padding.c

   1 #include <string.h>
   2 #include <assert.h>
   3 #include <unistd.h>
   4 #include "kstring.h"
   5 #include "sam_header.h"
   6 #include "sam.h"
   7 #include "bam.h"
   8 #include "faidx.h"
   9
  10 bam_header_t *bam_header_dup(const bam_header_t *h0); /*in sam.c*/
  11
  12 static void replace_cigar(bam1_t *b, int n, uint32_t *cigar)
  13 {
  14         if (n != b->core.n_cigar) {
  15                 int o = b->core.l_qname + b->core.n_cigar * 4;
  16                 if (b->data_len + (n - b->core.n_cigar) * 4 > b->m_data) {
  17                         b->m_data = b->data_len + (n - b->core.n_cigar) * 4;
  18                         kroundup32(b->m_data);
  19                         b->data = (uint8_t*)realloc(b->data, b->m_data);
  20                 }
  21                 memmove(b->data + b->core.l_qname + n * 4, b->data + o, b->data_len - o);
  22                 memcpy(b->data + b->core.l_qname, cigar, n * 4);
  23                 b->data_len += (n - b->core.n_cigar) * 4;
  24                 b->core.n_cigar = n;
  25         } else memcpy(b->data + b->core.l_qname, cigar, n * 4);
  26 }
  27
  28 #define write_cigar(_c, _n, _m, _v) do { \
  29                 if (_n == _m) { \
  30                         _m = _m? _m<<1 : 4; \
  31                         _c = (uint32_t*)realloc(_c, _m * 4); \
  32                 } \
  33                 _c[_n++] = (_v); \
  34         } while (0)
  35
  36 static void unpad_seq(bam1_t *b, kstring_t *s)
  37 {
  38         int k, j, i;
  39         int length;
  40         uint32_t *cigar = bam1_cigar(b);
  41         uint8_t *seq = bam1_seq(b);
  42         // b->core.l_qseq gives length of the SEQ entry (including soft clips, S)
  43         // We need the padded length after alignment from the CIGAR (excluding
  44         // soft clips S, but including pads from CIGAR D operations)
  45         length = 0;
  46         for (k = 0; k < b->core.n_cigar; ++k) {
  47                 int op, ol;
  48                 op= bam_cigar_op(cigar[k]);
  49                 ol = bam_cigar_oplen(cigar[k]);
  50                 if (op == BAM_CMATCH || op == BAM_CEQUAL || op == BAM_CDIFF || op == BAM_CDEL)
  51                         length += ol;
  52         }
  53         ks_resize(s, length);
  54         for (k = 0, s->l = 0, j = 0; k < b->core.n_cigar; ++k) {
  55                 int op, ol;
  56                 op = bam_cigar_op(cigar[k]);
  57                 ol = bam_cigar_oplen(cigar[k]);
  58                 if (op == BAM_CMATCH || op == BAM_CEQUAL || op == BAM_CDIFF) {
  59                         for (i = 0; i < ol; ++i, ++j) s->s[s->l++] = bam1_seqi(seq, j);
  60                 } else if (op == BAM_CSOFT_CLIP) {
  61                         j += ol;
  62                 } else if (op == BAM_CHARD_CLIP) {
  63                         /* do nothing */
  64                 } else if (op == BAM_CDEL) {
  65                         for (i = 0; i < ol; ++i) s->s[s->l++] = 0;
  66                 } else {
  67                         fprintf(stderr, "[depad] ERROR: Didn't expect CIGAR op %c in read %s\n", BAM_CIGAR_STR[op], bam1_qname(b));
  68                         assert(-1);
  69                 }
  70         }
  71         assert(length == s->l);
  72 }
  73
  74 int load_unpadded_ref(faidx_t *fai, char *ref_name, int ref_len, kstring_t *seq)
  75 {
  76         char base;
  77         char *fai_ref = 0;
  78         int fai_ref_len = 0, k;
  79
  80         fai_ref = fai_fetch(fai, ref_name, &fai_ref_len);
  81         if (fai_ref_len != ref_len) {
  82                 fprintf(stderr, "[depad] ERROR: FASTA sequence %s length %i, expected %i\n", ref_name, fai_ref_len, ref_len);
  83                 free(fai_ref);
  84                 return -1;
  85         }
  86         ks_resize(seq, ref_len);
  87         seq->l = 0;
  88         for (k = 0; k < ref_len; ++k) {
  89                 base = fai_ref[k];
  90                 if (base == '-' || base == '*') {
  91                         // Map gaps to null to match unpad_seq function
  92                         seq->s[seq->l++] = 0;
  93                 } else {
  94                         int i = bam_nt16_table[(int)base];
  95                         if (i == 0 || i==16) { // Equals maps to 0, anything unexpected to 16
  96                                 fprintf(stderr, "[depad] ERROR: Invalid character %c (ASCII %i) in FASTA sequence %s\n", base, (int)base, ref_name);
