 0; // i.e. 0M /* If had same operator either side, combine them in post_op */ if (pre_op == post_op) { /* If CIGAR M, could treat as simple integers since BAM_CMATCH is zero*/ cigar2[i] = bam_cigar_gen(bam_cigar_oplen(cigar2[i-2]) + bam_cigar_oplen(cigar2[i]), post_op); cigar2[i-2] = 0; // i.e. 0M } } } /* Remove the zero'd operators (0M) */ for (i = k = 0; i < n2; ++i) if (cigar2[i]) cigar2[k++] = cigar2[i]; n2 = k; replace_cigar(b, n2, cigar2); b->core.pos = posmap[b->core.pos]; if (b->core.mtid < 0 || b->core.mpos < 0) { /* Nice case, no mate to worry about*/ // fprintf(stderr, "[depad] Read '%s' mate not mapped\n", bam1_qname(b)); /* TODO - Warning if FLAG says mate should be mapped? */ /* Clean up funny input where mate position is given but mate reference is missing: */ b->core.mtid = -1; b->core.mpos = -1; } else if (b->core.mtid == b->core.tid) { /* Nice case, same reference */ // fprintf(stderr, "[depad] Read '%s' mate mapped to same ref\n", bam1_qname(b)); b->core.mpos = posmap[b->core.mpos]; } else { /* Nasty case, Must load alternative posmap */ // fprintf(stderr, "[depad] Loading reference '%s' temporarily\n", h->target_name[b->core.mtid]); if (!fai) { fprintf(stderr, "[depad] ERROR: Needed reference %s sequence for mate (and no FASTA file)\n", h->target_name[b->core.mtid]); return -1; } /* Temporarily load the other reference sequence */ if (load_unpadded_ref(fai, h->target_name[b->core.mtid], h->target_len[b->core.mtid], &r)) { fprintf(stderr, "[depad] ERROR: Failed to load '%s' from reference FASTA\n", h->target_name[b->core.mtid]); return -1; } posmap = update_posmap(posmap, r); b->core.mpos = posmap[b->core.mpos]; /* Restore the reference and posmap*/ if (load_unpadded_ref(fai, h->target_name[b->core.tid], h->target_len[b->core.tid], &r)) { fprintf(stderr, "[depad] ERROR: Failed to load '%s' from reference FASTA\n", h->target_name[b->core.tid]); return -1; } posmap = update_posmap(posmap, r); } } samwrite(out, b); } if (read_ret < -1) { fprintf(stderr, "[depad] truncated file.\n"); ret = 1; } free(r.s); free(q.s); free(posmap); bam_destroy1(b); return ret; } bam_header_t * fix_header(bam_header_t *old, faidx_t *fai) { int i = 0, unpadded_len = 0; bam_header_t *header = 0 ; header = bam_header_dup(old); for (i = 0; i < old->n_targets; ++i) { unpadded_len = get_unpadded_len(fai, old->target_name[i], old->target_len[i]); if (unpadded_len < 0) { fprintf(stderr, "[depad] ERROR getting unpadded length of '%s', padded length %i\n", old->target_name[i], old->target_len[i]); } else { header->target_len[i] = unpadded_len; //fprintf(stderr, "[depad] Recalculating '%s' length %i -> %i\n", old->target_name[i], old->target_len[i], header->target_len[i]); } } /* Duplicating the header allocated new buffer for header string */ /* After modifying the @SQ lines it will only get smaller, since */ /* the LN entries will be the same or shorter, and we'll remove */ /* any MD entries (MD5 checksums). */ assert(strlen(old->text) == strlen(header->text)); assert (0==strcmp(old->text, header->text)); const char *text; text = old->text; header->text[0] = '\0'; /* Resuse the allocated buffer */ char * newtext = header->text; char * end=NULL; while (text[0]=='@') { end = strchr(text, '\n'); assert(end != 0); if (text[1]=='S' && text[2]=='Q' && text[3]=='\t') { /* TODO - edit the @SQ line here to remove MD and fix LN. */ /* For now just remove the @SQ line, and samtools will */ /* automatically generate a minimal replacement with LN. */ /* However, that discards any other tags like AS, SP, UR. */ //fprintf(stderr, "[depad] Removing @SQ line\n"); } else { /* Copy this line to the new header */ strncat(newtext, text, end - text + 1); } text = end + 1; } assert (text[0]=='\0'); /* Check