X-Git-Url: https://git.donarmstrong.com/?a=blobdiff_plain;f=bam_plcmd.c;h=c665a923a8a2ec1eaab957444012689ffd8fab16;hb=8b0f98e2ac96b5ab07fd482cbaaa06d458254a90;hp=0140c6670c1facfbd6b3084bff5cf812ab4f08ab;hpb=f93dae0d03856955f9424e8b2aaf261304ca647e;p=samtools.git

diff --git a/bam_plcmd.c b/bam_plcmd.c
index 0140c66..c665a92 100644
--- a/bam_plcmd.c
+++ b/bam_plcmd.c
@@ -1,14 +1,27 @@
+#include <math.h>
 #include <stdio.h>
 #include <unistd.h>
 #include <ctype.h>
-#include "bam.h"
+#include "sam.h"
 #include "faidx.h"
 #include "bam_maqcns.h"
 #include "khash.h"
-KHASH_SET_INIT_INT64(64)
+#include "glf.h"
+#include "kstring.h"
 
-#define BAM_PLF_SIMPLE 0x01
-#define BAM_PLF_CNS 0x02
+typedef int *indel_list_t;
+KHASH_MAP_INIT_INT64(64, indel_list_t)
+
+#define BAM_PLF_SIMPLE     0x01
+#define BAM_PLF_CNS        0x02
+#define BAM_PLF_INDEL_ONLY 0x04
+#define BAM_PLF_GLF        0x08
+#define BAM_PLF_VAR_ONLY   0x10
+#define BAM_PLF_2ND        0x20
+#define BAM_PLF_RANBASE    0x40
+#define BAM_PLF_1STBASE    0x80
+#define BAM_PLF_ALLBASE    0x100
+#define BAM_PLF_READPOS    0x200
 
 typedef struct {
 	bam_header_t *h;
@@ -17,94 +30,274 @@ typedef struct {
 	faidx_t *fai;
 	khash_t(64) *hash;
 	uint32_t format;
-	int tid, len;
+	int tid, len, last_pos;
+	int mask;
+    int max_depth;  // for indel calling, ignore reads with the depth too high. 0 for unlimited
 	char *ref;
+	glfFile fp_glf; // for glf output only
 } pu_data_t;
 
-char **bam_load_pos(const char *fn, int *_n);
+char **__bam_get_lines(const char *fn, int *_n);
 void bam_init_header_hash(bam_header_t *header);
 int32_t bam_get_tid(const bam_header_t *header, const char *seq_name);
 
 static khash_t(64) *load_pos(const char *fn, bam_header_t *h)
 {
-	int n, tmp, i;
-	char **list, *s;
-	uint64_t x;
+	char **list;
+	int i, j, n, *fields, max_fields;
 	khash_t(64) *hash;
 	bam_init_header_hash(h);
-	list = bam_load_pos(fn, &n);
+	list = __bam_get_lines(fn, &n);
 	hash = kh_init(64);
+	max_fields = 0; fields = 0;
 	for (i = 0; i < n; ++i) {
-		x = (uint64_t)bam_get_tid(h, list[i]) << 32;
-		s = list[i];
-		while (*s++);
-		x |= *((uint32_t*)s) - 1;
-		kh_put(64, hash, x, &tmp);
-		free(list[i]);
-	}
-	free(list);
+		char *str = list[i];
+		int chr, n_fields, ret;
+		khint_t k;
+		uint64_t x;
+		n_fields = ksplit_core(str, 0, &max_fields, &fields);
+		if (n_fields < 2) continue;
+		chr = bam_get_tid(h, str + fields[0]);
+		if (chr < 0) {
+			fprintf(stderr, "[load_pos] unknown reference sequence name: %s\n", str + fields[0]);
+			continue;
+		}
+		x = (uint64_t)chr << 32 | (atoi(str + fields[1]) - 1);
+		k = kh_put(64, hash, x, &ret);
+		if (ret == 0) {
+			fprintf(stderr, "[load_pos] position %s:%s has been loaded.\n", str+fields[0], str+fields[1]);
+			continue;
+		}
+		kh_val(hash, k) = 0;
+		if (n_fields > 2) {
+			// count
+			for (j = 2; j < n_fields; ++j) {
+				char *s = str + fields[j];
+				if ((*s != '+' && *s != '-') || !isdigit(s[1])) break;
+ 			}
+			if (j > 2) { // update kh_val()
+				int *q, y, z;
+				q = kh_val(hash, k) = (int*)calloc(j - 1, sizeof(int));
