12 static inline int printw(int c, FILE *fp)
16 if (c == 0) return fputc('0', fp);
17 for (l = 0, x = c < 0? -c : c; x > 0; x /= 10) buf[l++] = x%10 + '0';
18 if (c < 0) buf[l++] = '-';
20 for (x = 0; x < l/2; ++x) {
21 int y = buf[x]; buf[x] = buf[l-1-x]; buf[l-1-x] = y;
27 static inline void pileup_seq(const bam_pileup1_t *p, int pos, int ref_len, const char *ref)
32 putchar(p->b->core.qual > 93? 126 : p->b->core.qual + 33);
35 int c = bam_nt16_rev_table[bam1_seqi(bam1_seq(p->b), p->qpos)];
37 int rb = pos < ref_len? ref[pos] : 'N';
38 if (c == '=' || bam_nt16_table[c] == bam_nt16_table[rb]) c = bam1_strand(p->b)? ',' : '.';
39 else c = bam1_strand(p->b)? tolower(c) : toupper(c);
41 if (c == '=') c = bam1_strand(p->b)? ',' : '.';
42 else c = bam1_strand(p->b)? tolower(c) : toupper(c);
45 } else putchar(p->is_refskip? (bam1_strand(p->b)? '<' : '>') : '*');
47 putchar('+'); printw(p->indel, stdout);
48 for (j = 1; j <= p->indel; ++j) {
49 int c = bam_nt16_rev_table[bam1_seqi(bam1_seq(p->b), p->qpos + j)];
50 putchar(bam1_strand(p->b)? tolower(c) : toupper(c));
52 } else if (p->indel < 0) {
53 printw(p->indel, stdout);
54 for (j = 1; j <= -p->indel; ++j) {
55 int c = (ref && (int)pos+j < ref_len)? ref[pos+j] : 'N';
56 putchar(bam1_strand(p->b)? tolower(c) : toupper(c));
59 if (p->is_tail) putchar('$');
67 #define MPLP_NO_COMP 0x20
68 #define MPLP_NO_ORPHAN 0x40
69 #define MPLP_REALN 0x80
70 #define MPLP_NO_INDEL 0x400
71 #define MPLP_REDO_BAQ 0x800
72 #define MPLP_ILLUMINA13 0x1000
73 #define MPLP_IGNORE_RG 0x2000
74 #define MPLP_PRINT_POS 0x4000
75 #define MPLP_PRINT_MAPQ 0x8000
76 #define MPLP_PER_SAMPLE 0x10000
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, fmt_flag;
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_REDO_BAQ)? 7 : 3);
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");
214 fprintf(stderr, "[%s] failed to open %s: %s\n", __func__, fn[i], strerror(errno));
217 data[i]->conf = conf;
218 h_tmp = bam_header_read(data[i]->fp);
219 data[i]->h = i? h : h_tmp; // for i==0, "h" has not been set yet
220 bam_smpl_add(sm, fn[i], (conf->flag&MPLP_IGNORE_RG)? 0 : h_tmp->text);
221 rghash = bcf_call_add_rg(rghash, h_tmp->text, conf->pl_list);
225 idx = bam_index_load(fn[i]);
227 fprintf(stderr, "[%s] fail to load index for %s\n", __func__, fn[i]);
230 if (bam_parse_region(h_tmp, conf->reg, &tid, &beg, &end) < 0) {
231 fprintf(stderr, "[%s] malformatted region or wrong seqname for %s\n", __func__, fn[i]);
234 if (i == 0) tid0 = tid, beg0 = beg, end0 = end;
235 data[i]->iter = bam_iter_query(idx, tid, beg, end);
236 bam_index_destroy(idx);
238 if (i == 0) h = h_tmp;
240 // FIXME: to check consistency
241 bam_header_destroy(h_tmp);
245 gplp.n_plp = calloc(sm->n, sizeof(int));
246 gplp.m_plp = calloc(sm->n, sizeof(int));
247 gplp.plp = calloc(sm->n, sizeof(void*));
249 fprintf(stderr, "[%s] %d samples in %d input files\n", __func__, sm->n, n);
250 // write the VCF header
251 if (conf->flag & MPLP_GLF) {
253 bh = calloc(1, sizeof(bcf_hdr_t));
254 s.l = s.m = 0; s.s = 0;
255 bp = bcf_open("-", (conf->flag&MPLP_NO_COMP)? "wu" : "w");
256 for (i = 0; i < h->n_targets; ++i) {
