11
static inline int printw(int c, FILE *fp)
15
if (c == 0) return fputc('0', fp);
16
for (l = 0, x = c < 0? -c : c; x > 0; x /= 10) buf[l++] = x%10 + '0';
17
if (c < 0) buf[l++] = '-';
19
for (x = 0; x < l/2; ++x) {
20
int y = buf[x]; buf[x] = buf[l-1-x]; buf[l-1-x] = y;
26
static inline void pileup_seq(const bam_pileup1_t *p, int pos, int ref_len, const char *ref)
31
putchar(p->b->core.qual > 93? 126 : p->b->core.qual + 33);
34
int c = bam_nt16_rev_table[bam1_seqi(bam1_seq(p->b), p->qpos)];
36
int rb = pos < ref_len? ref[pos] : 'N';
37
if (c == '=' || bam_nt16_table[c] == bam_nt16_table[rb]) c = bam1_strand(p->b)? ',' : '.';
38
else c = bam1_strand(p->b)? tolower(c) : toupper(c);
40
if (c == '=') c = bam1_strand(p->b)? ',' : '.';
41
else c = bam1_strand(p->b)? tolower(c) : toupper(c);
44
} else putchar(p->is_refskip? (bam1_strand(p->b)? '<' : '>') : '*');
46
putchar('+'); printw(p->indel, stdout);
47
for (j = 1; j <= p->indel; ++j) {
48
int c = bam_nt16_rev_table[bam1_seqi(bam1_seq(p->b), p->qpos + j)];
49
putchar(bam1_strand(p->b)? tolower(c) : toupper(c));
51
} else if (p->indel < 0) {
52
printw(p->indel, stdout);
53
for (j = 1; j <= -p->indel; ++j) {
54
int c = (ref && (int)pos+j < ref_len)? ref[pos+j] : 'N';
55
putchar(bam1_strand(p->b)? tolower(c) : toupper(c));
58
if (p->is_tail) putchar('$');
66
#define MPLP_NO_COMP 0x20
67
#define MPLP_NO_ORPHAN 0x40
68
#define MPLP_REALN 0x80
69
#define MPLP_FMT_DP 0x100
70
#define MPLP_FMT_SP 0x200
71
#define MPLP_NO_INDEL 0x400
72
#define MPLP_EXT_BAQ 0x800
73
#define MPLP_ILLUMINA13 0x1000
74
#define MPLP_IGNORE_RG 0x2000
75
#define MPLP_PRINT_POS 0x4000
76
#define MPLP_PRINT_MAPQ 0x8000
78
void *bed_read(const char *fn);
79
void bed_destroy(void *_h);
80
int bed_overlap(const void *_h, const char *chr, int beg, int end);
83
int max_mq, min_mq, flag, min_baseQ, capQ_thres, max_depth, max_indel_depth;
84
int openQ, extQ, tandemQ, min_support; // for indels
85
double min_frac; // for indels
97
const mplp_conf_t *conf;
106
static int mplp_func(void *data, bam1_t *b)
108
extern int bam_realn(bam1_t *b, const char *ref);
109
extern int bam_prob_realn_core(bam1_t *b, const char *ref, int);
110
extern int bam_cap_mapQ(bam1_t *b, char *ref, int thres);
111
mplp_aux_t *ma = (mplp_aux_t*)data;
115
ret = ma->iter? bam_iter_read(ma->fp, ma->iter, b) : bam_read1(ma->fp, b);
117
if (b->core.tid < 0 || (b->core.flag&BAM_FUNMAP)) { // exclude unmapped reads
121
if (ma->conf->bed) { // test overlap
122
skip = !bed_overlap(ma->conf->bed, ma->h->target_name[b->core.tid], b->core.pos, bam_calend(&b->core, bam1_cigar(b)));