+				q[0] = j - 2; z = j; y = 1;
+				for (j = 2; j < z; ++j)
+					q[y++] = atoi(str + fields[j]);
+			}
+		}
+		free(str);
+	}
+	free(list); free(fields);
 	return hash;
 }
 
-static int pileup_func(uint32_t tid, uint32_t pos, int n, const bam_pileup1_t *pu, void *data)
+// an analogy to pileup_func() below
+static int glt3_func(uint32_t tid, uint32_t pos, int n, const bam_pileup1_t *pu, void *data)
 {
 	pu_data_t *d = (pu_data_t*)data;
 	bam_maqindel_ret_t *r = 0;
-	int i, j, rb;
-	uint32_t x;
-	if (d->hash && kh_get(64, d->hash, (uint64_t)tid<<32|pos) == kh_end(d->hash)) return 0;
+	int rb, *proposed_indels = 0;
+	glf1_t *g;
+	glf3_t *g3;
+
+	if (d->fai == 0) {
+		fprintf(stderr, "[glt3_func] reference sequence is required for generating GLT. Abort!\n");
+		exit(1);
+	}
+	if (d->hash) { // only output a list of sites
+		khint_t k = kh_get(64, d->hash, (uint64_t)tid<<32|pos);
+		if (k == kh_end(d->hash)) return 0;
+		proposed_indels = kh_val(d->hash, k);
+	}
+	g3 = glf3_init1();
 	if (d->fai && (int)tid != d->tid) {
+		if (d->ref) { // then write the end mark
+			g3->rtype = GLF3_RTYPE_END;
+			glf3_write1(d->fp_glf, g3);
+		}
+		glf3_ref_write(d->fp_glf, d->h->target_name[tid], d->h->target_len[tid]); // write reference
 		free(d->ref);
 		d->ref = fai_fetch(d->fai, d->h->target_name[tid], &d->len);
 		d->tid = tid;
+		d->last_pos = 0;
+	}
+	rb = (d->ref && (int)pos < d->len)? d->ref[pos] : 'N';
+	g = bam_maqcns_glfgen(n, pu, bam_nt16_table[rb], d->c);
+	memcpy(g3, g, sizeof(glf1_t));
+	g3->rtype = GLF3_RTYPE_SUB;
+	g3->offset = pos - d->last_pos;
+	d->last_pos = pos;
+	glf3_write1(d->fp_glf, g3);
+    if (pos < d->len) {
+        int m = (!d->max_depth || d->max_depth>n) ? n : d->max_depth;
+		if (proposed_indels)
+			r = bam_maqindel(m, pos, d->ido, pu, d->ref, proposed_indels[0], proposed_indels+1);
+		else r = bam_maqindel(m, pos, d->ido, pu, d->ref, 0, 0);
+	}
+	if (r) { // then write indel line
+		int het = 3 * n, min;
+		min = het;
+		if (min > r->gl[0]) min = r->gl[0];
+		if (min > r->gl[1]) min = r->gl[1];
+		g3->ref_base = 0;
+		g3->rtype = GLF3_RTYPE_INDEL;
+		memset(g3->lk, 0, 10);
+		g3->lk[0] = r->gl[0] - min < 255? r->gl[0] - min : 255;
+		g3->lk[1] = r->gl[1] - min < 255? r->gl[1] - min : 255;
+		g3->lk[2] = het - min < 255? het - min : 255;
+		g3->offset = 0;
+		g3->indel_len[0] = r->indel1;
+		g3->indel_len[1] = r->indel2;
+		g3->min_lk = min < 255? min : 255;
+		g3->max_len = (abs(r->indel1) > abs(r->indel2)? abs(r->indel1) : abs(r->indel2)) + 1;
+		g3->indel_seq[0] = strdup(r->s[0]+1);
+		g3->indel_seq[1] = strdup(r->s[1]+1);
+		glf3_write1(d->fp_glf, g3);
+		bam_maqindel_ret_destroy(r);
+	}
+	free(g);
+	glf3_destroy1(g3);
+	return 0;
+}
+
+static void pileup_seq(const bam_pileup1_t *p, int pos, int ref_len, const char *ref)
+{
+	if (p->is_head) printf("^%c", p->b->core.qual > 93? 126 : p->b->core.qual + 33);
+	if (!p->is_del) {
+		int j, rb, c = bam_nt16_rev_table[bam1_seqi(bam1_seq(p->b), p->qpos)];
+		rb = (ref && pos < ref_len)? ref[pos] : 'N';
+		if (c == '=' || toupper(c) == toupper(rb)) c = bam1_strand(p->b)? ',' : '.';