257 kputs(h->target_name[i], &s);
261 bh->name = malloc(s.l);
262 memcpy(bh->name, s.s, s.l);
264 for (i = 0; i < sm->n; ++i) {
265 kputs(sm->smpl[i], &s); kputc('\0', &s);
268 bh->sname = malloc(s.l);
269 memcpy(bh->sname, s.s, s.l);
270 bh->txt = malloc(strlen(BAM_VERSION) + 64);
271 bh->l_txt = 1 + sprintf(bh->txt, "##samtoolsVersion=%s\n", BAM_VERSION);
274 bcf_hdr_write(bp, bh);
275 bca = bcf_call_init(-1., conf->min_baseQ);
276 bcr = calloc(sm->n, sizeof(bcf_callret1_t));
277 bca->rghash = rghash;
278 bca->openQ = conf->openQ, bca->extQ = conf->extQ, bca->tandemQ = conf->tandemQ;
279 bca->min_frac = conf->min_frac;
280 bca->min_support = conf->min_support;
281 bca->per_sample_flt = conf->flag & MPLP_PER_SAMPLE;
283 if (tid0 >= 0 && conf->fai) { // region is set
284 ref = faidx_fetch_seq(conf->fai, h->target_name[tid0], 0, 0x7fffffff, &ref_len);
286 for (i = 0; i < n; ++i) data[i]->ref = ref, data[i]->ref_id = tid0;
287 } else ref_tid = -1, ref = 0;
288 iter = bam_mplp_init(n, mplp_func, (void**)data);
289 max_depth = conf->max_depth;
290 if (max_depth * sm->n > 1<<20)
291 fprintf(stderr, "(%s) Max depth is above 1M. Potential memory hog!\n", __func__);
292 if (max_depth * sm->n < 8000) {
293 max_depth = 8000 / sm->n;
294 fprintf(stderr, "<%s> Set max per-file depth to %d\n", __func__, max_depth);
296 max_indel_depth = conf->max_indel_depth * sm->n;
297 bam_mplp_set_maxcnt(iter, max_depth);
298 while (bam_mplp_auto(iter, &tid, &pos, n_plp, plp) > 0) {
299 if (conf->reg && (pos < beg0 || pos >= end0)) continue; // out of the region requested
300 if (conf->bed && tid >= 0 && !bed_overlap(conf->bed, h->target_name[tid], pos, pos+1)) continue;
301 if (tid != ref_tid) {
303 if (conf->fai) ref = faidx_fetch_seq(conf->fai, h->target_name[tid], 0, 0x7fffffff, &ref_len);
304 for (i = 0; i < n; ++i) data[i]->ref = ref, data[i]->ref_id = tid;
307 if (conf->flag & MPLP_GLF) {
308 int total_depth, _ref0, ref16;
309 bcf1_t *b = calloc(1, sizeof(bcf1_t));
310 for (i = total_depth = 0; i < n; ++i) total_depth += n_plp[i];
311 group_smpl(&gplp, sm, &buf, n, fn, n_plp, plp, conf->flag & MPLP_IGNORE_RG);
312 _ref0 = (ref && pos < ref_len)? ref[pos] : 'N';
313 ref16 = bam_nt16_table[_ref0];
314 for (i = 0; i < gplp.n; ++i)
315 bcf_call_glfgen(gplp.n_plp[i], gplp.plp[i], ref16, bca, bcr + i);
316 bcf_call_combine(gplp.n, bcr, ref16, &bc);
317 bcf_call2bcf(tid, pos, &bc, b, bcr, conf->fmt_flag, 0, 0);
318 bcf_write(bp, bh, b);
321 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) {
322 for (i = 0; i < gplp.n; ++i)
323 bcf_call_glfgen(gplp.n_plp[i], gplp.plp[i], -1, bca, bcr + i);
324 if (bcf_call_combine(gplp.n, bcr, -1, &bc) >= 0) {
325 b = calloc(1, sizeof(bcf1_t));
326 bcf_call2bcf(tid, pos, &bc, b, bcr, conf->fmt_flag, bca, ref);
327 bcf_write(bp, bh, b);
332 printf("%s\t%d\t%c", h->target_name[tid], pos + 1, (ref && pos < ref_len)? ref[pos] : 'N');
333 for (i = 0; i < n; ++i) {
335 for (j = cnt = 0; j < n_plp[i]; ++j) {
336 const bam_pileup1_t *p = plp[i] + j;
337 if (bam1_qual(p->b)[p->qpos] >= conf->min_baseQ) ++cnt;
339 printf("\t%d\t", cnt);
341 printf("*\t*"); // FIXME: printf() is very slow...
