1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464
|
#include <string.h>
#include <stdio.h>
#include <zlib.h>
#include <assert.h>
#include "bntseq.h"
#include "bwa.h"
#include "ksw.h"
#include "utils.h"
#include "kstring.h"
#include "kvec.h"
#ifdef USE_MALLOC_WRAPPERS
# include "malloc_wrap.h"
#endif
int bwa_verbose = 3;
char bwa_rg_id[256];
char *bwa_pg;
/************************
* Batch FASTA/Q reader *
************************/
#include "kseq.h"
KSEQ_DECLARE(gzFile)
static inline void trim_readno(kstring_t *s)
{
if (s->l > 2 && s->s[s->l-2] == '/' && isdigit(s->s[s->l-1]))
s->l -= 2, s->s[s->l] = 0;
}
static inline char *dupkstring(const kstring_t *str, int dupempty)
{
char *s = (str->l > 0 || dupempty)? malloc(str->l + 1) : NULL;
if (!s) return NULL;
memcpy(s, str->s, str->l);
s[str->l] = '\0';
return s;
}
static inline void kseq2bseq1(const kseq_t *ks, bseq1_t *s)
{ // TODO: it would be better to allocate one chunk of memory, but probably it does not matter in practice
s->name = dupkstring(&ks->name, 1);
s->comment = dupkstring(&ks->comment, 0);
s->seq = dupkstring(&ks->seq, 1);
s->qual = dupkstring(&ks->qual, 0);
s->l_seq = ks->seq.l;
}
bseq1_t *bseq_read(int chunk_size, int *n_, void *ks1_, void *ks2_)
{
kseq_t *ks = (kseq_t*)ks1_, *ks2 = (kseq_t*)ks2_;
int size = 0, m, n;
bseq1_t *seqs;
m = n = 0; seqs = 0;
while (kseq_read(ks) >= 0) {
if (ks2 && kseq_read(ks2) < 0) { // the 2nd file has fewer reads
fprintf(stderr, "[W::%s] the 2nd file has fewer sequences.\n", __func__);
break;
}
if (n >= m) {
m = m? m<<1 : 256;
seqs = realloc(seqs, m * sizeof(bseq1_t));
}
trim_readno(&ks->name);
kseq2bseq1(ks, &seqs[n]);
seqs[n].id = n;
size += seqs[n++].l_seq;
if (ks2) {
trim_readno(&ks2->name);
kseq2bseq1(ks2, &seqs[n]);
seqs[n].id = n;
size += seqs[n++].l_seq;
}
if (size >= chunk_size && (n&1) == 0) break;
}
if (size == 0) { // test if the 2nd file is finished
if (ks2 && kseq_read(ks2) >= 0)
fprintf(stderr, "[W::%s] the 1st file has fewer sequences.\n", __func__);
}
*n_ = n;
return seqs;
}
void bseq_classify(int n, bseq1_t *seqs, int m[2], bseq1_t *sep[2])
{
int i, has_last;
kvec_t(bseq1_t) a[2] = {{0,0,0}, {0,0,0}};
for (i = 1, has_last = 1; i < n; ++i) {
if (has_last) {
if (strcmp(seqs[i].name, seqs[i-1].name) == 0) {
kv_push(bseq1_t, a[1], seqs[i-1]);
kv_push(bseq1_t, a[1], seqs[i]);
has_last = 0;
} else kv_push(bseq1_t, a[0], seqs[i-1]);
} else has_last = 1;
}
if (has_last) kv_push(bseq1_t, a[0], seqs[i-1]);
sep[0] = a[0].a, m[0] = a[0].n;
sep[1] = a[1].a, m[1] = a[1].n;
}
/*****************
* CIGAR related *
*****************/
void bwa_fill_scmat(int a, int b, int8_t mat[25])
{
int i, j, k;
for (i = k = 0; i < 4; ++i) {
for (j = 0; j < 4; ++j)