+		else c = bam1_strand(p->b)? tolower(c) : toupper(c);
+		putchar(c);
+		if (p->indel > 0) {
+			printf("+%d", p->indel);
+			for (j = 1; j <= p->indel; ++j) {
+				c = bam_nt16_rev_table[bam1_seqi(bam1_seq(p->b), p->qpos + j)];
+				putchar(bam1_strand(p->b)? tolower(c) : toupper(c));
+			}
+		} else if (p->indel < 0) {
+			printf("%d", p->indel);
+			for (j = 1; j <= -p->indel; ++j) {
+				c = (ref && (int)pos+j < ref_len)? ref[pos+j] : 'N';
+				putchar(bam1_strand(p->b)? tolower(c) : toupper(c));
+			}
+		}
+	} else putchar('*');
+	if (p->is_tail) putchar('$');
+}
+
+static int pileup_func(uint32_t tid, uint32_t pos, int n, const bam_pileup1_t *pu, void *data)
+{
+	pu_data_t *d = (pu_data_t*)data;
+	bam_maqindel_ret_t *r = 0;
+	int i, rb, rms_mapq = -1, *proposed_indels = 0;
+	uint64_t rms_aux;
+	uint32_t cns = 0;
+
+	// if GLF is required, suppress -c completely
+	if (d->format & BAM_PLF_GLF) return glt3_func(tid, pos, n, pu, data);
+	// if d->hash is initialized, only output the sites in the hash table
+	if (d->hash) {
+		khint_t k = kh_get(64, d->hash, (uint64_t)tid<<32|pos);
+		if (k == kh_end(d->hash)) return 0;
+		proposed_indels = kh_val(d->hash, k);
+	}
+	// update d->ref if necessary
+	if (d->fai && (int)tid != d->tid) {
+		free(d->ref);
+		d->ref = faidx_fetch_seq(d->fai, d->h->target_name[tid], 0, 0x7fffffff, &d->len);
+		d->tid = tid;
 	}
 	rb = (d->ref && (int)pos < d->len)? d->ref[pos] : 'N';
+	// when the indel-only mode is asked for, return if no reads mapped with indels
+	if (d->format & BAM_PLF_INDEL_ONLY) {
+		for (i = 0; i < n; ++i)
+			if (pu[i].indel != 0) break;
+		if (i == n) return 0;
+	}
+	// call the consensus and indel
+	if (d->format & BAM_PLF_CNS) { // call consensus
+		if (d->format & (BAM_PLF_RANBASE|BAM_PLF_1STBASE)) { // use a random base or the 1st base as the consensus call
+			const bam_pileup1_t *p = (d->format & BAM_PLF_1STBASE)? pu : pu + (int)(drand48() * n);
+			int q = bam1_qual(p->b)[p->qpos];
+			int mapQ = p->b->core.qual < d->c->cap_mapQ? p->b->core.qual : d->c->cap_mapQ;
+			uint32_t b = bam1_seqi(bam1_seq(p->b), p->qpos);
+			cns = b<<28 | 0xf<<24 | mapQ<<16 | q<<8;
+		} else if (d->format & BAM_PLF_ALLBASE) { // collapse all bases
+			uint64_t rmsQ = 0;
+			uint32_t b = 0;
+			for (i = 0; i < n; ++i) {
+				const bam_pileup1_t *p = pu + i;
+				int q = p->b->core.qual < d->c->cap_mapQ? p->b->core.qual : d->c->cap_mapQ;
+				b |= bam1_seqi(bam1_seq(p->b), p->qpos);
+				rmsQ += q * q;
+			}
+			rmsQ = (uint64_t)(sqrt((double)rmsQ / n) + .499);
+			cns = b<<28 | 0xf<<24 | rmsQ<<16 | 60<<8;
+		} else cns = bam_maqcns_call(n, pu, d->c);
+	}
+    if ((d->format & (BAM_PLF_CNS|BAM_PLF_INDEL_ONLY)) && d->ref && pos < d->len) { // call indels
+        int m = (!d->max_depth || d->max_depth>n) ? n : d->max_depth;
+        if (proposed_indels) // the first element gives the size of the array
+            r = bam_maqindel(m, pos, d->ido, pu, d->ref, proposed_indels[0], proposed_indels+1);
+        else r = bam_maqindel(m, pos, d->ido, pu, d->ref, 0, 0);