342 if (conf->flag & MPLP_PRINT_POS) printf("\t*");
344 for (j = 0; j < n_plp[i]; ++j) {
345 const bam_pileup1_t *p = plp[i] + j;
346 if (bam1_qual(p->b)[p->qpos] >= conf->min_baseQ)
347 pileup_seq(plp[i] + j, pos, ref_len, ref);
350 for (j = 0; j < n_plp[i]; ++j) {
351 const bam_pileup1_t *p = plp[i] + j;
352 int c = bam1_qual(p->b)[p->qpos];
353 if (c >= conf->min_baseQ) {
354 c = c + 33 < 126? c + 33 : 126;
358 if (conf->flag & MPLP_PRINT_MAPQ) {
360 for (j = 0; j < n_plp[i]; ++j) {
361 int c = plp[i][j].b->core.qual + 33;
362 if (c > 126) c = 126;
366 if (conf->flag & MPLP_PRINT_POS) {
368 for (j = 0; j < n_plp[i]; ++j) {
369 if (j > 0) putchar(',');
370 printf("%d", plp[i][j].qpos + 1); // FIXME: printf() is very slow...
380 bam_smpl_destroy(sm); free(buf.s);
381 for (i = 0; i < gplp.n; ++i) free(gplp.plp[i]);
382 free(gplp.plp); free(gplp.n_plp); free(gplp.m_plp);
383 bcf_call_del_rghash(rghash);
384 bcf_hdr_destroy(bh); bcf_call_destroy(bca); free(bc.PL); free(bcr);
385 bam_mplp_destroy(iter);
386 bam_header_destroy(h);
387 for (i = 0; i < n; ++i) {
388 bam_close(data[i]->fp);
389 if (data[i]->iter) bam_iter_destroy(data[i]->iter);
392 free(data); free(plp); free(ref); free(n_plp);
396 #define MAX_PATH_LEN 1024
397 static int read_file_list(const char *file_list,int *n,char **argv[])
399 char buf[MAX_PATH_LEN];
407 FILE *fh = fopen(file_list,"r");
410 fprintf(stderr,"%s: %s\n", file_list,strerror(errno));
414 files = calloc(nfiles,sizeof(char*));
416 while ( fgets(buf,MAX_PATH_LEN,fh) )
418 // allow empty lines and trailing spaces
420 while ( len>0 && isspace(buf[len-1]) ) len--;
421 if ( !len ) continue;
423 // check sanity of the file list
425 if (stat(buf, &sb) != 0)
427 // no such file, check if it is safe to print its name
428 int i, safe_to_print = 1;
429 for (i=0; i<len; i++)
430 if (!isprint(buf[i])) { safe_to_print = 0; break; }
432 fprintf(stderr,"The file list \"%s\" appears broken, could not locate: %s\n", file_list,buf);
434 fprintf(stderr,"Does the file \"%s\" really contain a list of files and do all exist?\n", file_list);
439 files = realloc(files,nfiles*sizeof(char*));
440 files[nfiles-1] = strdup(buf);
445 fprintf(stderr,"No files read from %s\n", file_list);
454 int bam_mpileup(int argc, char *argv[])