we didn't overflow the buffer */ assert (strlen(header->text) <= strlen(old->text)); if (strlen(header->text) < header->l_text) { //fprintf(stderr, "[depad] Reallocating header buffer\n"); assert (newtext == header->text); newtext = malloc(strlen(header->text) + 1); strcpy(newtext, header->text); free(header->text); header->text = newtext; header->l_text = strlen(newtext); } //fprintf(stderr, "[depad] Here is the new header (pending @SQ lines),\n\n%s\n(end)\n", header->text); return header; } static int usage(int is_long_help); int main_pad2unpad(int argc, char *argv[]) { samfile_t *in = 0, *out = 0; bam_header_t *h = 0; faidx_t *fai = 0; int c, is_bamin = 1, compress_level = -1, is_bamout = 1, is_long_help = 0; char in_mode[5], out_mode[5], *fn_out = 0, *fn_list = 0, *fn_ref = 0; int ret=0; /* parse command-line options */ strcpy(in_mode, "r"); strcpy(out_mode, "w"); while ((c = getopt(argc, argv, "Sso:u1T:?")) >= 0) { switch (c) { case 'S': is_bamin = 0; break; case 's': assert(compress_level == -1); is_bamout = 0; break; case 'o': fn_out = strdup(optarg); break; case 'u': assert(is_bamout == 1); compress_level = 0; break; case '1': assert(is_bamout == 1); compress_level = 1; break; case 'T': fn_ref = strdup(optarg); break; case '?': is_long_help = 1; break; default: return usage(is_long_help); } } if (argc == optind) return usage(is_long_help); if (is_bamin) strcat(in_mode, "b"); if (is_bamout) strcat(out_mode, "b"); strcat(out_mode, "h"); if (compress_level >= 0) { char tmp[2]; tmp[0] = compress_level + '0'; tmp[1] = '\0'; strcat(out_mode, tmp); } // Load FASTA reference (also needed for SAM -> BAM if missing header) if (fn_ref) { fn_list = samfaipath(fn_ref); fai = fai_load(fn_ref); } // open file handlers if ((in = samopen(argv[optind], in_mode, fn_list)) == 0) { fprintf(stderr, "[depad] failed to open \"%s\" for reading.\n", argv[optind]); ret = 1; goto depad_end; } if (in->header == 0) { fprintf(stderr, "[depad] failed to read the header from \"%s\".\n", argv[optind]); ret = 1; goto depad_end; } if (in->header->text == 0 || in->header->l_text == 0) { fprintf(stderr, "[depad] Warning - failed to read any header text from \"%s\".\n", argv[optind]); assert (0 == in->header->l_text); assert (0 == in->header->text); } if (fn_ref) { h = fix_header(in->header, fai); } else { fprintf(stderr, "[depad] Warning - reference lengths will not be corrected without FASTA reference\n"); h = in->header; } if ((out = samopen(fn_out? fn_out : "-", out_mode, h)) == 0) { fprintf(stderr, "[depad] failed to open \"%s\" for writing.\n", fn_out? fn_out : "standard output"); ret = 1; goto depad_end; } // Do the depad ret = bam_pad2unpad(in, out, fai); depad_end: // close files, free and return if (fai) fai_destroy(fai); if (h != in->header) bam_header_destroy(h); samclose(in); samclose(out); free(fn_list); free(fn_out); return ret; } static int usage(int is_long_help) { fprintf(stderr, "\n"); fprintf(stderr, "Usage: samtools depad \n\n"); fprintf(stderr, "Options: -s output is SAM (default is BAM)\n"); fprintf(stderr, " -S input is SAM (default is BAM)\n"); fprintf(stderr, " -u uncompressed BAM output (can't use with -s)\n"); fprintf(stderr, " -1 fast compression BAM output (can't use with -s)\n"); fprintf(stderr, " -T FILE reference sequence file [null]\n"); fprintf(stderr, " -o FILE output file name [stdout]\n"); fprintf(stderr, " -? longer help\n"); fprintf(stderr, "\n"); if (is_long_help) fprintf(stderr, "Notes:\n\ \n\ 1. Requires embedded reference sequences (before the reads for that reference),\n\ with the future aim to also support a FASTA padded reference sequence file.\n\ \n\ 2. The input padded alignment read's CIGAR strings must not use P or I operators.\n\ \n"); return 1; }