125
if (ma->conf->rghash) { // exclude read groups
126
uint8_t *rg = bam_aux_get(b, "RG");
127
skip = (rg && bcf_str2id(ma->conf->rghash, (const char*)(rg+1)) >= 0);
130
if (ma->conf->flag & MPLP_ILLUMINA13) {
132
uint8_t *qual = bam1_qual(b);
133
for (i = 0; i < b->core.l_qseq; ++i)
134
qual[i] = qual[i] > 31? qual[i] - 31 : 0;
136
has_ref = (ma->ref && ma->ref_id == b->core.tid)? 1 : 0;
138
if (has_ref && (ma->conf->flag&MPLP_REALN)) bam_prob_realn_core(b, ma->ref, (ma->conf->flag & MPLP_EXT_BAQ)? 3 : 1);
139
if (has_ref && ma->conf->capQ_thres > 10) {
140
int q = bam_cap_mapQ(b, ma->ref, ma->conf->capQ_thres);
142
else if (b->core.qual > q) b->core.qual = q;
144
else if (b->core.qual < ma->conf->min_mq) skip = 1;
145
else if ((ma->conf->flag&MPLP_NO_ORPHAN) && (b->core.flag&1) && !(b->core.flag&2)) skip = 1;
150
static void group_smpl(mplp_pileup_t *m, bam_sample_t *sm, kstring_t *buf,
151
int n, char *const*fn, int *n_plp, const bam_pileup1_t **plp, int ignore_rg)
154
memset(m->n_plp, 0, m->n * sizeof(int));
155
for (i = 0; i < n; ++i) {
156
for (j = 0; j < n_plp[i]; ++j) {
157
const bam_pileup1_t *p = plp[i] + j;
160
q = ignore_rg? 0 : bam_aux_get(p->b, "RG");
161
if (q) id = bam_smpl_rg2smid(sm, fn[i], (char*)q+1, buf);
162
if (id < 0) id = bam_smpl_rg2smid(sm, fn[i], 0, buf);
163
if (id < 0 || id >= m->n) {
164
assert(q); // otherwise a bug
165
fprintf(stderr, "[%s] Read group %s used in file %s but absent from the header or an alignment missing read group.\n", __func__, (char*)q+1, fn[i]);
168
if (m->n_plp[id] == m->m_plp[id]) {
169
m->m_plp[id] = m->m_plp[id]? m->m_plp[id]<<1 : 8;
170
m->plp[id] = realloc(m->plp[id], sizeof(bam_pileup1_t) * m->m_plp[id]);
172
m->plp[id][m->n_plp[id]++] = *p;
177
static int mpileup(mplp_conf_t *conf, int n, char **fn)
179
extern void *bcf_call_add_rg(void *rghash, const char *hdtext, const char *list);
180
extern void bcf_call_del_rghash(void *rghash);
182
int i, tid, pos, *n_plp, tid0 = -1, beg0 = 0, end0 = 1u<<29, ref_len, ref_tid = -1, max_depth, max_indel_depth;
183
const bam_pileup1_t **plp;
189
bcf_callaux_t *bca = 0;
190
bcf_callret1_t *bcr = 0;
195
bam_sample_t *sm = 0;
199
memset(&gplp, 0, sizeof(mplp_pileup_t));
200
memset(&buf, 0, sizeof(kstring_t));
201
memset(&bc, 0, sizeof(bcf_call_t));
202
data = calloc(n, sizeof(void*));
203
plp = calloc(n, sizeof(void*));
204
n_plp = calloc(n, sizeof(int*));
205
sm = bam_smpl_init();
207
// read the header and initialize data
208
for (i = 0; i < n; ++i) {
210
data[i] = calloc(1, sizeof(mplp_aux_t));