457 const char *file_list = NULL;
459 int nfiles = 0, use_orphan = 0;
461 memset(&mplp, 0, sizeof(mplp_conf_t));
465 mplp.max_depth = 250; mplp.max_indel_depth = 250;
466 mplp.openQ = 40; mplp.extQ = 20; mplp.tandemQ = 100;
467 mplp.min_frac = 0.002; mplp.min_support = 1;
468 mplp.flag = MPLP_NO_ORPHAN | MPLP_REALN;
469 while ((c = getopt(argc, argv, "Agf:r:l:M:q:Q:uaRC:BDSd:L:b:P:po:e:h:Im:F:EG:6OsV")) >= 0) {
472 mplp.fai = fai_load(optarg);
473 if (mplp.fai == 0) return 1;
475 case 'd': mplp.max_depth = atoi(optarg); break;
476 case 'r': mplp.reg = strdup(optarg); break;
477 case 'l': mplp.bed = bed_read(optarg); break;
478 case 'P': mplp.pl_list = strdup(optarg); break;
479 case 'p': mplp.flag |= MPLP_PER_SAMPLE; break;
480 case 'g': mplp.flag |= MPLP_GLF; break;
481 case 'u': mplp.flag |= MPLP_NO_COMP | MPLP_GLF; break;
482 case 'a': mplp.flag |= MPLP_NO_ORPHAN | MPLP_REALN; break;
483 case 'B': mplp.flag &= ~MPLP_REALN; break;
484 case 'D': mplp.fmt_flag |= B2B_FMT_DP; break;
485 case 'S': mplp.fmt_flag |= B2B_FMT_SP; break;
486 case 'V': mplp.fmt_flag |= B2B_FMT_DV; break;
487 case 'I': mplp.flag |= MPLP_NO_INDEL; break;
488 case 'E': mplp.flag |= MPLP_REDO_BAQ; break;
489 case '6': mplp.flag |= MPLP_ILLUMINA13; break;
490 case 'R': mplp.flag |= MPLP_IGNORE_RG; break;
491 case 's': mplp.flag |= MPLP_PRINT_MAPQ; break;
492 case 'O': mplp.flag |= MPLP_PRINT_POS; break;
493 case 'C': mplp.capQ_thres = atoi(optarg); break;
494 case 'M': mplp.max_mq = atoi(optarg); break;
495 case 'q': mplp.min_mq = atoi(optarg); break;
496 case 'Q': mplp.min_baseQ = atoi(optarg); break;
497 case 'b': file_list = optarg; break;
498 case 'o': mplp.openQ = atoi(optarg); break;
499 case 'e': mplp.extQ = atoi(optarg); break;
500 case 'h': mplp.tandemQ = atoi(optarg); break;
501 case 'A': use_orphan = 1; break;
502 case 'F': mplp.min_frac = atof(optarg); break;
503 case 'm': mplp.min_support = atoi(optarg); break;
504 case 'L': mplp.max_indel_depth = atoi(optarg); break;
508 mplp.rghash = bcf_str2id_init();
509 if ((fp_rg = fopen(optarg, "r")) == 0)
510 fprintf(stderr, "(%s) Fail to open file %s. Continue anyway.\n", __func__, optarg);
511 while (!feof(fp_rg) && fscanf(fp_rg, "%s", buf) > 0) // this is not a good style, but forgive me...
