X-Git-Url: https://git.donarmstrong.com/?a=blobdiff_plain;f=bam_plcmd.c;h=f0e63ce62cd91cc6cada955eaa19c9b2493e1872;hb=6ee6fd65332260af63fbfbf0a625ea3847371eda;hp=38680df3357a42b80cf251a3b8c796e057d17d32;hpb=5fa181a401f891a049e3c43d03917e9452d1d739;p=samtools.git

diff --git a/bam_plcmd.c b/bam_plcmd.c
index 38680df..f0e63ce 100644
--- a/bam_plcmd.c
+++ b/bam_plcmd.c
@@ -1,7 +1,8 @@
+#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"
@@ -15,6 +16,13 @@ KHASH_MAP_INIT_INT64(64, indel_list_t)
 #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
+#define BAM_PLF_NOBAQ      0x400
 
 typedef struct {
 	bam_header_t *h;
@@ -25,8 +33,10 @@ typedef struct {
 	uint32_t format;
 	int tid, len, last_pos;
 	int mask;
+	int capQ_thres, min_baseQ;
+    int max_depth;  // for indel calling, ignore reads with the depth too high. 0 for unlimited
 	char *ref;
-	glfFile fp; // for glf output only
+	glfFile fp_glf; // for glf output only
 } pu_data_t;
 
 char **__bam_get_lines(const char *fn, int *_n);
@@ -81,6 +91,21 @@ static khash_t(64) *load_pos(const char *fn, bam_header_t *h)
 	return hash;
 }
 
+static inline int printw(int c, FILE *fp)
+{
+	char buf[16];
+	int l, x;
+	if (c == 0) return fputc('0', fp);
+	for (l = 0, x = c < 0? -c : c; x > 0; x /= 10) buf[l++] = x%10 + '0';
+	if (c < 0) buf[l++] = '-';
+	buf[l] = 0;
+	for (x = 0; x < l/2; ++x) {
+		int y = buf[x]; buf[x] = buf[l-1-x]; buf[l-1-x] = y;
+	}
+	fputs(buf, fp);
+	return 0;
+}
+
 // 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)
 {
@@ -103,9 +128,9 @@ static int glt3_func(uint32_t tid, uint32_t pos, int n, const bam_pileup1_t *pu,
 	if (d->fai && (int)tid != d->tid) {
 		if (d->ref) { // then write the end mark
 			g3->rtype = GLF3_RTYPE_END;
-			glf3_write1(d->fp, g3);
+			glf3_write1(d->fp_glf, g3);
 		}
-		glf3_ref_write(d->fp, d->h->target_name[tid], d->h->target_len[tid]); // write reference
+		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;
@@ -117,10 +142,13 @@ static int glt3_func(uint32_t tid, uint32_t pos, int n, const bam_pileup1_t *pu,
 	g3->rtype = GLF3_RTYPE_SUB;
 	g3->offset = pos - d->last_pos;
 	d->last_pos = pos;
-	glf3_write1(d->fp, g3);
-	if (proposed_indels)
-		r = bam_maqindel(n, pos, d->ido, pu, d->ref, proposed_indels[0], proposed_indels+1);
-	else r = bam_maqindel(n, pos, d->ido, pu, d->ref, 0, 0);
+	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;
@@ -139,7 +167,7 @@ static int glt3_func(uint32_t tid, uint32_t pos, int n, const bam_pileup1_t *pu,
 		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, g3);
+		glf3_write1(d->fp_glf, g3);
 		bam_maqindel_ret_destroy(r);
 	}
 	free(g);
@@ -147,80 +175,151 @@ static int glt3_func(uint32_t tid, uint32_t pos, int n, const bam_pileup1_t *pu,
 	return 0;
 }
 
+static inline void pileup_seq(const bam_pileup1_t *p, int pos, int ref_len, const char *ref)
+{
+	int j;
+	if (p->is_head) {
+		putchar('^');
+		putchar(p->b->core.qual > 93? 126 : p->b->core.qual + 33);
+	}
+	if (!p->is_del) {
+		int 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);
+	} else putchar(p->is_refskip? (bam1_strand(p->b)? '<' : '>') : '*');
+	if (p->indel > 0) {
+		putchar('+'); printw(p->indel, stdout);
+		for (j = 1; j <= p->indel; ++j) {
+			int 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) {
+		printw(p->indel, stdout);