211
data[i]->fp = strcmp(fn[i], "-") == 0? bam_dopen(fileno(stdin), "r") : bam_open(fn[i], "r");
212
data[i]->conf = conf;
213
h_tmp = bam_header_read(data[i]->fp);
214
data[i]->h = i? h : h_tmp; // for i==0, "h" has not been set yet
215
bam_smpl_add(sm, fn[i], (conf->flag&MPLP_IGNORE_RG)? 0 : h_tmp->text);
216
rghash = bcf_call_add_rg(rghash, h_tmp->text, conf->pl_list);
220
idx = bam_index_load(fn[i]);
222
fprintf(stderr, "[%s] fail to load index for %d-th input.\n", __func__, i+1);
225
if (bam_parse_region(h_tmp, conf->reg, &tid, &beg, &end) < 0) {
226
fprintf(stderr, "[%s] malformatted region or wrong seqname for %d-th input.\n", __func__, i+1);
229
if (i == 0) tid0 = tid, beg0 = beg, end0 = end;
230
data[i]->iter = bam_iter_query(idx, tid, beg, end);
231
bam_index_destroy(idx);
233
if (i == 0) h = h_tmp;
235
// FIXME: to check consistency
236
bam_header_destroy(h_tmp);
240
gplp.n_plp = calloc(sm->n, sizeof(int));
241
gplp.m_plp = calloc(sm->n, sizeof(int));
242
gplp.plp = calloc(sm->n, sizeof(void*));
244
fprintf(stderr, "[%s] %d samples in %d input files\n", __func__, sm->n, n);
245
// write the VCF header
246
if (conf->flag & MPLP_GLF) {
248
bh = calloc(1, sizeof(bcf_hdr_t));
249
s.l = s.m = 0; s.s = 0;
250
bp = bcf_open("-", (conf->flag&MPLP_NO_COMP)? "wu" : "w");
251
for (i = 0; i < h->n_targets; ++i) {
252
kputs(h->target_name[i], &s);
256
bh->name = malloc(s.l);
257
memcpy(bh->name, s.s, s.l);
259
for (i = 0; i < sm->n; ++i) {
260
kputs(sm->smpl[i], &s); kputc('\0', &s);
263
bh->sname = malloc(s.l);
264
memcpy(bh->sname, s.s, s.l);
265
bh->txt = malloc(strlen(BAM_VERSION) + 64);
266
bh->l_txt = 1 + sprintf(bh->txt, "##samtoolsVersion=%s\n", BAM_VERSION);
269
bcf_hdr_write(bp, bh);
270
bca = bcf_call_init(-1., conf->min_baseQ);
271
bcr = calloc(sm->n, sizeof(bcf_callret1_t));
272
bca->rghash = rghash;
273
bca->openQ = conf->openQ, bca->extQ = conf->extQ, bca->tandemQ = conf->tandemQ;
274
bca->min_frac = conf->min_frac;
275
bca->min_support = conf->min_support;
277
if (tid0 >= 0 && conf->fai) { // region is set
278
ref = faidx_fetch_seq(conf->fai, h->target_name[tid0], 0, 0x7fffffff, &ref_len);
280
for (i = 0; i < n; ++i) data[i]->ref = ref, data[i]->ref_id = tid0;
281
} else ref_tid = -1, ref = 0;
282
iter = bam_mplp_init(n, mplp_func, (void**)data);
283
max_depth = conf->max_depth;
284
if (max_depth * sm->n > 1<<20)
285
fprintf(stderr, "(%s) Max depth is above 1M. Potential memory hog!\n", __func__);
286
if (max_depth * sm->n < 8000) {
287
max_depth = 8000 / sm->n;
288