  97                                 free(fai_ref);
  98                                 return -1;
  99                         }
 100                         seq->s[seq->l++] = i;
 101                 }
 102         }
 103         assert(ref_len == seq->l);
 104         free(fai_ref);
 105         return 0;
 106 }
 107
 108 int get_unpadded_len(faidx_t *fai, char *ref_name, int padded_len)
 109 {
 110         char base;
 111         char *fai_ref = 0;
 112         int fai_ref_len = 0, k;
 113         int bases=0, gaps=0;
 114
 115         fai_ref = fai_fetch(fai, ref_name, &fai_ref_len);
 116         if (fai_ref_len != padded_len) {
 117                 fprintf(stderr, "[depad] ERROR: FASTA sequence '%s' length %i, expected %i\n", ref_name, fai_ref_len, padded_len);
 118                 free(fai_ref);
 119                 return -1;
 120         }
 121         for (k = 0; k < padded_len; ++k) {
 122                 //fprintf(stderr, "[depad] checking base %i of %i or %i\n", k+1, ref_len, strlen(fai_ref));
 123                 base = fai_ref[k];
 124                 if (base == '-' || base == '*') {
 125                         gaps += 1;
 126                 } else {
 127                         int i = bam_nt16_table[(int)base];
 128                         if (i == 0 || i==16) { // Equals maps to 0, anything unexpected to 16
 129                                 fprintf(stderr, "[depad] ERROR: Invalid character %c (ASCII %i) in FASTA sequence '%s'\n", base, (int)base, ref_name);
 130                                 free(fai_ref);
 131                                 return -1;
 132                         }
 133                         bases += 1;
 134                 }
 135         }
 136         free(fai_ref);
 137         assert (padded_len == bases + gaps);
 138         return bases;
 139 }
 140
 141 inline int * update_posmap(int *posmap, kstring_t ref)
 142 {
 143         int i, k;
 144         posmap = realloc(posmap, ref.m * sizeof(int));
 145         for (i = k = 0; i < ref.l; ++i) {
 146                 posmap[i] = k;
 147                 if (ref.s[i]) ++k;
 148         }
 149         return posmap;
 150 }
 151
 152 int bam_pad2unpad(samfile_t *in, samfile_t *out, faidx_t *fai)
 153 {
 154         bam_header_t *h = 0;
 155         bam1_t *b = 0;
 156         kstring_t r, q;
 157         int r_tid = -1;
 158         uint32_t *cigar2 = 0;
 159         int ret = 0, n2 = 0, m2 = 0, *posmap = 0;
 160
 161         b = bam_init1();
 162         r.l = r.m = q.l = q.m = 0; r.s = q.s = 0;
 163         int read_ret;
 164         h = in->header;
 165         while ((read_ret = samread(in, b)) >= 0) { // read one alignment from `in'
 166                 uint32_t *cigar = bam1_cigar(b);
 167                 n2 = 0;
 168                 if (b->core.pos == 0 && b->core.tid >= 0 && strcmp(bam1_qname(b), h->target_name[b->core.tid]) == 0) {
 169                         // fprintf(stderr, "[depad] Found embedded reference '%s'\n", bam1_qname(b));
 170                         r_tid = b->core.tid;
 171                         unpad_seq(b, &r);
 172                         if (h->target_len[r_tid] != r.l) {
 173                                 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);
 174                                 return -1;
 175                         }
 176                         if (fai) {
 177                                 // Check the embedded reference matches the FASTA file
 178                                 if (load_unpadded_ref(fai, h->target_name[b->core.tid], h->target_len[b->core.tid], &q)) {
 179                                         fprintf(stderr, "[depad] ERROR: Failed to load embedded reference '%s' from FASTA\n", h->target_name[b->core.tid]);
 180                                         return -1;
 181                                 }
 182                                 assert(r.l == q.l);
 183                                 int i;
 184                                 for (i = 0; i < r.l; ++i) {
 185                                         if (r.s[i] != q.s[i]) {
 186                                                 // Show gaps as ASCII 45
 187                                                 fprintf(stderr, "[depad] ERROR: Embedded sequence and reference FASTA don't match for %s base %i, '%c' vs '%c'\n",
 188                                                         h->target_name[b->core.tid], i+1,
