/* padding.c -- depad subcommand. Copyright (C) 2011, 2012 Broad Institute. Copyright (C) 2014-2016, 2019-2020 Genome Research Ltd. Portions copyright (C) 2012, 2013 Peter Cock, The James Hutton Institute. Author: Heng Li Permission is hereby granted, free of charge, to any person obtaining a copy of this software and associated documentation files (the "Software"), to deal in the Software without restriction, including without limitation the rights to use, copy, modify, merge, publish, distribute, sublicense, and/or sell copies of the Software, and to permit persons to whom the Software is furnished to do so, subject to the following conditions: The above copyright notice and this permission notice shall be included in all copies or substantial portions of the Software. THE SOFTWARE IS PROVIDED "AS IS", WITHOUT WARRANTY OF ANY KIND, EXPRESS OR IMPLIED, INCLUDING BUT NOT LIMITED TO THE WARRANTIES OF MERCHANTABILITY, FITNESS FOR A PARTICULAR PURPOSE AND NONINFRINGEMENT. IN NO EVENT SHALL THE AUTHORS OR COPYRIGHT HOLDERS BE LIABLE FOR ANY CLAIM, DAMAGES OR OTHER LIABILITY, WHETHER IN AN ACTION OF CONTRACT, TORT OR OTHERWISE, ARISING FROM, OUT OF OR IN CONNECTION WITH THE SOFTWARE OR THE USE OR OTHER DEALINGS IN THE SOFTWARE. */ #include #include #include #include #include #include #include #include #include "sam_opts.h" #include "samtools.h" #define bam_reg2bin(b,e) hts_reg2bin((b),(e), 14, 5) static int replace_cigar(bam1_t *b, uint32_t n, uint32_t *cigar) { int diff = 0; if (n != b->core.n_cigar) { int o = b->core.l_qname + b->core.n_cigar * 4; if (n > b->core.n_cigar) { diff = (n - b->core.n_cigar) * 4; if ((INT_MAX - b->l_data)/4 < (n - b->core.n_cigar)) { fprintf(stderr, "[depad] ERROR: BAM record too big\n"); return -1; } if (b->l_data + diff > b->m_data) { b->m_data = b->l_data + diff; kroundup32(b->m_data); uint8_t *tmp = (uint8_t*)realloc(b->data, b->m_data); if (!tmp) { fprintf(stderr, "[depad] ERROR: Memory allocation failure.\n"); return -1; } b->data = tmp; } } else { diff = -(int)((b->core.n_cigar - n) * 4); } memmove(b->data + b->core.l_qname + n * 4, b->data + o, b->l_data - o); b->core.n_cigar = n; } memcpy(b->data + b->core.l_qname, cigar, n * 4); b->l_data += diff; return 0; } #define write_cigar(_c, _n, _m, _v) do { \ if (_n == _m) { \ _m = _m? _m<<1 : 4; \ _c = (uint32_t*)realloc(_c, _m * 4); \ if (!(_c)) { \ fprintf(stderr, "[depad] ERROR: Memory allocation failure.\n"); \ return -1; \ } \ } \ _c[_n++] = (_v); \ } while (0) static int unpad_seq(bam1_t *b, kstring_t *s) { // Returns 0 on success, -1 on an error int k, j, i; int length; int cigar_n_warning = 0; /* Make this a global and limit to one CIGAR N warning? */ uint32_t *cigar = bam_get_cigar(b); uint8_t *seq = bam_get_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 = bam_cigar2rlen(b->core.n_cigar, cigar); 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++] = bam_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 if (op == BAM_CREF_SKIP) { /* Treat CIGAR N as D (not ideal, but better than ignoring it) */ for (i = 0; i < ol; ++i) s->s[s->l++] = 0; if (0 == cigar_n_warning) { cigar_n_warning = -1; fprintf(stderr, "[depad] WARNING: CIGAR op N treated as op D in read %s\n", bam_get_qname(b)); } } else { fprintf(stderr, "[depad] ERROR: Didn't expect CIGAR op %c in read %s\n", BAM_CIGAR_STR[op], bam_get_qname(b)); return -1; } } return length != s->l; } int load_unpadded_ref(faidx_t *fai, const char *ref_name, hts_pos_t ref_len, kstring_t *seq) { char base; char *fai_ref = 0; hts_pos_t fai_ref_len = 0, k; fai_ref = fai_fetch64(fai, ref_name, &fai_ref_len); if (fai_ref_len != ref_len) { fprintf(stderr, "[depad] ERROR: FASTA sequence %s length %"PRIhts_pos", expected %"PRIhts_pos"\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 = seq_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; } hts_pos_t get_unpadded_len(faidx_t *fai, const char *ref_name, hts_pos_t padded_len) { char base; char *fai_ref = 0; hts_pos_t fai_ref_len = 0, k; hts_pos_t bases=0, gaps=0; fai_ref = fai_fetch64(fai, ref_name, &fai_ref_len); if (fai_ref_len != padded_len) { fprintf(stderr, "[depad] ERROR: FASTA sequence '%s' length %"PRIhts_pos", expected %"PRIhts_pos"\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 = seq_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; } static 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 *in, samFile *out, sam_hdr_t *h, faidx_t *fai) { bam1_t *b = 0; kstring_t r, q; int r_tid = -1; uint32_t *cigar2 = 0; int ret = 0, *posmap = 0; uint32_t n2 = 0, m2 = 0; b = bam_init1(); if (!b) { fprintf(stderr, "[depad] Couldn't allocate bam struct\n"); return -1; } r.l = r.m = q.l = q.m = 0; r.s = q.s = 0; int read_ret; while ((read_ret = sam_read1(in, h, b)) >= 0) { // read one alignment from `in' // Cannot depad unmapped CRAM data if (b->core.flag & BAM_FUNMAP) goto next_seq; uint32_t *cigar = bam_get_cigar(b); n2 = 0; if (b->core.pos == 0 && b->core.tid >= 0 && strcmp(bam_get_qname(b), sam_hdr_tid2name(h, b->core.tid)) == 0) { // fprintf(stderr, "[depad] Found embedded reference '%s'\n", bam_get_qname(b)); r_tid = b->core.tid; if (0!=unpad_seq(b, &r)) { fprintf(stderr, "[depad] ERROR: Problem parsing SEQ and/or CIGAR in reference %s\n", bam_get_qname(b)); return -1; }; if (sam_hdr_tid2len(h, r_tid) != r.l) { fprintf(stderr, "[depad] ERROR: (Padded) length of '%s' is %"PRId64" in BAM header, but %zu in embedded reference\n", bam_get_qname(b), (int64_t) sam_hdr_tid2len(h, r_tid), r.l); return -1; } if (fai) { // Check the embedded reference matches the FASTA file if (load_unpadded_ref(fai, sam_hdr_tid2name(h, b->core.tid), sam_hdr_tid2len(h, b->core.tid), &q)) { fprintf(stderr, "[depad] ERROR: Failed to load embedded reference '%s' from FASTA\n", sam_hdr_tid2name(h, 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", sam_hdr_tid2name(h, b->core.tid), i+1, r.s[i] ? seq_nt16_str[(int)r.s[i]] : 45, q.s[i] ? seq_nt16_str[(int)q.s[i]] : 45); return -1; } } } write_cigar(cigar2, n2, m2, bam_cigar_gen(b->core.l_qseq, BAM_CMATCH)); if (replace_cigar(b, n2, cigar2) < 0) return -1; 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", bam_get_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], bam_get_qname(b)); } else if (fai) { if (load_unpadded_ref(fai, sam_hdr_tid2name(h, b->core.tid), sam_hdr_tid2len(h, b->core.tid), &r)) { fprintf(stderr, "[depad] ERROR: Failed to load '%s' from reference FASTA\n", sam_hdr_tid2name(h, 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", sam_hdr_tid2name(h, b->core.tid)); return -1; } if (0!=unpad_seq(b, &q)) { fprintf(stderr, "[depad] ERROR: Problem parsing SEQ and/or CIGAR in read %s\n", bam_get_qname(b)); return -1; }; 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)); k = 0; } else if (q.s[0] == BAM_CPAD) { // Join 'k' CPAD to our first cigar op CPAD too. for (k = 0; k+1 < b->core.pos && !r.s[b->core.pos - k - 1]; ++k); } else { k = 0; } /* Count consecutive CIGAR operators to turn into a CIGAR string */ for (i = 1, k++, 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; if (replace_cigar(b, n2, cigar2) < 0) return -1; } /* Even unmapped reads can have a POS value, e.g. if their mate was mapped */ if (b->core.pos != -1) 