mat[k++] = i == j? a : -b;
mat[k++] = -1; // ambiguous base
}
for (j = 0; j < 5; ++j) mat[k++] = -1;
}
// Generate CIGAR when the alignment end points are known
uint32_t *bwa_gen_cigar2(const int8_t mat[25], int o_del, int e_del, int o_ins, int e_ins, int w_, int64_t l_pac, const uint8_t *pac, int l_query, uint8_t *query, int64_t rb, int64_t re, int *score, int *n_cigar, int *NM)
{
uint32_t *cigar = 0;
uint8_t tmp, *rseq;
int i;
int64_t rlen;
kstring_t str;
const char *int2base;
if (n_cigar) *n_cigar = 0;
if (NM) *NM = -1;
if (l_query <= 0 || rb >= re || (rb < l_pac && re > l_pac)) return 0; // reject if negative length or bridging the forward and reverse strand
rseq = bns_get_seq(l_pac, pac, rb, re, &rlen);
if (re - rb != rlen) goto ret_gen_cigar; // possible if out of range
if (rb >= l_pac) { // then reverse both query and rseq; this is to ensure indels to be placed at the leftmost position
for (i = 0; i < l_query>>1; ++i)
tmp = query[i], query[i] = query[l_query - 1 - i], query[l_query - 1 - i] = tmp;
for (i = 0; i < rlen>>1; ++i)
tmp = rseq[i], rseq[i] = rseq[rlen - 1 - i], rseq[rlen - 1 - i] = tmp;
}
if (l_query == re - rb && w_ == 0) { // no gap; no need to do DP
// UPDATE: we come to this block now... FIXME: due to an issue in mem_reg2aln(), we never come to this block. This does not affect accuracy, but it hurts performance.
if (n_cigar) {
cigar = malloc(4);
cigar[0] = l_query<<4 | 0;
*n_cigar = 1;
}
for (i = 0, *score = 0; i < l_query; ++i)
*score += mat[rseq[i]*5 + query[i]];
} else {
int w, max_gap, max_ins, max_del, min_w;
// set the band-width
max_ins = (int)((double)(((l_query+1)>>1) * mat[0] - o_ins) / e_ins + 1.);
max_del = (int)((double)(((l_query+1)>>1) * mat[0] - o_del) / e_del + 1.);
max_gap = max_ins > max_del? max_ins : max_del;
max_gap = max_gap > 1? max_gap : 1;
w = (max_gap + abs((int)rlen - l_query) + 1) >> 1;
w = w < w_? w : w_;
min_w = abs((int)rlen - l_query) + 3;
w = w > min_w? w : min_w;
// NW alignment
if (bwa_verbose >= 4) {
printf("* Global bandwidth: %d\n", w);
printf("* Global ref: "); for (i = 0; i < rlen; ++i) putchar("ACGTN"[(int)rseq[i]]); putchar('\n');
printf("* Global query: "); for (i = 0; i < l_query; ++i) putchar("ACGTN"[(int)query[i]]); putchar('\n');
}
*score = ksw_global2(l_query, query, rlen, rseq, 5, mat, o_del, e_del, o_ins, e_ins, w, n_cigar, &cigar);
}
if (NM && n_cigar) {// compute NM and MD
int k, x, y, u, n_mm = 0, n_gap = 0;
str.l = str.m = *n_cigar * 4; str.s = (char*)cigar; // append MD to CIGAR
int2base = rb < l_pac? "ACGTN" : "TGCAN";
for (k = 0, x = y = u = 0; k < *n_cigar; ++k) {
int op, len;
cigar = (uint32_t*)str.s;
op = cigar[k]&0xf, len = cigar[k]>>4;
if (op == 0) { // match
for (i = 0; i < len; ++i) {