fprintf(stderr, "<%s> Set max per-file depth to %d\n", __func__, max_depth);
290
max_indel_depth = conf->max_indel_depth * sm->n;
291
bam_mplp_set_maxcnt(iter, max_depth);
292
while (bam_mplp_auto(iter, &tid, &pos, n_plp, plp) > 0) {
293
if (conf->reg && (pos < beg0 || pos >= end0)) continue; // out of the region requested
294
if (conf->bed && tid >= 0 && !bed_overlap(conf->bed, h->target_name[tid], pos, pos+1)) continue;
295
if (tid != ref_tid) {
297
if (conf->fai) ref = faidx_fetch_seq(conf->fai, h->target_name[tid], 0, 0x7fffffff, &ref_len);
298
for (i = 0; i < n; ++i) data[i]->ref = ref, data[i]->ref_id = tid;
301
if (conf->flag & MPLP_GLF) {
302
int total_depth, _ref0, ref16;
303
bcf1_t *b = calloc(1, sizeof(bcf1_t));
304
for (i = total_depth = 0; i < n; ++i) total_depth += n_plp[i];
305
group_smpl(&gplp, sm, &buf, n, fn, n_plp, plp, conf->flag & MPLP_IGNORE_RG);
306
_ref0 = (ref && pos < ref_len)? ref[pos] : 'N';
307
ref16 = bam_nt16_table[_ref0];
308
for (i = 0; i < gplp.n; ++i)
309
bcf_call_glfgen(gplp.n_plp[i], gplp.plp[i], ref16, bca, bcr + i);
310
bcf_call_combine(gplp.n, bcr, ref16, &bc);
311
bcf_call2bcf(tid, pos, &bc, b, (conf->flag&(MPLP_FMT_DP|MPLP_FMT_SP))? bcr : 0,
312
(conf->flag&MPLP_FMT_SP), 0, 0);
313
bcf_write(bp, bh, b);
316
if (!(conf->flag&MPLP_NO_INDEL) && total_depth < max_indel_depth && bcf_call_gap_prep(gplp.n, gplp.n_plp, gplp.plp, pos, bca, ref, rghash) >= 0) {
317
for (i = 0; i < gplp.n; ++i)
318
bcf_call_glfgen(gplp.n_plp[i], gplp.plp[i], -1, bca, bcr + i);
319
if (bcf_call_combine(gplp.n, bcr, -1, &bc) >= 0) {
320
b = calloc(1, sizeof(bcf1_t));
321
bcf_call2bcf(tid, pos, &bc, b, (conf->flag&(MPLP_FMT_DP|MPLP_FMT_SP))? bcr : 0,
322
(conf->flag&MPLP_FMT_SP), bca, ref);
323
bcf_write(bp, bh, b);
328
printf("%s\t%d\t%c", h->target_name[tid], pos + 1, (ref && pos < ref_len)? ref[pos] : 'N');
329
for (i = 0; i < n; ++i) {
331
printf("\t%d\t", n_plp[i]);
333
printf("*\t*"); // FIXME: printf() is very slow...
334
if (conf->flag & MPLP_PRINT_POS) printf("\t*");
336
for (j = 0; j < n_plp[i]; ++j)
337
pileup_seq(plp[i] + j, pos, ref_len, ref);
339
for (j = 0; j < n_plp[i]; ++j) {
340
const bam_pileup1_t *p = plp[i] + j;
341
int c = bam1_qual(p->b)[p->qpos] + 33;
342
if (c > 126) c = 126;
345
if (conf->flag & MPLP_PRINT_MAPQ) {
347
for (j = 0; j < n_plp[i]; ++j) {
348
int c = plp[i][j].b->core.qual + 33;
349
if (c > 126) c = 126;
353
if (conf->flag & MPLP_PRINT_POS) {
355
for (j = 0; j < n_plp[i]; ++j) {
356
if (j > 0) putchar(',');
357
printf("%d", plp[i][j].qpos + 1); // FIXME: printf() is very slow...
