1 #include <math.h>
2 #include <stdio.h>
3 #include <unistd.h>
4 #include <ctype.h>
5 #include <string.h>
6 #include <errno.h>
7 #include "sam.h"
8 #include "faidx.h"
9 #include "kstring.h"
10 
printw(int c,FILE * fp)11 static inline int printw(int c, FILE *fp)
12 {
13 	char buf[16];
14 	int l, x;
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++] = '-';
18 	buf[l] = 0;
19 	for (x = 0; x < l/2; ++x) {
20 		int y = buf[x]; buf[x] = buf[l-1-x]; buf[l-1-x] = y;
21 	}
22 	fputs(buf, fp);
23 	return 0;
24 }
25 
pileup_seq(const bam_pileup1_t * p,int pos,int ref_len,const char * ref)26 static inline void pileup_seq(const bam_pileup1_t *p, int pos, int ref_len, const char *ref)
27 {
28 	int j;
29 	if (p->is_head) {
30 		putchar('^');
31 		putchar(p->b->core.qual > 93? 126 : p->b->core.qual + 33);
32 	}
33 	if (!p->is_del) {
34 		int c = bam_nt16_rev_table[bam1_seqi(bam1_seq(p->b), p->qpos)];
35 		if (ref) {
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);
39 		} else {
40 			if (c == '=') c = bam1_strand(p->b)? ',' : '.';
41 			else c = bam1_strand(p->b)? tolower(c) : toupper(c);
42 		}
43 		putchar(c);
44 	} else putchar(p->is_refskip? (bam1_strand(p->b)? '<' : '>') : '*');
45 	if (p->indel > 0) {
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));
50 		}
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));
56 		}
57 	}
58 	if (p->is_tail) putchar('$');
59 }
60 
61 #include <assert.h>
62 #include "bam2bcf.h"
63 #include "sample.h"
64 
65 #define MPLP_GLF   0x10
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
77 
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);
81 
82 typedef struct {
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
86 	char *reg, *pl_list;
87 	faidx_t *fai;
88 	void *bed, *rghash;
89 } mplp_conf_t;
90 
91 typedef struct {
92 	bamFile fp;
93 	bam_iter_t iter;
94 	bam_header_t *h;
95 	int ref_id;
96 	char *ref;
97 	const mplp_conf_t *conf;
98 } mplp_aux_t;
99 
100 typedef struct {
101 	int n;
102 	int *n_plp, *m_plp;
103 	bam_pileup1_t **plp;
104 } mplp_pileup_t;
105 
mplp_func(void * data,bam1_t * b)106 static int mplp_func(void *data, bam1_t *b)
107 {
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;
112 	int ret, skip = 0;
113 	do {
114 		int has_ref;
115 		ret = ma->iter? bam_iter_read(ma->fp, ma->iter, b) : bam_read1(ma->fp, b);
116 		if (ret < 0) break;
117 		if (b->core.tid < 0 || (b->core.flag&BAM_FUNMAP)) { // exclude unmapped reads
118 			skip = 1;
119 			continue;
120 		}
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)));
123 			if (skip) continue;
124 		}
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);
128 			if (skip) continue;
129 		}
130 		if (ma->conf->flag & MPLP_ILLUMINA13) {
131 			int i;
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;
135 		}
136 		has_ref = (ma->ref && ma->ref_id == b->core.tid)? 1 : 0;
137 		skip = 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);
141 			if (q < 0) skip = 1;
142 			else if (b->core.qual > q) b->core.qual = q;
143 		}
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;
146 	} while (skip);
147 	return ret;
148 }
149 
group_smpl(mplp_pileup_t * m,bam_sample_t * sm,kstring_t * buf,int n,char * const * fn,int * n_plp,const bam_pileup1_t ** plp,int ignore_rg)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)
152 {
153 	int i, j;
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;
158 			uint8_t *q;
159 			int id = -1;
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]);
166 				exit(1);
167 			}
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]);
171 			}
172 			m->plp[id][m->n_plp[id]++] = *p;
173 		}
174 	}
175 }
176 
mpileup(mplp_conf_t * conf,int n,char ** fn)177 static int mpileup(mplp_conf_t *conf, int n, char **fn)
178 {
179 	extern void *bcf_call_add_rg(void *rghash, const char *hdtext, const char *list);
180 	extern void bcf_call_del_rghash(void *rghash);
181 	mplp_aux_t **data;
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;
184 	bam_mplp_t iter;
185 	bam_header_t *h = 0;
186 	char *ref;
187 	void *rghash = 0;
188 
189 	bcf_callaux_t *bca = 0;
190 	bcf_callret1_t *bcr = 0;
191 	bcf_call_t bc;
192 	bcf_t *bp = 0;
193 	bcf_hdr_t *bh = 0;
194 
195 	bam_sample_t *sm = 0;
196 	kstring_t buf;
197 	mplp_pileup_t gplp;
198 
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();
206 
207 	// read the header and initialize data
208 	for (i = 0; i < n; ++i) {
209 		bam_header_t *h_tmp;
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);
217 		if (conf->reg) {
218 			int beg, end;
219 			bam_index_t *idx;
220 			idx = bam_index_load(fn[i]);
221 			if (idx == 0) {
222 				fprintf(stderr, "[%s] fail to load index for %d-th input.\n", __func__, i+1);
223 				exit(1);
224 			}
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);
227 				exit(1);
228 			}
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);
232 		}
233 		if (i == 0) h = h_tmp;
234 		else {