 189                                                         r.s[i] ? bam_nt16_rev_table[(int)r.s[i]] : 45,
 190                                                         q.s[i] ? bam_nt16_rev_table[(int)q.s[i]] : 45);
 191                                                 return -1;
 192                                         }
 193                                 }
 194                         }
 195                         write_cigar(cigar2, n2, m2, bam_cigar_gen(b->core.l_qseq, BAM_CMATCH));
 196                         replace_cigar(b, n2, cigar2);
 197                         posmap = update_posmap(posmap, r);
 198                         b->core.bin = bam_reg2bin(0, bam_calend(&b->core, bam1_cigar(b)));
 199                 } else if (b->core.n_cigar > 0) {
 200                         int i, k, op;
 201                         if (b->core.tid < 0) {
 202                                 fprintf(stderr, "[depad] ERROR: Read '%s' has CIGAR but no RNAME\n", bam1_qname(b));
 203                                 return -1;
 204                         } else if (b->core.tid == r_tid) {
 205                                 ; // good case, reference available
 206                                 //fprintf(stderr, "[depad] Have ref '%s' for read '%s'\n", h->target_name[b->core.tid], bam1_qname(b));
 207                         } else if (fai) {
 208                                 if (load_unpadded_ref(fai, h->target_name[b->core.tid], h->target_len[b->core.tid], &r)) {
 209                                         fprintf(stderr, "[depad] ERROR: Failed to load '%s' from reference FASTA\n", h->target_name[b->core.tid]);
 210                                         return -1;
 211                                 }
 212                                 posmap = update_posmap(posmap, r);
 213                                 r_tid = b->core.tid;
 214                                 // fprintf(stderr, "[depad] Loaded %s from FASTA file\n", h->target_name[b->core.tid]);
 215                         } else {
 216                                 fprintf(stderr, "[depad] ERROR: Missing %s embedded reference sequence (and no FASTA file)\n", h->target_name[b->core.tid]);
 217                                 return -1;
 218                         }
 219                         unpad_seq(b, &q);
 220                         if (bam_cigar_op(cigar[0]) == BAM_CSOFT_CLIP) {
 221                                 write_cigar(cigar2, n2, m2, cigar[0]);
 222                         } else if (bam_cigar_op(cigar[0]) == BAM_CHARD_CLIP) {
 223                                 write_cigar(cigar2, n2, m2, cigar[0]);
 224                                 if (b->core.n_cigar > 2 && bam_cigar_op(cigar[1]) == BAM_CSOFT_CLIP) {
 225                                         write_cigar(cigar2, n2, m2, cigar[1]);
 226                                 }
 227                         }
 228                         /* Determine CIGAR operator for each base in the aligned read */
 229                         for (i = 0, k = b->core.pos; i < q.l; ++i, ++k)
 230                                 q.s[i] = q.s[i]? (r.s[k]? BAM_CMATCH : BAM_CINS) : (r.s[k]? BAM_CDEL : BAM_CPAD);
 231                         /* Include any pads if starts with an insert */
 232                         if (q.s[0] == BAM_CINS) {
 233                                 for (k = 0; k+1 < b->core.pos && !r.s[b->core.pos - k - 1]; ++k);
 234                                 if (k) write_cigar(cigar2, n2, m2, bam_cigar_gen(k, BAM_CPAD));
 235                         }
 236                         /* Count consecutive CIGAR operators to turn into a CIGAR string */
 237                         for (i = k = 1, op = q.s[0]; i < q.l; ++i) {
 238                                 if (op != q.s[i]) {
 239                                         write_cigar(cigar2, n2, m2, bam_cigar_gen(k, op));
 240                                         op = q.s[i]; k = 1;
 241                                 } else ++k;
 242                         }
 243                         write_cigar(cigar2, n2, m2, bam_cigar_gen(k, op));
 244                         if (bam_cigar_op(cigar[b->core.n_cigar-1]) == BAM_CSOFT_CLIP) {
 245                                 write_cigar(cigar2, n2, m2, cigar[b->core.n_cigar-1]);
 246                         } else if (bam_cigar_op(cigar[b->core.n_cigar-1]) == BAM_CHARD_CLIP) {