+	}
+	// when only variant sites are asked for, test if the site is a variant
+	if ((d->format & BAM_PLF_CNS) && (d->format & BAM_PLF_VAR_ONLY)) {
+		if (!(bam_nt16_table[rb] != 15 && cns>>28 != bam_nt16_table[rb])) { // not a SNP
+			if (!(r && (r->gt == 2 || strcmp(r->s[r->gt], "*")))) { // not an indel
+				if (r) bam_maqindel_ret_destroy(r);
+				return 0;
+			}
+		}
+	}
+	// print the first 3 columns
 	printf("%s\t%d\t%c\t", d->h->target_name[tid], pos + 1, rb);
-	if (d->format & BAM_PLF_CNS) { // consensus
+	// print consensus information if required
+	if (d->format & BAM_PLF_CNS) {
 		int ref_q, rb4 = bam_nt16_table[rb];
-		x = bam_maqcns_call(n, pu, d->c);
 		ref_q = 0;
-		if (rb4 != 15 && x>>28 != 15 && x>>28 != rb4) { // a SNP
-			ref_q = ((x>>24&0xf) == rb4)? x>>8&0xff : (x>>8&0xff) + (x&0xff);
+		if (rb4 != 15 && cns>>28 != 15 && cns>>28 != rb4) { // a SNP
+			ref_q = ((cns>>24&0xf) == rb4)? cns>>8&0xff : (cns>>8&0xff) + (cns&0xff);
 			if (ref_q > 255) ref_q = 255;
 		}
-		printf("%c\t%d\t%d\t%d\t", bam_nt16_rev_table[x>>28], x>>8&0xff, ref_q, x>>16&0xff);
-		if (d->ref) // indel calling
-			r = bam_maqindel(n, pos, d->ido, pu, d->ref);
+		rms_mapq = cns>>16&0xff;
+		printf("%c\t%d\t%d\t%d\t", bam_nt16_rev_table[cns>>28], cns>>8&0xff, ref_q, rms_mapq);
 	}
-	// pileup strings
+	// print pileup sequences
 	printf("%d\t", n);
+	rms_aux = 0; // we need to recalculate rms_mapq when -c is not flagged on the command line
 	for (i = 0; i < n; ++i) {
 		const bam_pileup1_t *p = pu + i;
-		if (p->is_head) printf("^%c", p->b->core.qual > 93? 126 : p->b->core.qual + 33);
-		if (!p->is_del) {
-			int c = bam_nt16_rev_table[bam1_seqi(bam1_seq(p->b), p->qpos)];
-			if (toupper(c) == toupper(rb)) c = bam1_strand(p->b)? ',' : '.';
-			else bam1_strand(p->b)? tolower(c) : toupper(c);
-			putchar(c);
-			if (p->indel > 0) {
-				printf("+%d", p->indel);
-				for (j = 1; j <= p->indel; ++j) {
-					c = bam_nt16_rev_table[bam1_seqi(bam1_seq(p->b), p->qpos + j)];
-					putchar(bam1_strand(p->b)? tolower(c) : toupper(c));
-				}
-			} else if (p->indel < 0) {
-				printf("%d", p->indel);
-				for (j = 1; j <= -p->indel; ++j) {
-					c = (d->ref && (int)pos+j < d->len)? d->ref[pos+j] : 'N';
-					putchar(bam1_strand(p->b)? tolower(c) : toupper(c));
-				}
-			}
-		} else putchar('*');
-		if (p->is_tail) putchar('$');
+		int tmp = p->b->core.qual < d->c->cap_mapQ? p->b->core.qual : d->c->cap_mapQ;
+		rms_aux += tmp * tmp;
+		pileup_seq(p, pos, d->len, d->ref);
 	}
+	// finalize rms_mapq
+	rms_aux = (uint64_t)(sqrt((double)rms_aux / n) + .499);
+	if (rms_mapq < 0) rms_mapq = rms_aux;
 	putchar('\t');
+	// print quality
 	for (i = 0; i < n; ++i) {
 		const bam_pileup1_t *p = pu + i;
 		int c = bam1_qual(p->b)[p->qpos] + 33;
 		if (c > 126) c = 126;
 		putchar(c);
 	}
+	if (d->format & BAM_PLF_2ND) { // print 2nd calls and qualities
+		const unsigned char *q;
+		putchar('\t');
+		for (i = 0; i < n; ++i) {
+			const bam_pileup1_t *p = pu + i;
+			q = bam_aux_get(p->b, "E2");
+			putchar(q? q[p->qpos + 1] : 'N');
+		}