367
bam_smpl_destroy(sm); free(buf.s);
368
for (i = 0; i < gplp.n; ++i) free(gplp.plp[i]);
369
free(gplp.plp); free(gplp.n_plp); free(gplp.m_plp);
370
bcf_call_del_rghash(rghash);
371
bcf_hdr_destroy(bh); bcf_call_destroy(bca); free(bc.PL); free(bcr);
372
bam_mplp_destroy(iter);
373
bam_header_destroy(h);
374
for (i = 0; i < n; ++i) {
375
bam_close(data[i]->fp);
376
if (data[i]->iter) bam_iter_destroy(data[i]->iter);
379
free(data); free(plp); free(ref); free(n_plp);
383
#define MAX_PATH_LEN 1024
384
static int read_file_list(const char *file_list,int *n,char **argv[])
386
char buf[MAX_PATH_LEN];
390
FILE *fh = fopen(file_list,"r");
393
fprintf(stderr,"%s: %s\n", file_list,strerror(errno));
397
// Speed is not an issue here, determine the number of files by reading the file twice
399
while ( fgets(buf,MAX_PATH_LEN,fh) ) nfiles++;
401
if ( fseek(fh, 0L, SEEK_SET) )
403
fprintf(stderr,"%s: %s\n", file_list,strerror(errno));
407
files = calloc(nfiles,sizeof(char*));
409
while ( fgets(buf,MAX_PATH_LEN,fh) )
412
while ( len>0 && isspace(buf[len-1]) ) len--;
413
if ( !len ) continue;
415
files[nfiles] = malloc(sizeof(char)*(len+1));
416
strncpy(files[nfiles],buf,len);
417
files[nfiles][len] = 0;
423
fprintf(stderr,"No files read from %s\n", file_list);
432
int bam_mpileup(int argc, char *argv[])
435
const char *file_list = NULL;
437
int nfiles = 0, use_orphan = 0;
439
memset(&mplp, 0, sizeof(mplp_conf_t));
440
#define MPLP_PRINT_POS 0x4000
444
mplp.max_depth = 250; mplp.max_indel_depth = 250;
445
mplp.openQ = 40; mplp.extQ = 20; mplp.tandemQ = 100;
446
mplp.min_frac = 0.002; mplp.min_support = 1;
447
mplp.flag = MPLP_NO_ORPHAN | MPLP_REALN;
448
while ((c = getopt(argc, argv, "Agf:r:l:M:q:Q:uaRC:BDSd:L:b:P:o:e:h:Im:F:EG:6Os")) >= 0) {
451
mplp.fai = fai_load(optarg);
452
if (mplp.fai == 0) return 1;
454
case 'd': mplp.max_depth = atoi(optarg); break;
455
case 'r': mplp.reg = strdup(optarg); break;
456
case 'l': mplp.bed = bed_read(optarg); break;
457
case 'P': mplp.pl_list = strdup(optarg); break;
458
case 'g': mplp.flag |= MPLP_GLF; break;
459
case 'u': mplp.flag |= MPLP_NO_COMP | MPLP_GLF; break;
460
case 'a': mplp.flag |= MPLP_NO_ORPHAN | MPLP_REALN; break;
461
case 'B': mplp.flag &= ~MPLP_REALN; break;
462
case 'D': mplp.flag |= MPLP_FMT_DP; break;
463
case 'S': mplp.flag |= MPLP_FMT_SP; break;
464
case 'I': mplp.flag |= MPLP_NO_INDEL; break;
465
case 'E': mplp.flag |= MPLP_EXT_BAQ; break;
466
case '6': mplp.flag |= MPLP_ILLUMINA13; break;
467
case 'R': mplp.flag |= MPLP_IGNORE_RG; break;
468
case 's': mplp.flag |= MPLP_PRINT_MAPQ; break;
469
case 'O': mplp.flag |= MPLP_PRINT_POS; break;
470
case 'C': mplp.capQ_thres = atoi(optarg); break;
471
case 'M': mplp.max_mq = atoi(optarg); break;
472
case 'q': mplp.min_mq = atoi(optarg); break;
473
case 'Q': mplp.min_baseQ = atoi(optarg); break;
474
case 'b': file_list = optarg; break;
475
case 'o': mplp.openQ = atoi(optarg); break;
476
case 'e': mplp.extQ = atoi(optarg); break;
477
case 'h': mplp.tandemQ = atoi(optarg); break;
478
case 'A': use_orphan = 1; break;
479
case 'F': mplp.min_frac = atof(optarg); break;
480
case 'm': mplp.min_support = atoi(optarg); break;
481
case 'L': mplp.max_indel_depth = atoi(optarg); break;
485
mplp.rghash = bcf_str2id_init();
486
if ((fp_rg = fopen(optarg, "r")) == 0)
487
fprintf(stderr, "(%s) Fail to open file %s. Continue anyway.\n", __func__, optarg);
488
while (!feof(fp_rg) && fscanf(fp_rg, "%s", buf) > 0) // this is not a good style, but forgive me...