 247                                 if (b->core.n_cigar > 2 && bam_cigar_op(cigar[b->core.n_cigar-2]) == BAM_CSOFT_CLIP) {
 248                                         write_cigar(cigar2, n2, m2, cigar[b->core.n_cigar-2]);
 249                                 }
 250                                 write_cigar(cigar2, n2, m2, cigar[b->core.n_cigar-1]);
 251                         }
 252                         /* Remove redundant P operators between M/X/=/D operators, e.g. 5M2P10M -> 15M */
 253                         int pre_op, post_op;
 254                         for (i = 2; i < n2; ++i)
 255                                 if (bam_cigar_op(cigar2[i-1]) == BAM_CPAD) {
 256                                         pre_op = bam_cigar_op(cigar2[i-2]);
 257                                         post_op = bam_cigar_op(cigar2[i]);
 258                                         /* Note don't need to check for X/= as code above will use M only */
 259                                         if ((pre_op == BAM_CMATCH || pre_op == BAM_CDEL) && (post_op == BAM_CMATCH || post_op == BAM_CDEL)) {
 260                                                 /* This is a redundant P operator */
 261                                                 cigar2[i-1] = 0; // i.e. 0M
 262                                                 /* If had same operator either side, combine them in post_op */
 263                                                 if (pre_op == post_op) {
 264                                                         /* If CIGAR M, could treat as simple integers since BAM_CMATCH is zero*/
 265                                                         cigar2[i] = bam_cigar_gen(bam_cigar_oplen(cigar2[i-2]) + bam_cigar_oplen(cigar2[i]), post_op);
 266                                                         cigar2[i-2] = 0; // i.e. 0M
 267                                                 }
 268                                         }
 269                                 }
 270                         /* Remove the zero'd operators (0M) */
 271                         for (i = k = 0; i < n2; ++i)
 272                                 if (cigar2[i]) cigar2[k++] = cigar2[i];
 273                         n2 = k;
 274                         replace_cigar(b, n2, cigar2);
 275                         b->core.pos = posmap[b->core.pos];
 276                         b->core.bin = bam_reg2bin(b->core.pos, bam_calend(&b->core.pos, bam1_cigar(b)));
 277                         if (b->core.mtid < 0 || b->core.mpos < 0) {
 278                                 /* Nice case, no mate to worry about*/
 279                                 // fprintf(stderr, "[depad] Read '%s' mate not mapped\n", bam1_qname(b));
 280                                 /* TODO - Warning if FLAG says mate should be mapped? */
 281                                 /* Clean up funny input where mate position is given but mate reference is missing: */
 282                                 b->core.mtid = -1;
 283                                 b->core.mpos = -1;
 284                         } else if (b->core.mtid == b->core.tid) {
 285                                 /* Nice case, same reference */
 286                                 // fprintf(stderr, "[depad] Read '%s' mate mapped to same ref\n", bam1_qname(b));
 287                                 b->core.mpos = posmap[b->core.mpos];
 288                         } else {
 289                                 /* Nasty case, Must load alternative posmap */
 290                                 // fprintf(stderr, "[depad] Loading reference '%s' temporarily\n", h->target_name[b->core.mtid]);
 291                                 if (!fai) {
 292                                         fprintf(stderr, "[depad] ERROR: Needed reference %s sequence for mate (and no FASTA file)\n", h->target_name[b->core.mtid]);
 293                                         return -1;
 294                                 }
 295                                 /* Temporarily load the other reference sequence */
 296                                 if (load_unpadded_ref(fai, h->target_name[b->core.mtid], h->target_len[b->core.mtid], &r)) {
 297                                         fprintf(stderr, "[depad] ERROR: Failed to load '%s' from reference FASTA\n", h->target_name[b->core.mtid]);
 298                                         return -1;
 299                                 }
 300                                 posmap = update_posmap(posmap, r);