+		putchar('\t');
+		for (i = 0; i < n; ++i) {
+			const bam_pileup1_t *p = pu + i;
+			q = bam_aux_get(p->b, "U2");
+			putchar(q? q[p->qpos + 1] : '!');
+		}
+	}
+	// print mapping quality if -s is flagged on the command line
 	if (d->format & BAM_PLF_SIMPLE) {
 		putchar('\t');
 		for (i = 0; i < n; ++i) {
@@ -113,10 +306,28 @@ static int pileup_func(uint32_t tid, uint32_t pos, int n, const bam_pileup1_t *p
 			putchar(c);
 		}
 	}
+	// print read position
+	if (d->format & BAM_PLF_READPOS) {
+		putchar('\t');
+		for (i = 0; i < n; ++i) {
+			int x = pu[i].qpos;
+			int l = pu[i].b->core.l_qseq;
+			printf("%d,", x < l/2? x+1 : -((l-1)-x+1));
+		}
+	}
 	putchar('\n');
-	if (r) { // then print indel line
-		printf("%s\t%d\t*\t%s/%s\t", d->h->target_name[tid], pos + 1, r->s1, r->s2);
-		printf("%d\t%d\t%d\t%d\n", r->cnt1, r->cnt2, r->cnt_ambi, r->cnt_anti);
+	// print the indel line if r has been calculated. This only happens if:
+	// a) -c or -i are flagged, AND b) the reference sequence is available
+	if (r) {
+		printf("%s\t%d\t*\t", d->h->target_name[tid], pos + 1);
+		if (r->gt < 2) printf("%s/%s\t", r->s[r->gt], r->s[r->gt]);
+		else printf("%s/%s\t", r->s[0], r->s[1]);
+		printf("%d\t%d\t", r->q_cns, r->q_ref);
+		printf("%d\t%d\t", rms_mapq, n);
+		printf("%s\t%s\t", r->s[0], r->s[1]);
+		//printf("%d\t%d\t", r->gl[0], r->gl[1]);
+		printf("%d\t%d\t%d\t", r->cnt1, r->cnt2, r->cnt_anti);
+		printf("%d\t%d\n", r->cnt_ref, r->cnt_ambi);
 		bam_maqindel_ret_destroy(r);
 	}
 	return 0;
@@ -124,71 +335,251 @@ static int pileup_func(uint32_t tid, uint32_t pos, int n, const bam_pileup1_t *p
 
 int bam_pileup(int argc, char *argv[])
 {
-	int c;
+	int c, is_SAM = 0;
 	char *fn_list = 0, *fn_fa = 0, *fn_pos = 0;
 	pu_data_t *d = (pu_data_t*)calloc(1, sizeof(pu_data_t));
-	d->tid = -1;
+    d->max_depth = 0;
+	d->tid = -1; d->mask = BAM_DEF_MASK;
 	d->c = bam_maqcns_init();
-	while ((c = getopt(argc, argv, "st:f:cT:N:r:l:")) >= 0) {
+	d->c->is_soap = 1; // change the default model
+	d->ido = bam_maqindel_opt_init();
+	while ((c = getopt(argc, argv, "st:f:cT:N:r:l:d:im:gI:G:vM:S2aR:PA")) >= 0) {
 		switch (c) {
+		case 'a': d->c->is_soap = 1; break;
+		case 'A': d->c->is_soap = 0; break;
 		case 's': d->format |= BAM_PLF_SIMPLE; break;
 		case 't': fn_list = strdup(optarg); break;
 		case 'l': fn_pos = strdup(optarg); break;
 		case 'f': fn_fa = strdup(optarg); break;
 		case 'T': d->c->theta = atof(optarg); break;
 		case 'N': d->c->n_hap = atoi(optarg); break;
-		case 'r': d->c->het_rate = atoi(optarg); break;
+		case 'r': d->c->het_rate = atof(optarg); d->ido->r_snp = d->c->het_rate; break;
+		case 'M': d->c->cap_mapQ = atoi(optarg); break;
+		case 'd': d->max_depth = atoi(optarg); break;
 		case 'c': d->format |= BAM_PLF_CNS; break;
+		case 'i': d->format |= BAM_PLF_INDEL_ONLY; break;
+		case 'v': d->format |= BAM_PLF_VAR_ONLY; break;
+		case 'm': d->mask = strtol(optarg, 0, 0); break;
+		case 'g': d->format |= BAM_PLF_GLF; break;
+		case '2': d->format |= BAM_PLF_2ND; break;
+		case 'P': d->format |= BAM_PLF_READPOS; break;