+		for (j = 1; j <= -p->indel; ++j) {
+			int c = (ref && (int)pos+j < ref_len)? ref[pos+j] : 'N';
+			putchar(bam1_strand(p->b)? tolower(c) : toupper(c));
+		}
+	}
+	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, j, rb, max_mapq = 0, *proposed_indels = 0;
-	uint32_t x;
+	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) { // only output a list of sites
+	// 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);
 	}
-	if (d->fai && (int)tid != d->tid) { // then update d->ref
+	// update d->ref if necessary
+	if (d->fai && (int)tid != d->tid) {
 		free(d->ref);
-		d->ref = fai_fetch(d->fai, d->h->target_name[tid], &d->len);
+		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;
 	}
-	printf("%s\t%d\t%c\t", d->h->target_name[tid], pos + 1, rb);
-	if (d->format & BAM_PLF_CNS) { // consensus
-		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 (ref_q > 255) ref_q = 255;
+	// 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 {
+			glf1_t *g = bam_maqcns_glfgen(n, pu, bam_nt16_table[rb], d->c);
+			cns = g->depth == 0? (0xfu<<28 | 0xf<<24) : glf2cns(g, (int)(d->c->q_r + .499));
+			free(g);
 		}
-		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->format & (BAM_PLF_CNS|BAM_PLF_INDEL_ONLY)) && d->ref) {
-		if (proposed_indels)
-			r = bam_maqindel(n, pos, d->ido, pu, d->ref, proposed_indels[0], proposed_indels+1);
-		else r = bam_maqindel(n, pos, d->ido, pu, d->ref, 0, 0);
+    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 != 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;
+			}
+		}
 	}
-	// pileup strings
-	printf("%d\t", n);
+	// print the first 3 columns
+	fputs(d->h->target_name[tid], stdout); putchar('\t');
+	printw(pos+1, stdout); putchar('\t'); putchar(rb); putchar('\t');
+	// print consensus information if required
+	if (d->format & BAM_PLF_CNS) {
+		putchar(bam_nt16_rev_table[cns>>28]); putchar('\t');
+		printw(cns>>8&0xff, stdout); putchar('\t');
+		printw(cns&0xff, stdout); putchar('\t');
+		printw(cns>>16&0xff, stdout); putchar('\t');
+	}
+	// print pileup sequences
+	printw(n, stdout); putchar('\t');
+	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 (max_mapq < p->b->core.qual) max_mapq = p->b->core.qual;
-		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 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 = (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) {
@@ -229,16 +328,28 @@ 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;
+			printw(x < l/2? x+1 : -((l-1)-x+1), stdout); putchar(',');
+		}
+	}
 	putchar('\n');
-	if (r) { // then print indel line
+	// 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", max_mapq, n);
+		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\n", r->cnt1, r->cnt2, r->cnt_anti);
+		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;
@@ -246,101 +357,446 @@ 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->mask = BAM_DEF_MASK;
+    d->max_depth = 1024; d->tid = -1; d->mask = BAM_DEF_MASK; d->min_baseQ = 13;
 	d->c = bam_maqcns_init();
+	d->c->errmod = BAM_ERRMOD_MAQ2; // change the default model
 	d->ido = bam_maqindel_opt_init();
-	while ((c = getopt(argc, argv, "st:f:cT:N:r:l:im:gI:G:")) >= 0) {
+	while ((c = getopt(argc, argv, "st:f:cT:N:r:l:d:im:gI:G:vM:S2aR:PAQ:C:B")) >= 0) {
 		switch (c) {
+		case 'Q': d->c->min_baseQ = atoi(optarg); break;