 301                                 b->core.mpos = posmap[b->core.mpos];
 302                                 /* Restore the reference and posmap*/
 303                                 if (load_unpadded_ref(fai, h->target_name[b->core.tid], h->target_len[b->core.tid], &r)) {
 304                                         fprintf(stderr, "[depad] ERROR: Failed to load '%s' from reference FASTA\n", h->target_name[b->core.tid]);
 305                                         return -1;
 306                                 }
 307                                 posmap = update_posmap(posmap, r);
 308                         }
 309                 }
 310                 samwrite(out, b);
 311         }
 312         if (read_ret < -1) {
 313                 fprintf(stderr, "[depad] truncated file.\n");
 314                 ret = 1;
 315         }
 316         free(r.s); free(q.s); free(posmap);
 317         bam_destroy1(b);
 318         return ret;
 319 }
 320
 321 bam_header_t * fix_header(bam_header_t *old, faidx_t *fai)
 322 {
 323         int i = 0, unpadded_len = 0;
 324         bam_header_t *header = 0 ;
 325
 326         header = bam_header_dup(old);
 327         for (i = 0; i < old->n_targets; ++i) {
 328                 unpadded_len = get_unpadded_len(fai, old->target_name[i], old->target_len[i]);
 329                 if (unpadded_len < 0) {
 330                         fprintf(stderr, "[depad] ERROR getting unpadded length of '%s', padded length %i\n", old->target_name[i], old->target_len[i]);
 331                 } else {
 332                         header->target_len[i] = unpadded_len;
 333                         //fprintf(stderr, "[depad] Recalculating '%s' length %i -> %i\n", old->target_name[i], old->target_len[i], header->target_len[i]);
 334                 }
 335         }
 336         /* Duplicating the header allocated new buffer for header string */
 337         /* After modifying the @SQ lines it will only get smaller, since */
 338         /* the LN entries will be the same or shorter, and we'll remove */
 339         /* any MD entries (MD5 checksums). */
 340         assert(strlen(old->text) == strlen(header->text));
 341         assert (0==strcmp(old->text, header->text));
 342         const char *text;
 343         text = old->text;
 344         header->text[0] = '\0'; /* Resuse the allocated buffer */
 345         char * newtext = header->text;
 346         char * end=NULL;
 347         while (text[0]=='@') {
 348                 end = strchr(text, '\n');
 349                 assert(end != 0);
 350                 if (text[1]=='S' && text[2]=='Q' && text[3]=='\t') {
 351                         /* TODO - edit the @SQ line here to remove MD and fix LN. */
 352                         /* For now just remove the @SQ line, and samtools will */
 353                         /* automatically generate a minimal replacement with LN. */
 354                         /* However, that discards any other tags like AS, SP, UR. */
 355                         //fprintf(stderr, "[depad] Removing @SQ line\n");
 356                 } else {
 357                         /* Copy this line to the new header */
 358                         strncat(newtext, text, end - text + 1);
 359                 }
 360                 text = end + 1;
 361         }
 362         assert (text[0]=='\0');
 363         /* Check we didn't overflow the buffer */
 364         assert (strlen(header->text) <= strlen(old->text));
 365         if (strlen(header->text) < header->l_text) {
 366                 //fprintf(stderr, "[depad] Reallocating header buffer\n");
 367                 assert (newtext == header->text);
 368                 newtext = malloc(strlen(header->text) + 1);
 369                 strcpy(newtext, header->text);
 370                 free(header->text);
 371                 header->text = newtext;
 372                 header->l_text = strlen(newtext);
 373         }
 374         //fprintf(stderr, "[depad] Here is the new header (pending @SQ lines),\n\n%s\n(end)\n", header->text);
 375         return header;
 376 }
 377
 378 static int usage(int is_long_help);
 379
 380 int main_pad2unpad(int argc, char *argv[])
 381 {
 382         samfile_t *in = 0, *out = 0;
 383         bam_header_t *h = 0;
 384         faidx_t *fai = 0;
 385         int c, is_bamin = 1, compress_level = -1, is_bamout = 1, is_long_help = 0;