512 bcf_str2id_add(mplp.rghash, strdup(buf));
518 if (use_orphan) mplp.flag &= ~MPLP_NO_ORPHAN;
520 fprintf(stderr, "\n");
521 fprintf(stderr, "Usage: samtools mpileup [options] in1.bam [in2.bam [...]]\n\n");
522 fprintf(stderr, "Input options:\n\n");
523 fprintf(stderr, " -6 assume the quality is in the Illumina-1.3+ encoding\n");
524 fprintf(stderr, " -A count anomalous read pairs\n");
525 fprintf(stderr, " -B disable BAQ computation\n");
526 fprintf(stderr, " -b FILE list of input BAM filenames, one per line [null]\n");
527 fprintf(stderr, " -C INT parameter for adjusting mapQ; 0 to disable [0]\n");
528 fprintf(stderr, " -d INT max per-BAM depth to avoid excessive memory usage [%d]\n", mplp.max_depth);
529 fprintf(stderr, " -E recalculate extended BAQ on the fly thus ignoring existing BQs\n");
530 fprintf(stderr, " -f FILE faidx indexed reference sequence file [null]\n");
531 fprintf(stderr, " -G FILE exclude read groups listed in FILE [null]\n");
532 fprintf(stderr, " -l FILE list of positions (chr pos) or regions (BED) [null]\n");
533 fprintf(stderr, " -M INT cap mapping quality at INT [%d]\n", mplp.max_mq);
534 fprintf(stderr, " -r STR region in which pileup is generated [null]\n");
535 fprintf(stderr, " -R ignore RG tags\n");
536 fprintf(stderr, " -q INT skip alignments with mapQ smaller than INT [%d]\n", mplp.min_mq);
537 fprintf(stderr, " -Q INT skip bases with baseQ/BAQ smaller than INT [%d]\n", mplp.min_baseQ);
538 fprintf(stderr, "\nOutput options:\n\n");
539 fprintf(stderr, " -D output per-sample DP in BCF (require -g/-u)\n");
540 fprintf(stderr, " -g generate BCF output (genotype likelihoods)\n");
541 fprintf(stderr, " -O output base positions on reads (disabled by -g/-u)\n");
542 fprintf(stderr, " -s output mapping quality (disabled by -g/-u)\n");
543 fprintf(stderr, " -S output per-sample strand bias P-value in BCF (require -g/-u)\n");
544 fprintf(stderr, " -u generate uncompress BCF output\n");
545 fprintf(stderr, "\nSNP/INDEL genotype likelihoods options (effective with `-g' or `-u'):\n\n");
546 fprintf(stderr, " -e INT Phred-scaled gap extension seq error probability [%d]\n", mplp.extQ);
547 fprintf(stderr, " -F FLOAT minimum fraction of gapped reads for candidates [%g]\n", mplp.min_frac);
548 fprintf(stderr, " -h INT coefficient for homopolymer errors [%d]\n", mplp.tandemQ);
549 fprintf(stderr, " -I do not perform indel calling\n");
550 fprintf(stderr, " -L INT max per-sample depth for INDEL calling [%d]\n", mplp.max_indel_depth);
551 fprintf(stderr, " -m INT minimum gapped reads for indel candidates [%d]\n", mplp.min_support);
552 fprintf(stderr, " -o INT Phred-scaled gap open sequencing error probability [%d]\n", mplp.openQ);
553 fprintf(stderr, " -p apply -m and -F per-sample to increase sensitivity\n");
554 fprintf(stderr, " -P STR comma separated list of platforms for indels [all]\n");
555 fprintf(stderr, "\n");
556 fprintf(stderr, "Notes: Assuming diploid individuals.\n\n");
561 if ( read_file_list(file_list,&nfiles,&fn) ) return 1;
562 mpileup(&mplp,nfiles,fn);
563 for (c=0; c<nfiles; c++) free(fn[c]);
565 } else mpileup(&mplp, argc - optind, argv + optind);
566 if (mplp.rghash) bcf_str2id_thorough_destroy(mplp.rghash);
567 free(mplp.reg); free(mplp.pl_list);
568 if (mplp.fai) fai_destroy(mplp.fai);
569 if (mplp.bed) bed_destroy(mplp.bed);