+		case 'I': d->ido->q_indel = atoi(optarg); break;
+		case 'G': d->ido->r_indel = atof(optarg); break;
+		case 'S': is_SAM = 1; break;
+		case 'R':
+			if (strcmp(optarg, "random") == 0) d->format |= BAM_PLF_RANBASE;
+			else if (strcmp(optarg, "first") == 0) d->format |= BAM_PLF_1STBASE;
+			else if (strcmp(optarg, "all") == 0) d->format |= BAM_PLF_ALLBASE;
+			else fprintf(stderr, "[bam_pileup] unrecognized -R\n");
+			break;
 		default: fprintf(stderr, "Unrecognizd option '-%c'.\n", c); return 1;
 		}
 	}
+	if (fn_list) is_SAM = 1;
 	if (optind == argc) {
 		fprintf(stderr, "\n");
-		fprintf(stderr, "Usage:  bamtk pileup [options] <in.bam>|<in.sam>\n\n");
+		fprintf(stderr, "Usage:  samtools pileup [options] <in.bam>|<in.sam>\n\n");
 		fprintf(stderr, "Option: -s        simple (yet incomplete) pileup format\n");
-		fprintf(stderr, "        -t FILE   list of reference sequences (assume the input is in SAM)\n");
+		fprintf(stderr, "        -S        the input is in SAM\n");
+		fprintf(stderr, "        -A        use the MAQ model for SNP calling\n");
+		fprintf(stderr, "        -2        output the 2nd best call and quality\n");
+		fprintf(stderr, "        -i        only show lines/consensus with indels\n");
+		fprintf(stderr, "        -m INT    filtering reads with bits in INT [%d]\n", d->mask);
+		fprintf(stderr, "        -M INT    cap mapping quality at INT [%d]\n", d->c->cap_mapQ);
+        fprintf(stderr, "        -d INT    limit maximum depth for indels [unlimited]\n");
+		fprintf(stderr, "        -t FILE   list of reference sequences (force -S)\n");
 		fprintf(stderr, "        -l FILE   list of sites at which pileup is output\n");
 		fprintf(stderr, "        -f FILE   reference sequence in the FASTA format\n\n");
-		fprintf(stderr, "        -c        output the maq consensus sequence\n");
-		fprintf(stderr, "        -T FLOAT  theta in maq consensus calling model (for -c only) [%f]\n", d->c->theta);
-		fprintf(stderr, "        -N INT    number of haplotypes in the sample (for -c only) [%d]\n", d->c->n_hap);
-		fprintf(stderr, "        -r FLOAT  prior of a difference between any two haplotypes (for -c only) [%f]\n\n",
-				d->c->het_rate);
+		fprintf(stderr, "        -c        output the SOAPsnp consensus sequence\n");
+		fprintf(stderr, "        -v        print variants only (for -c)\n");
+		fprintf(stderr, "        -g        output in the GLFv3 format (suppressing -c/-i/-s)\n");
+		fprintf(stderr, "        -T FLOAT  theta in maq consensus calling model (for -c/-g) [%f]\n", d->c->theta);
+		fprintf(stderr, "        -N INT    number of haplotypes in the sample (for -c/-g) [%d]\n", d->c->n_hap);
+		fprintf(stderr, "        -r FLOAT  prior of a difference between two haplotypes (for -c/-g) [%f]\n", d->c->het_rate);
+		fprintf(stderr, "        -G FLOAT  prior of an indel between two haplotypes (for -c/-g) [%f]\n", d->ido->r_indel);
+		fprintf(stderr, "        -I INT    phred prob. of an indel in sequencing/prep. (for -c/-g) [%d]\n", d->ido->q_indel);
+		fprintf(stderr, "\n");
 		free(fn_list); free(fn_fa); free(d);