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", bam_get_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", bam_get_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", sam_hdr_tid2name(h, b->core.mtid)); return -1; } /* Temporarily load the other reference sequence */ if (load_unpadded_ref(fai, sam_hdr_tid2name(h, b->core.mtid), sam_hdr_tid2len(h, b->core.mtid), &r)) { fprintf(stderr, "[depad] ERROR: Failed to load '%s' from reference FASTA\n", sam_hdr_tid2name(h, 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, sam_hdr_tid2name(h, b->core.tid), sam_hdr_tid2len(h, b->core.tid), &r)) { fprintf(stderr, "[depad] ERROR: Failed to load '%s' from reference FASTA\n", sam_hdr_tid2name(h, b->core.tid)); return -1; } posmap = update_posmap(posmap, r); } /* Most reads will have been moved so safest to always recalculate the BIN value */ b->core.bin = bam_reg2bin(b->core.pos, bam_endpos(b)); next_seq: if (sam_write1(out, h, b) < 0) { print_error_errno("depad", "error writing to output"); return -1; } } if (read_ret < -1) { fprintf(stderr, "[depad] truncated file.\n"); ret = 1; } free(r.s); free(q.s); free(posmap); free(cigar2); bam_destroy1(b); return ret; } sam_hdr_t * fix_header(sam_hdr_t *old, faidx_t *fai) { int i = 0, ret = 0; hts_pos_t unpadded_len = 0; sam_hdr_t *header = sam_hdr_dup(old); if (!header) return NULL; int nref = sam_hdr_nref(old); char len_buf[64]; for (i = 0; i < nref; ++i) { unpadded_len = get_unpadded_len(fai, sam_hdr_tid2name(old, i), sam_hdr_tid2len(old, i)); if (unpadded_len < 0) { fprintf(stderr, "[depad] ERROR getting unpadded length of '%s', padded length %"PRIhts_pos"\n", sam_hdr_tid2name(old, i), (hts_pos_t) sam_hdr_tid2len(old, i)); } else if (unpadded_len > sam_hdr_tid2len(old, i)) { fprintf(stderr, "[depad] New unpadded length of '%s' is larger than the padded length (%"PRIhts_pos" > %"PRIhts_pos")\n", sam_hdr_tid2name(old, i), unpadded_len, (hts_pos_t) sam_hdr_tid2len(old, i)); ret = 1; } else { sprintf(len_buf, "%"PRIhts_pos"", unpadded_len); if ((ret |= sam_hdr_update_line(header, "SQ", "SN", sam_hdr_tid2name(header, i), "LN", len_buf, NULL))) fprintf(stderr, "[depad] Error updating length of '%s' from %"PRIhts_pos" to %"PRIhts_pos"\n", sam_hdr_tid2name(header, i), (hts_pos_t) sam_hdr_tid2len(header, i), unpadded_len); //fprintf(stderr, "[depad] Recalculating '%s' length %i -> %i\n", old->target_name[i], old->target_len[i], header->target_len[i]); } } if (ret) { sam_hdr_destroy(header); return NULL; } return header; } static int usage(int is_long_help); int main_pad2unpad(int argc, char *argv[]) { samFile *in = 0, *out = 0; sam_hdr_t *h = 0, *h_fix = 0; faidx_t *fai = 0; int c, compress_level = -1, is_long_help = 0, no_pg = 0; char in_mode[5], out_mode[6], *fn_out = 0, *fn_fai = 0, *fn_out_idx = NULL; int ret=0; char *arg_list = NULL; sam_global_args ga = SAM_GLOBAL_ARGS_INIT; static const struct option lopts[] = { SAM_OPT_GLOBAL_OPTIONS('-', 0, 0, 0, 'T', '-'), {"no-PG", no_argument, NULL, 1}, { NULL, 0, NULL, 0 } }; /* parse command-line options */ strcpy(in_mode, "r"); strcpy(out_mode, "w"); while ((c = getopt_long(argc, argv, "SCso:u1T:?", lopts, NULL)) >= 0) { switch (c) { case 'S': break; case 'C': hts_parse_format(&ga.out, "cram"); break; case 's': assert(compress_level == -1); hts_parse_format(&ga.out, "sam"); break; case 'o': fn_out = strdup(optarg); break; case 'u': compress_level = 0; if (ga.out.format == unknown_format) hts_parse_format(&ga.out, "bam"); break; case '1': compress_level = 1; if (ga.out.format == unknown_format) hts_parse_format(&ga.out, "bam"); break; case 1: no_pg = 1; break; case '?': is_long_help = 1; break; default: if (parse_sam_global_opt(c, optarg, lopts, &ga) == 0) break; fprintf(stderr, "[bam_fillmd] unrecognized option '-%c'\n\n", c); return usage(is_long_help); } } if (argc == optind) return usage(is_long_help); 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 (ga.reference) { fn_fai = fai_path(ga.reference); fai = fai_load3(ga.reference, fn_fai, NULL, FAI_CREATE); } // open file handlers if ((in = sam_open_format(argv[optind], in_mode, &ga.in)) == 0) { print_error_errno("depad", "failed to open \"%s\" for reading", argv[optind]); ret = 1; goto depad_end; } if (fn_fai && hts_set_fai_filename(in, fn_fai) != 0) { fprintf(stderr, "[depad] failed to load reference file \"%s\".\n", fn_fai); ret = 1; goto depad_end; } if ((h = sam_hdr_read(in)) == 0) { fprintf(stderr, "[depad] failed to read the header from \"%s\".\n", argv[optind]); ret = 1; goto depad_end; } if (fai) { if (!(h_fix = fix_header(h, fai))){ fprintf(stderr, "[depad] failed to fix the header from\n"); ret = 1; goto depad_end; } } else { fprintf(stderr, "[depad] Warning - reference lengths will not be corrected without FASTA reference\n"); h_fix = h; } char wmode[2]; strcat(out_mode, sam_open_mode(wmode, fn_out, NULL)==0 ? wmode : "b"); if ((out = sam_open_format(fn_out? fn_out : "-", out_mode, &ga.out)) == 0) { print_error_errno("depad", "failed to open \"%s\" for writing", fn_out? fn_out : "standard output"); ret = 1; goto depad_end; } // Reference-based CRAM won't work unless we also create a new reference. // We could embed this, but for now we take the easy option. if (ga.out.format == cram) hts_set_opt(out, CRAM_OPT_NO_REF, 1); if (!no_pg) { if(!(arg_list = stringify_argv(argc+1, argv-1))) { fprintf(stderr, "[depad] failed to create arg_list\n"); ret = 1; goto depad_end; } if (sam_hdr_add_pg(h_fix, "samtools", "VN", samtools_version(), arg_list ? "CL": NULL, arg_list ? arg_list : NULL, NULL)) { fprintf(stderr, "[depad] failed to add PG line to header\n"); ret = 1; goto depad_end; } } if (sam_hdr_write(out, h_fix) != 0) { fprintf(stderr, "[depad] failed to write header.\n"); ret = 1; goto depad_end; } if (ga.write_index) { if (!(fn_out_idx = auto_index(out, fn_out, h_fix))) { ret = 1; goto depad_end; } } // Do the depad if (bam_pad2unpad(in, out, h, fai) != 0) ret = 1; if (ga.write_index) { if (sam_idx_save(out) < 0) { print_error_errno("depad", "writing index failed"); ret = 1; } } depad_end: // close files, free and return free(arg_list); if (fai) fai_destroy(fai); if (h) sam_hdr_destroy(h); if (h_fix && h_fix != h) sam_hdr_destroy(h_fix); if (in) sam_close(in); if (out && sam_close(out) < 0) { fprintf(stderr, "[depad] error on closing output file.\n"); ret = 1; } free(fn_fai); free(fn_out); if (fn_out_idx) free(fn_out_idx); sam_global_args_free(&ga); return ret; } static int usage(int is_long_help) { fprintf(stderr, "\n"); fprintf(stderr, "Usage: samtools depad \n\n"); fprintf(stderr, "Options:\n"); fprintf(stderr, " -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, --reference FILE\n"); fprintf(stderr, " Padded reference sequence file [null]\n"); fprintf(stderr, " -o FILE Output file name [stdout]\n"); fprintf(stderr, " --no-PG do not add a PG line\n"); fprintf(stderr, " -? Longer help\n"); sam_global_opt_help(stderr, "-...--.."); if (is_long_help) fprintf(stderr, "Notes:\n" "\n" "1. Requires embedded reference sequences (before the reads for that reference),\n" " or ideally a FASTA file of the padded reference sequences (via a -T option).\n" "\n" "2. Input padded alignment reads' CIGAR strings must not use P or I operators.\n" "\n"); return 1; }