+		case 'C': d->capQ_thres = atoi(optarg); break;
+		case 'B': d->format |= BAM_PLF_NOBAQ; break;
+		case 'a': d->c->errmod = BAM_ERRMOD_SOAP; break;
+		case 'A': d->c->errmod = BAM_ERRMOD_MAQ; 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 = atof(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 'm': d->mask = atoi(optarg); 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 (d->c->errmod != BAM_ERRMOD_MAQ2) d->c->theta += 0.02;
+	if (d->c->theta > 1.0) d->c->theta = 1.0;
+	if (fn_list) is_SAM = 1;
 	if (optind == argc) {
 		fprintf(stderr, "\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, "        -S        the input is in SAM\n");
+		fprintf(stderr, "        -B        disable BAQ computation\n");
+		fprintf(stderr, "        -A        use the original MAQ model for SNP calling (DEPRECATED)\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, "        -t FILE   list of reference sequences (assume the input is in SAM)\n");
+		fprintf(stderr, "        -Q INT    min base quality (possibly capped by BAQ) [%d]\n", d->c->min_baseQ);
+		fprintf(stderr, "        -C INT    coefficient for adjusting mapQ of poor mappings [%d]\n", d->capQ_thres);
+		fprintf(stderr, "        -m INT    filtering reads with bits in INT [0x%x]\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 [%d]\n", d->max_depth);
+		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, "        -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, "        -c        compute the consensus sequence\n");
+		fprintf(stderr, "        -v        print variants only (for -c)\n");
+		fprintf(stderr, "        -g        output in the GLFv3 format (DEPRECATED)\n");
+		fprintf(stderr, "        -T FLOAT  theta in maq consensus calling model (for -c) [%.4g]\n", d->c->theta);
+		fprintf(stderr, "        -N INT    number of haplotypes in the sample (for -c) [%d]\n", d->c->n_hap);
+		fprintf(stderr, "        -r FLOAT  prior of a difference between two haplotypes (for -c) [%.4g]\n", d->c->het_rate);
+		fprintf(stderr, "        -G FLOAT  prior of an indel between two haplotypes (for -c) [%.4g]\n", d->ido->r_indel);
+		fprintf(stderr, "        -I INT    phred prob. of an indel in sequencing/prep. (for -c) [%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);
-	if (d->format & (BAM_PLF_CNS|BAM_PLF_GLF)) bam_maqcns_prepare(d->c);
-	if (d->format & BAM_PLF_GLF) {
+	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 = bgzf_fdopen(fileno(stdout), "w");
-		glf3_header_write(d->fp, h);
+		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_list) { // the input is SAM
-		tamFile fp;
-		bam1_t *b;
-		int ret;
-		bam_plbuf_t *buf = bam_plbuf_init(pileup_func, d);
-		bam_plbuf_set_mask(buf, d->mask);
-		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 { // the input is BAM
-		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->h == 0) {
-			fprintf(stderr, "[bam_pileup] fail to read the BAM header. Abort!\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, d->mask, pileup_func, d);
-		bam_close(fp);
+		{ // run pileup
+			extern int bam_prob_realn(bam1_t *b, const char *ref);
+			extern int bam_cap_mapQ(bam1_t *b, char *ref, int thres);
+			bam1_t *b;
+			int ret, tid, pos, n_plp;
+			bam_plp_t iter;
+			const bam_pileup1_t *plp;
+			b = bam_init1();
+			iter = bam_plp_init(0, 0);
+			bam_plp_set_mask(iter, d->mask);