 		return 1;
 	}
+	if (d->format & (BAM_PLF_RANBASE|BAM_PLF_1STBASE|BAM_PLF_ALLBASE)) d->format |= BAM_PLF_CNS;
 	if (fn_fa) d->fai = fai_load(fn_fa);
-	free(fn_fa);
-	bam_maqcns_prepare(d->c);
-	d->ido = bam_maqindel_opt_init();
-	if (fn_list) {
-		tamFile fp;
-		bam1_t *b;
-		int ret;
-		bam_plbuf_t *buf = bam_plbuf_init(pileup_func, d);
-		d->h = sam_header_read2(fn_list);
-		if (fn_pos) d->hash = load_pos(fn_pos, d->h);
-		fp = sam_open(argv[optind]);
-		b = (bam1_t*)calloc(1, sizeof(bam1_t));
-		while ((ret = sam_read1(fp, d->h, b)) >= 0)
-			bam_plbuf_push(b, buf);
-		bam_plbuf_push(0, buf);
-		bam_plbuf_destroy(buf);
-		bam_destroy1(b);
-		sam_close(fp);
-	} else {
-		bamFile fp;
-		fp = (strcmp(argv[optind], "-") == 0)? bam_dopen(fileno(stdin), "r") : bam_open(argv[optind], "r");
-		d->h = bam_header_read(fp);
+	if (d->format & (BAM_PLF_CNS|BAM_PLF_GLF)) bam_maqcns_prepare(d->c); // consensus calling
+	if (d->format & BAM_PLF_GLF) { // for glf output
+		glf3_header_t *h;
+		h = glf3_header_init();
+		d->fp_glf = bgzf_fdopen(fileno(stdout), "w");
+		glf3_header_write(d->fp_glf, h);
+		glf3_header_destroy(h);
+	}
+	if (d->fai == 0 && (d->format & (BAM_PLF_CNS|BAM_PLF_INDEL_ONLY)))
+		fprintf(stderr, "[bam_pileup] indels will not be called when -f is absent.\n");
+	if (fn_fa && is_SAM && fn_list == 0) fn_list = samfaipath(fn_fa);
+
+	{
+		samfile_t *fp;
+		fp = is_SAM? samopen(argv[optind], "r", fn_list) : samopen(argv[optind], "rb", 0);
+		if (fp == 0 || fp->header == 0) {
+			fprintf(stderr, "[bam_pileup] fail to read the header: non-exisiting file or wrong format.\n");
+			return 1;
+		}
+		d->h = fp->header;
 		if (fn_pos) d->hash = load_pos(fn_pos, d->h);
-		bam_pileup_file(fp, pileup_func, d);
-		bam_close(fp);
+		sampileup(fp, d->mask, pileup_func, d);
+		samclose(fp); // d->h will be destroyed here
 	}
-	free(fn_pos); free(fn_list);
-	kh_destroy(64, d->hash);
-	bam_header_destroy(d->h);
+
+	// free
+	if (d->format & BAM_PLF_GLF) bgzf_close(d->fp_glf);
+	if (fn_pos) { // free the hash table
+		khint_t k;
+		for (k = kh_begin(d->hash); k < kh_end(d->hash); ++k)
+			if (kh_exist(d->hash, k)) free(kh_val(d->hash, k));
+		kh_destroy(64, d->hash);
+	}
+	free(fn_pos); free(fn_list); free(fn_fa);
 	if (d->fai) fai_destroy(d->fai);
 	bam_maqcns_destroy(d->c);
 	free(d->ido); free(d->ref); free(d);
 	return 0;
 }
+
+/***********
+ * mpileup *
+ ***********/
+
+typedef struct {
+	char *reg, *fn_pos;
+	faidx_t *fai;
+	kh_64_t *hash;
+} mplp_conf_t;
+
+typedef struct {
+	bamFile fp;
+	bam_iter_t iter;
+} mplp_aux_t;
+
+static int mplp_func(void *data, bam1_t *b)
+{
+	mplp_aux_t *ma = (mplp_aux_t*)data;
+	if (ma->iter) return bam_iter_read(ma->fp, ma->iter, b);
+	return bam_read1(ma->fp, b);
+}
+
+static int mpileup(mplp_conf_t *conf, int n, char **fn)
+{
+	mplp_aux_t **data;
+	int i, tid, pos, *n_plp, beg0 = 0, end0 = 1u<<29, ref_len, ref_tid;
+	const bam_pileup1_t **plp;
+	bam_mplp_t iter;
+	bam_header_t *h = 0;
+	char *ref;
+	khash_t(64) *hash = 0;
+	// allocate
+	data = calloc(n, sizeof(void*));