if (query[x + i] != rseq[y + i]) {
kputw(u, &str);
kputc(int2base[rseq[y+i]], &str);
++n_mm; u = 0;
} else ++u;
}
x += len; y += len;
} else if (op == 2) { // deletion
if (k > 0 && k < *n_cigar - 1) { // don't do the following if D is the first or the last CIGAR
kputw(u, &str); kputc('^', &str);
for (i = 0; i < len; ++i)
kputc(int2base[rseq[y+i]], &str);
u = 0; n_gap += len;
}
y += len;
} else if (op == 1) x += len, n_gap += len; // insertion
}
kputw(u, &str); kputc(0, &str);
*NM = n_mm + n_gap;
cigar = (uint32_t*)str.s;
}
if (rb >= l_pac) // reverse back query
for (i = 0; i < l_query>>1; ++i)
tmp = query[i], query[i] = query[l_query - 1 - i], query[l_query - 1 - i] = tmp;
ret_gen_cigar:
free(rseq);
return cigar;
}
uint32_t *bwa_gen_cigar(const int8_t mat[25], int q, int r, int w_, int64_t l_pac, const uint8_t *pac, int l_query, uint8_t *query, int64_t rb, int64_t re, int *score, int *n_cigar, int *NM)
{
return bwa_gen_cigar2(mat, q, r, q, r, w_, l_pac, pac, l_query, query, rb, re, score, n_cigar, NM);
}
/*********************
* Full index reader *
*********************/
char *bwa_idx_infer_prefix(const char *hint)
{
char *prefix;
int l_hint;
FILE *fp;
l_hint = strlen(hint);
prefix = malloc(l_hint + 3 + 4 + 1);
strcpy(prefix, hint);
strcpy(prefix + l_hint, ".64.bwt");
if ((fp = fopen(prefix, "rb")) != 0) {
fclose(fp);
prefix[l_hint + 3] = 0;
return prefix;
} else {
strcpy(prefix + l_hint, ".bwt");
if ((fp = fopen(prefix, "rb")) == 0) {
free(prefix);
return 0;
} else {
fclose(fp);
prefix[l_hint] = 0;
return prefix;
}
}
}
bwt_t *bwa_idx_load_bwt(const char *hint)
{
char *tmp, *prefix;
bwt_t *bwt;
prefix = bwa_idx_infer_prefix(hint);
if (prefix == 0) {
if (bwa_verbose >= 1) fprintf(stderr, "[E::%s] fail to locate the index files\n", __func__);
return 0;
}
tmp = calloc(strlen(prefix) + 5, 1);
strcat(strcpy(tmp, prefix), ".bwt"); // FM-index
bwt = bwt_restore_bwt(tmp);
strcat(strcpy(tmp, prefix), ".sa"); // partial suffix array (SA)
bwt_restore_sa(tmp, bwt);
free(tmp); free(prefix);
return bwt;
}
bwaidx_t *bwa_idx_load_from_disk(const char *hint, int which)
{
bwaidx_t *idx;
char *prefix;
prefix = bwa_idx_infer_prefix(hint);
if (prefix == 0) {
if (bwa_verbose >= 1) fprintf(stderr, "[E::%s] fail to locate the index files\n", __func__);
return 0;
}
idx = calloc(1, sizeof(bwaidx_t));
if (which & BWA_IDX_BWT) idx->bwt = bwa_idx_load_bwt(hint);
if (which & BWA_IDX_BNS) {
int i, c;
idx->bns = bns_restore(prefix);
for (i = c = 0; i < idx->bns->n_seqs; ++i)
if (idx->bns->anns[i].is_alt) ++c;
if (bwa_verbose >= 3)
fprintf(stderr, "[M::%s] read %d ALT contigs\n", __func__, c);
if (which & BWA_IDX_PAC) {
idx->pac = calloc(idx->bns->l_pac/4+1, 1);