489
bcf_str2id_add(mplp.rghash, strdup(buf));
495
if (use_orphan) mplp.flag &= ~MPLP_NO_ORPHAN;
497
fprintf(stderr, "\n");
498
fprintf(stderr, "Usage: samtools mpileup [options] in1.bam [in2.bam [...]]\n\n");
499
fprintf(stderr, "Input options:\n\n");
500
fprintf(stderr, " -6 assume the quality is in the Illumina-1.3+ encoding\n");
501
fprintf(stderr, " -A count anomalous read pairs\n");
502
fprintf(stderr, " -B disable BAQ computation\n");
503
fprintf(stderr, " -b FILE list of input BAM files [null]\n");
504
fprintf(stderr, " -C INT parameter for adjusting mapQ; 0 to disable [0]\n");
505
fprintf(stderr, " -d INT max per-BAM depth to avoid excessive memory usage [%d]\n", mplp.max_depth);
506
fprintf(stderr, " -E extended BAQ for higher sensitivity but lower specificity\n");
507
fprintf(stderr, " -f FILE faidx indexed reference sequence file [null]\n");
508
fprintf(stderr, " -G FILE exclude read groups listed in FILE [null]\n");
509
fprintf(stderr, " -l FILE list of positions (chr pos) or regions (BED) [null]\n");
510
fprintf(stderr, " -M INT cap mapping quality at INT [%d]\n", mplp.max_mq);
511
fprintf(stderr, " -r STR region in which pileup is generated [null]\n");
512
fprintf(stderr, " -R ignore RG tags\n");
513
fprintf(stderr, " -q INT skip alignments with mapQ smaller than INT [%d]\n", mplp.min_mq);
514
fprintf(stderr, " -Q INT skip bases with baseQ/BAQ smaller than INT [%d]\n", mplp.min_baseQ);
515
fprintf(stderr, "\nOutput options:\n\n");
516
fprintf(stderr, " -D output per-sample DP in BCF (require -g/-u)\n");
517
fprintf(stderr, " -g generate BCF output (genotype likelihoods)\n");
518
fprintf(stderr, " -O output base positions on reads (disabled by -g/-u)\n");
519
fprintf(stderr, " -s output mapping quality (disabled by -g/-u)\n");
520
fprintf(stderr, " -S output per-sample strand bias P-value in BCF (require -g/-u)\n");
521
fprintf(stderr, " -u generate uncompress BCF output\n");
522
fprintf(stderr, "\nSNP/INDEL genotype likelihoods options (effective with `-g' or `-u'):\n\n");
523
fprintf(stderr, " -e INT Phred-scaled gap extension seq error probability [%d]\n", mplp.extQ);
524
fprintf(stderr, " -F FLOAT minimum fraction of gapped reads for candidates [%g]\n", mplp.min_frac);
525
fprintf(stderr, " -h INT coefficient for homopolymer errors [%d]\n", mplp.tandemQ);
526
fprintf(stderr, " -I do not perform indel calling\n");
527
fprintf(stderr, " -L INT max per-sample depth for INDEL calling [%d]\n", mplp.max_indel_depth);
528
fprintf(stderr, " -m INT minimum gapped reads for indel candidates [%d]\n", mplp.min_support);
529
fprintf(stderr, " -o INT Phred-scaled gap open sequencing error probability [%d]\n", mplp.openQ);
530
fprintf(stderr, " -P STR comma separated list of platforms for indels [all]\n");
531
fprintf(stderr, "\n");
532
fprintf(stderr, "Notes: Assuming diploid individuals.\n\n");
536
if ( read_file_list(file_list,&nfiles,&fn) ) return 1;
537
mpileup(&mplp,nfiles,fn);
538
for (c=0; c<nfiles; c++) free(fn[c]);
540
} else mpileup(&mplp, argc - optind, argv + optind);
541
if (mplp.rghash) bcf_str2id_thorough_destroy(mplp.rghash);
542
free(mplp.reg); free(mplp.pl_list);
543
if (mplp.fai) fai_destroy(mplp.fai);
544
if (mplp.bed) bed_destroy(mplp.bed);