 386         char in_mode[5], out_mode[5], *fn_out = 0, *fn_list = 0, *fn_ref = 0;
 387         int ret=0;
 388
 389         /* parse command-line options */
 390         strcpy(in_mode, "r"); strcpy(out_mode, "w");
 391         while ((c = getopt(argc, argv, "Sso:u1T:?")) >= 0) {
 392                 switch (c) {
 393                 case 'S': is_bamin = 0; break;
 394                 case 's': assert(compress_level == -1); is_bamout = 0; break;
 395                 case 'o': fn_out = strdup(optarg); break;
 396                 case 'u': assert(is_bamout == 1); compress_level = 0; break;
 397                 case '1': assert(is_bamout == 1); compress_level = 1; break;
 398                 case 'T': fn_ref = strdup(optarg); break;
 399                 case '?': is_long_help = 1; break;
 400                 default: return usage(is_long_help);
 401                 }
 402         }
 403         if (argc == optind) return usage(is_long_help);
 404
 405         if (is_bamin) strcat(in_mode, "b");
 406         if (is_bamout) strcat(out_mode, "b");
 407         strcat(out_mode, "h");
 408         if (compress_level >= 0) {
 409                 char tmp[2];
 410                 tmp[0] = compress_level + '0'; tmp[1] = '\0';
 411                 strcat(out_mode, tmp);
 412         }
 413
 414         // Load FASTA reference (also needed for SAM -> BAM if missing header)
 415         if (fn_ref) {
 416                 fn_list = samfaipath(fn_ref);
 417                 fai = fai_load(fn_ref);
 418         }
 419         // open file handlers
 420         if ((in = samopen(argv[optind], in_mode, fn_list)) == 0) {
 421                 fprintf(stderr, "[depad] failed to open \"%s\" for reading.\n", argv[optind]);
 422                 ret = 1;
 423                 goto depad_end;
 424         }
 425         if (in->header == 0) {
 426                 fprintf(stderr, "[depad] failed to read the header from \"%s\".\n", argv[optind]);
 427                 ret = 1;
 428                 goto depad_end;
 429         }
 430         if (in->header->text == 0 || in->header->l_text == 0) {
 431                 fprintf(stderr, "[depad] Warning - failed to read any header text from \"%s\".\n", argv[optind]);
 432                 assert (0 == in->header->l_text);
 433                 assert (0 == in->header->text);
 434         }
 435         if (fn_ref) {
 436                 h = fix_header(in->header, fai);
 437         } else {
 438                 fprintf(stderr, "[depad] Warning - reference lengths will not be corrected without FASTA reference\n");
 439                 h = in->header;
 440         }
 441         if ((out = samopen(fn_out? fn_out : "-", out_mode, h)) == 0) {
 442                 fprintf(stderr, "[depad] failed to open \"%s\" for writing.\n", fn_out? fn_out : "standard output");
 443                 ret = 1;
 444                 goto depad_end;
 445         }
 446
 447         // Do the depad
 448         ret = bam_pad2unpad(in, out, fai);
 449
 450 depad_end:
 451         // close files, free and return
 452         if (fai) fai_destroy(fai);
 453         if (h != in->header) bam_header_destroy(h);
 454         samclose(in);
 455         samclose(out);
 456         free(fn_list); free(fn_out);
 457         return ret;
 458 }
 459
 460 static int usage(int is_long_help)
 461 {
 462         fprintf(stderr, "\n");
 463         fprintf(stderr, "Usage:   samtools depad <in.bam>\n\n");
 464         fprintf(stderr, "Options: -s       output is SAM (default is BAM)\n");
 465         fprintf(stderr, "         -S       input is SAM (default is BAM)\n");
 466         fprintf(stderr, "         -u       uncompressed BAM output (can't use with -s)\n");
 467         fprintf(stderr, "         -1       fast compression BAM output (can't use with -s)\n");
 468         fprintf(stderr, "         -T FILE  reference sequence file [null]\n");
 469         fprintf(stderr, "         -o FILE  output file name [stdout]\n");
 470         fprintf(stderr, "         -?       longer help\n");
 471         fprintf(stderr, "\n");
 472         if (is_long_help)
 473                 fprintf(stderr, "Notes:\n\
 474 \n\
 475   1. Requires embedded reference sequences (before the reads for that reference),\n\
 476      with the future aim to also support a FASTA padded reference sequence file.\n\
 477 \n\
 478   2. The input padded alignment read's CIGAR strings must not use P or I operators.\n\
 479 \n");
 480         return 1;
 481 }