err_fread_noeof(idx->pac, 1, idx->bns->l_pac/4+1, idx->bns->fp_pac); // concatenated 2-bit encoded sequence
err_fclose(idx->bns->fp_pac);
idx->bns->fp_pac = 0;
}
}
free(prefix);
return idx;
}
bwaidx_t *bwa_idx_load(const char *hint, int which)
{
return bwa_idx_load_from_disk(hint, which);
}
void bwa_idx_destroy(bwaidx_t *idx)
{
if (idx == 0) return;
if (idx->mem == 0) {
if (idx->bwt) bwt_destroy(idx->bwt);
if (idx->bns) bns_destroy(idx->bns);
if (idx->pac) free(idx->pac);
} else {
free(idx->bwt); free(idx->bns->anns); free(idx->bns);
if (!idx->is_shm) free(idx->mem);
}
free(idx);
}
int bwa_mem2idx(int64_t l_mem, uint8_t *mem, bwaidx_t *idx)
{
int64_t k = 0, x;
int i;
// generate idx->bwt
x = sizeof(bwt_t); idx->bwt = malloc(x); memcpy(idx->bwt, mem + k, x); k += x;
x = idx->bwt->bwt_size * 4; idx->bwt->bwt = (uint32_t*)(mem + k); k += x;
x = idx->bwt->n_sa * sizeof(bwtint_t); idx->bwt->sa = (bwtint_t*)(mem + k); k += x;
// generate idx->bns and idx->pac
x = sizeof(bntseq_t); idx->bns = malloc(x); memcpy(idx->bns, mem + k, x); k += x;
x = idx->bns->n_holes * sizeof(bntamb1_t); idx->bns->ambs = (bntamb1_t*)(mem + k); k += x;
x = idx->bns->n_seqs * sizeof(bntann1_t); idx->bns->anns = malloc(x); memcpy(idx->bns->anns, mem + k, x); k += x;
for (i = 0; i < idx->bns->n_seqs; ++i) {
idx->bns->anns[i].name = (char*)(mem + k); k += strlen(idx->bns->anns[i].name) + 1;
idx->bns->anns[i].anno = (char*)(mem + k); k += strlen(idx->bns->anns[i].anno) + 1;
}
idx->pac = (uint8_t*)(mem + k); k += idx->bns->l_pac/4+1;
assert(k == l_mem);
idx->l_mem = k; idx->mem = mem;
return 0;
}
int bwa_idx2mem(bwaidx_t *idx)
{
int i;
int64_t k, x, tmp;
uint8_t *mem;
// copy idx->bwt
x = idx->bwt->bwt_size * 4;
mem = realloc(idx->bwt->bwt, sizeof(bwt_t) + x); idx->bwt->bwt = 0;
memmove(mem + sizeof(bwt_t), mem, x);
memcpy(mem, idx->bwt, sizeof(bwt_t)); k = sizeof(bwt_t) + x;
x = idx->bwt->n_sa * sizeof(bwtint_t); mem = realloc(mem, k + x); memcpy(mem + k, idx->bwt->sa, x); k += x;
free(idx->bwt->sa);
free(idx->bwt); idx->bwt = 0;
// copy idx->bns
tmp = idx->bns->n_seqs * sizeof(bntann1_t) + idx->bns->n_holes * sizeof(bntamb1_t);
for (i = 0; i < idx->bns->n_seqs; ++i) // compute the size of heap-allocated memory
tmp += strlen(idx->bns->anns[i].name) + strlen(idx->bns->anns[i].anno) + 2;
mem = realloc(mem, k + sizeof(bntseq_t) + tmp);
x = sizeof(bntseq_t); memcpy(mem + k, idx->bns, x); k += x;
x = idx->bns->n_holes * sizeof(bntamb1_t); memcpy(mem + k, idx->bns->ambs, x); k += x;
free(idx->bns->ambs);
x = idx->bns->n_seqs * sizeof(bntann1_t); memcpy(mem + k, idx->bns->anns, x); k += x;
for (i = 0; i < idx->bns->n_seqs; ++i) {
x = strlen(idx->bns->anns[i].name) + 1; memcpy(mem + k, idx->bns->anns[i].name, x); k += x;