+	plp = calloc(n, sizeof(void*));
+	n_plp = calloc(n, sizeof(int*));
+	// read the header and initialize data
+	for (i = 0; i < n; ++i) {
+		bam_header_t *h_tmp;
+		data[i] = calloc(1, sizeof(mplp_aux_t));
+		data[i]->fp = bam_open(fn[i], "r");
+		h_tmp = bam_header_read(data[i]->fp);
+		if (conf->reg) {
+			int beg, end;
+			bam_index_t *idx;
+			idx = bam_index_load(fn[i]);
+			if (idx == 0) {
+				fprintf(stderr, "[%s] fail to load index for %d-th input.\n", __func__, i+1);
+				exit(1);
+			}
+			if (bam_parse_region(h_tmp, conf->reg, &tid, &beg, &end) < 0) {
+				fprintf(stderr, "[%s] malformatted region or wrong seqname for %d-th input.\n", __func__, i+1);
+				exit(1);
+			}
+			if (i == 0) beg0 = beg, end0 = end;
+			data[i]->iter = bam_iter_query(idx, tid, beg, end);
+			bam_index_destroy(idx);
+		}
+		if (i == 0) h = h_tmp;
+		else {
+			// FIXME: to check consistency
+			bam_header_destroy(h_tmp);
+		}
+	}
+	if (conf->fn_pos) hash = load_pos(conf->fn_pos, h);
+	// mpileup
+	ref_tid = -1; ref = 0;
+	iter = bam_mplp_init(n, mplp_func, (void**)data);
+	while (bam_mplp_auto(iter, &tid, &pos, n_plp, plp) > 0) {
+		if (conf->reg && (pos < beg0 || pos >= end0)) continue; // out of the region requested
+		if (hash) {
+			khint_t k;
+			k = kh_get(64, hash, (uint64_t)tid<<32 | pos);
+			if (k == kh_end(hash)) continue;
+		}
+		if (tid != ref_tid) {
+			free(ref);
+			if (conf->fai) ref = fai_fetch(conf->fai, h->target_name[tid], &ref_len);
+			ref_tid = tid;
+		}
+		printf("%s\t%d\t%c", h->target_name[tid], pos + 1, (ref && pos < ref_len)? ref[pos] : 'N');
+		for (i = 0; i < n; ++i) {
+			int j;
+			printf("\t%d\t", n_plp[i]);
+			if (n_plp[i] == 0) printf("*\t*");
+			else {
+				for (j = 0; j < n_plp[i]; ++j)
+					pileup_seq(plp[i] + j, pos, ref_len, ref);
+				putchar('\t');
+				for (j = 0; j < n_plp[i]; ++j) {
+					const bam_pileup1_t *p = plp[i] + j;
+					int c = bam1_qual(p->b)[p->qpos] + 33;
+					if (c > 126) c = 126;
+					putchar(c);
+				}
+			}
+		}
+		putchar('\n');
+	}
+	if (hash) { // free the hash table
+		khint_t k;
+		for (k = kh_begin(hash); k < kh_end(hash); ++k)
+			if (kh_exist(hash, k)) free(kh_val(hash, k));
+		kh_destroy(64, hash);
+	}
+	bam_mplp_destroy(iter);
+	bam_header_destroy(h);
+	for (i = 0; i < n; ++i) {
+		bam_close(data[i]->fp);
+		if (data[i]->iter) bam_iter_destroy(data[i]->iter);
+		free(data[i]);
+	}
+	free(data); free(plp); free(ref); free(n_plp);
+	return 0;
+}
+
+int bam_mpileup(int argc, char *argv[])
+{
+	int c;
+	mplp_conf_t mplp;
+	memset(&mplp, 0, sizeof(mplp_conf_t));
+	while ((c = getopt(argc, argv, "f:r:l:")) >= 0) {
+		switch (c) {
+		case 'f':
+			mplp.fai = fai_load(optarg);
+			if (mplp.fai == 0) return 1;
+			break;
+		case 'r': mplp.reg = strdup(optarg); break;
+		case 'l': mplp.fn_pos = strdup(optarg); break;
+		}
+	}
+	if (argc == 1) {
+		fprintf(stderr, "Usage: samtools mpileup [-r reg] [-f in.fa] [-l pos] in1.bam [in2.bam [...]]\n");
+		return 1;
+	}
+	mpileup(&mplp, argc - optind, argv + optind);
+	free(mplp.reg);
+	if (mplp.fai) fai_destroy(mplp.fai);
+	return 0;
+}