235 			// FIXME: to check consistency
236 			bam_header_destroy(h_tmp);
237 		}
238 	}
239 	gplp.n = sm->n;
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*));
243 
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) {
247 		kstring_t s;
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);
253 			kputc('\0', &s);
254 		}
255 		bh->l_nm = s.l;
256 		bh->name = malloc(s.l);
257 		memcpy(bh->name, s.s, s.l);
258 		s.l = 0;
259 		for (i = 0; i < sm->n; ++i) {
260 			kputs(sm->smpl[i], &s); kputc('\0', &s);
261 		}
262 		bh->l_smpl = s.l;
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);
267 		free(s.s);
268 		bcf_hdr_sync(bh);
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;
276 	}
277 	if (tid0 >= 0 && conf->fai) { // region is set
278 		ref = faidx_fetch_seq(conf->fai, h->target_name[tid0], 0, 0x7fffffff, &ref_len);
279 		ref_tid = tid0;
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);
289 	}
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) {
296 			free(ref); ref = 0;
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;
299 			ref_tid = tid;
300 		}
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);
314 			bcf_destroy(b);
315 			// call indels
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);
324 					bcf_destroy(b);
325 				}
326 			}
327 		} else {
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) {
330 				int j;
331 				printf("\t%d\t", n_plp[i]);
332 				if (n_plp[i] == 0) {
333 					printf("*\t*"); // FIXME: printf() is very slow...
334 					if (conf->flag & MPLP_PRINT_POS) printf("\t*");
335 				} else {
336 					for (j = 0; j < n_plp[i]; ++j)
337 						pileup_seq(plp[i] + j, pos, ref_len, ref);
338 					putchar('\t');
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;
343 						putchar(c);
344 					}
345 					if (conf->flag & MPLP_PRINT_MAPQ) {
346 						putchar('\t');
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;
350 							putchar(c);
351 						}
352 					}
353 					if (conf->flag & MPLP_PRINT_POS) {
354 						putchar('\t');
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...
358 						}
359 					}
360 				}
361 			}
362 			putchar('\n');
363 		}
364 	}
365 
366 	bcf_close(bp);
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);
377 		free(data[i]);
378 	}
379 	free(data); free(plp); free(ref); free(n_plp);
380 	return 0;
381 }
382 
383 #define MAX_PATH_LEN 1024
read_file_list(const char * file_list,int * n,char ** argv[])384 static int read_file_list(const char *file_list,int *n,char **argv[])
385 {
386     char buf[MAX_PATH_LEN];
387     int len, nfiles;
388     char **files;
389 
390     FILE *fh = fopen(file_list,"r");
391     if ( !fh )
392     {
393         fprintf(stderr,"%s: %s\n", file_list,strerror(errno));
394         return 1;
395     }
396 
397     // Speed is not an issue here, determine the number of files by reading the file twice
398     nfiles = 0;
399     while ( fgets(buf,MAX_PATH_LEN,fh) ) nfiles++;
400 
401     if ( fseek(fh, 0L, SEEK_SET) )
402     {
403         fprintf(stderr,"%s: %s\n", file_list,strerror(errno));
404         return 1;
405     }
406 
407     files = calloc(nfiles,sizeof(char*));
408     nfiles = 0;
409     while ( fgets(buf,MAX_PATH_LEN,fh) )
410     {
411         len = strlen(buf);
412         while ( len>0 && isspace(buf[len-1]) ) len--;
413         if ( !len ) continue;
414 
415         files[nfiles] = malloc(sizeof(char)*(len+1));
416         strncpy(files[nfiles],buf,len);
417         files[nfiles][len] = 0;
418         nfiles++;
419     }
420     fclose(fh);
421     if ( !nfiles )
422     {
423         fprintf(stderr,"No files read from %s\n", file_list);
424         return 1;
425     }
426     *argv = files;
427     *n    = nfiles;
428     return 0;
429 }
430 #undef MAX_PATH_LEN
431 
bam_mpileup(int argc,char * argv[])432 int bam_mpileup(int argc, char *argv[])
433 {
434 	int c;
435     const char *file_list = NULL;
436     char **fn = NULL;
437     int nfiles = 0, use_orphan = 0;
438 	mplp_conf_t mplp;
439 	memset(&mplp, 0, sizeof(mplp_conf_t));
440 	#define MPLP_PRINT_POS 0x4000
441 	mplp.max_mq = 60;
442 	mplp.min_baseQ = 13;
443 	mplp.capQ_thres = 0;
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) {
449 		switch (c) {
450 		case 'f':
451 			mplp.fai = fai_load(optarg);
452 			if (mplp.fai == 0) return 1;
453 			break;
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;
482 		case 'G': {
483 				FILE *fp_rg;
484 				char buf[1024];
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));
490 				fclose(fp_rg);
491 			}
492 			break;
493 		}
494 	}
495 	if (use_orphan) mplp.flag &= ~MPLP_NO_ORPHAN;
496 	if (argc == 1) {
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");
533 		return 1;
534 	}
535     if (file_list) {
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]);
539         free(fn);
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);
545 	return 0;
546 }
547