x = strlen(idx->bns->anns[i].anno) + 1; memcpy(mem + k, idx->bns->anns[i].anno, x); k += x;
free(idx->bns->anns[i].name); free(idx->bns->anns[i].anno);
}
free(idx->bns->anns);
// copy idx->pac
x = idx->bns->l_pac/4+1;
mem = realloc(mem, k + x);
memcpy(mem + k, idx->pac, x); k += x;
free(idx->bns); idx->bns = 0;
free(idx->pac); idx->pac = 0;
return bwa_mem2idx(k, mem, idx);
}
/***********************
* SAM header routines *
***********************/
void bwa_print_sam_hdr(const bntseq_t *bns, const char *hdr_line)
{
int i, n_SQ = 0;
extern char *bwa_pg;
if (hdr_line) {
const char *p = hdr_line;
while ((p = strstr(p, "@SQ\t")) != 0) {
if (p == hdr_line || *(p-1) == '\n') ++n_SQ;
p += 4;
}
}
if (n_SQ == 0) {
for (i = 0; i < bns->n_seqs; ++i) {
err_printf("@SQ\tSN:%s\tLN:%d", bns->anns[i].name, bns->anns[i].len);
if (bns->anns[i].is_alt) err_printf("\tAH:*\n");
else err_fputc('\n', stdout);
}
} else if (n_SQ != bns->n_seqs && bwa_verbose >= 2)
fprintf(stderr, "[W::%s] %d @SQ lines provided with -H; %d sequences in the index. Continue anyway.\n", __func__, n_SQ, bns->n_seqs);
if (hdr_line) err_printf("%s\n", hdr_line);
if (bwa_pg) err_printf("%s\n", bwa_pg);
}
static char *bwa_escape(char *s)
{
char *p, *q;
for (p = q = s; *p; ++p) {
if (*p == '\\') {
++p;
if (*p == 't') *q++ = '\t';
else if (*p == 'n') *q++ = '\n';
else if (*p == 'r') *q++ = '\r';
else if (*p == '\\') *q++ = '\\';
} else *q++ = *p;
}
*q = '\0';
return s;
}
char *bwa_set_rg(const char *s)
{
char *p, *q, *r, *rg_line = 0;
memset(bwa_rg_id, 0, 256);
if (strstr(s, "@RG") != s) {
if (bwa_verbose >= 1) fprintf(stderr, "[E::%s] the read group line is not started with @RG\n", __func__);
goto err_set_rg;
}
if (strstr(s, "\t") != NULL) {
if (bwa_verbose >= 1) fprintf(stderr, "[E::%s] the read group line contained literal <tab> characters -- replace with escaped tabs: \\t\n", __func__);
goto err_set_rg;
}
rg_line = strdup(s);
bwa_escape(rg_line);
if ((p = strstr(rg_line, "\tID:")) == 0) {
if (bwa_verbose >= 1) fprintf(stderr, "[E::%s] no ID within the read group line\n", __func__);
goto err_set_rg;
}
p += 4;
for (q = p; *q && *q != '\t' && *q != '\n'; ++q);
if (q - p + 1 > 256) {
if (bwa_verbose >= 1) fprintf(stderr, "[E::%s] @RG:ID is longer than 255 characters\n", __func__);
goto err_set_rg;
}
for (q = p, r = bwa_rg_id; *q && *q != '\t' && *q != '\n'; ++q)
*r++ = *q;
return rg_line;
err_set_rg:
free(rg_line);
return 0;
}
char *bwa_insert_header(const char *s, char *hdr)
{
int len = 0;
if (s == 0 || s[0] != '@') return hdr;
if (hdr) {
len = strlen(hdr);
hdr = realloc(hdr, len + strlen(s) + 2);
hdr[len++] = '\n';
strcpy(hdr + len, s);
} else hdr = strdup(s);
bwa_escape(hdr + len);
return hdr;
}
|