/* padding.c -- depad subcommand. Copyright (C) 2011, 2012 Broad Institute. Copyright (C) 2014-2016 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 "sam_header.h" #include "sam_opts.h" #include "samtools.h" #define bam_reg2bin(b,e) hts_reg2bin((b),(e), 14, 5) // The one and only function needed from sam.c. // Explicitly here to avoid including bam.h translation layer. extern char *samfaipath(const char *fn_ref); 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->l_data + (n - b->core.n_cigar) * 4 > b->m_data) { b->m_data = b->l_data + (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->l_data - o); memcpy(b->data + b->core.l_qname, cigar, n * 4); b->l_data += (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 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, 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 = 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; } 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 = 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, bam_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, n2 = 0, m2 = 0, *posmap = 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), h->target_name[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 (h->target_len[r_tid] != r.l) { fprintf(stderr, "[depad] ERROR: (Padded) length of '%s' is %u in BAM header, but %llu in embedded reference\n", bam_get_qname(b), h->target_len[r_tid], (unsigned long long)(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] ? 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)); 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", 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, 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; } 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; replace_cigar(b, n2, cigar2); } /* 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", 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); } /* 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); bam_destroy1(b); return ret; } bam_hdr_t * fix_header(bam_hdr_t *old, faidx_t *fai) { int i = 0, unpadded_len = 0; bam_hdr_t *header = 0 ; unsigned short ln_found; header = bam_hdr_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') { const char *cp = text+3; char *name = strstr(text, "\tSN:"); char *name_end; if (!name) { fprintf(stderr, "Unable to find SN: header field\n"); return NULL; } name += 4; for (name_end = name; name_end != end && *name_end != '\t'; name_end++); strcat(newtext, "@SQ"); ln_found = 0; /* Parse the @SQ lines */ while (cp != end) { if (!ln_found && end-cp >= 2 && strncmp(cp, "LN", 2) == 0) { // Rewrite the length char len_buf[100]; int tid; unsigned int old_length, new_length; const char *old_cp = cp; ln_found = 1; while (cp != end && *cp++ != '\t'); old_length = (int)(cp - old_cp); for (tid = 0; tid < header->n_targets; tid++) { // may want to hash this, but new header API incoming. if (strncmp(name, header->target_name[tid], name_end - name) == 0) { new_length = sprintf(len_buf, "LN:%d", header->target_len[tid]); if (new_length <= old_length) { strcat(newtext, len_buf); } else { fprintf(stderr, "LN value of the reference is larger than the original!\n"); exit(1); } break; } } if (cp != end) strcat(newtext, "\t"); } else if (end-cp >= 2 && ((ln_found && strncmp(cp, "LN", 2) == 0) || strncmp(cp, "M5", 2) == 0 || strncmp(cp, "UR", 2) == 0)) { // skip secondary LNs // MD5 changed during depadding; ditch it. // URLs are also invalid. while (cp != end && *cp++ != '\t'); } else { // Otherwise copy this sub-field verbatim const char *cp_start = cp; while (cp != end && *cp++ != '\t'); strncat(newtext, cp_start, cp-cp_start); } } // Add newline, replacing trailing '\t' if last on line was the LN: char *text_end = newtext + strlen(newtext); if (text_end[-1] == '\t') text_end[-1] = '\n'; else *text_end++ = '\n', *text_end = '\0'; } 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 *in = 0, *out = 0; bam_hdr_t *h = 0, *h_fix = 0; faidx_t *fai = 0; int c, compress_level = -1, is_long_help = 0; char in_mode[5], out_mode[6], *fn_out = 0, *fn_list = 0; int ret=0; sam_global_args ga = SAM_GLOBAL_ARGS_INIT; static const struct option lopts[] = { SAM_OPT_GLOBAL_OPTIONS('-', 0, 0, 0, 'T', '-'), { 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 '?': 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_list = samfaipath(ga.reference); fai = fai_load(ga.reference); } // 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_list && hts_set_fai_filename(in, fn_list) != 0) { fprintf(stderr, "[depad] failed to load reference file \"%s\".\n", fn_list); 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) { h_fix = fix_header(h, fai); } 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 (sam_hdr_write(out, h_fix) != 0) { fprintf(stderr, "[depad] failed to write header.\n"); ret = 1; goto depad_end; } // Do the depad if (bam_pad2unpad(in, out, h, fai) != 0) ret = 1; depad_end: // close files, free and return if (fai) fai_destroy(fai); if (h) bam_hdr_destroy(h); if (in) sam_close(in); if (out && sam_close(out) < 0) { fprintf(stderr, "[depad] error on closing output file.\n"); ret = 1; } 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:\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, " -? 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; }