+			while ((ret = samread(fp, b)) >= 0) {
+				int skip = 0;
+				// update d->ref if necessary
+				if (d->fai && (int)b->core.tid != d->tid) {
+					free(d->ref);
+					d->ref = faidx_fetch_seq(d->fai, d->h->target_name[b->core.tid], 0, 0x7fffffff, &d->len);
+					d->tid = b->core.tid;
+				}
+				if (d->ref && (d->format&BAM_PLF_CNS) && !(d->format&BAM_PLF_NOBAQ)) bam_prob_realn(b, d->ref);
+				if (d->ref && (d->format&BAM_PLF_CNS) && d->capQ_thres > 10) {
+					int q = bam_cap_mapQ(b, d->ref, d->capQ_thres);
+					if (q < 0) skip = 1;
+					else if (b->core.qual > q) b->core.qual = q;
+				} else if (b->core.flag&BAM_FUNMAP) skip = 1;
+				else if ((d->format&BAM_PLF_CNS) && (b->core.flag&1) && !(b->core.flag&2)) skip = 1;
+				if (skip) continue;
+				bam_plp_push(iter, b);
+				while ((plp = bam_plp_next(iter, &tid, &pos, &n_plp)) != 0)
+					pileup_func(tid, pos, n_plp, plp, d);
+			}
+			bam_plp_push(iter, 0);
+			while ((plp = bam_plp_next(iter, &tid, &pos, &n_plp)) != 0)
+				pileup_func(tid, pos, n_plp, plp, d);
+			bam_plp_destroy(iter);
+			bam_destroy1(b);
+		}
+		samclose(fp); // d->h will be destroyed here
 	}
-	if (d->format & BAM_PLF_GLF) bgzf_close(d->fp);
+
+	// 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);
-	bam_header_destroy(d->h);
+	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 *
+ ***********/
+
+#include <assert.h>
+#include "bam2bcf.h"
+#include "sample.h"
+
+#define MPLP_GLF   0x10
+#define MPLP_NO_COMP 0x20
+#define MPLP_NO_ORPHAN 0x40
+#define MPLP_REALN   0x80
+
+typedef struct {
+	int max_mq, min_mq, flag, min_baseQ, capQ_thres;
+	char *reg, *fn_pos;
+	faidx_t *fai;
+	kh_64_t *hash;
+} mplp_conf_t;
+
+typedef struct {
+	bamFile fp;
+	bam_iter_t iter;
+	int min_mq, flag, ref_id, capQ_thres;
+	char *ref;
+} mplp_aux_t;
+
+typedef struct {
+	int n;
+	int *n_plp, *m_plp;
+	bam_pileup1_t **plp;
+} mplp_pileup_t;
+
+static int mplp_func(void *data, bam1_t *b)
+{
+	extern int bam_realn(bam1_t *b, const char *ref);
+	extern int bam_prob_realn(bam1_t *b, const char *ref);
+	extern int bam_cap_mapQ(bam1_t *b, char *ref, int thres);
+	mplp_aux_t *ma = (mplp_aux_t*)data;
+	int ret, skip = 0;
+	do {
+		int has_ref = (ma->ref && ma->ref_id == b->core.tid)? 1 : 0;
+		ret = ma->iter? bam_iter_read(ma->fp, ma->iter, b) : bam_read1(ma->fp, b);
+		if (ret < 0) break;
+		skip = 0;
+		if (has_ref && (ma->flag&MPLP_REALN)) bam_prob_realn(b, ma->ref);
+		if (has_ref && ma->capQ_thres > 10) {
+			int q = bam_cap_mapQ(b, ma->ref, ma->capQ_thres);
+			if (q < 0) skip = 1;
+			else if (b->core.qual > q) b->core.qual = q;
+		} else if (b->core.flag&BAM_FUNMAP) skip = 1;
+		else if (b->core.qual < ma->min_mq) skip = 1; 
+		else if ((ma->flag&MPLP_NO_ORPHAN) && (b->core.flag&1) && !(b->core.flag&2)) skip = 1;
+	} while (skip);
+	return ret;
+}
+
+static void 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 i, j;
+	memset(m->n_plp, 0, m->n * sizeof(int));
+	for (i = 0; i < n; ++i) {
+		for (j = 0; j < n_plp[i]; ++j) {
+			const bam_pileup1_t *p = plp[i] + j;
+			uint8_t *q;
+			int id = -1;
+			q = bam_aux_get(p->b, "RG");
+			if (q) id = bam_smpl_rg2smid(sm, fn[i], (char*)q+1, buf);
+			if (id < 0) id = bam_smpl_rg2smid(sm, fn[i], 0, buf);
+			assert(id >= 0 && id < m->n);
+			if (m->n_plp[id] == m->m_plp[id]) {
+				m->m_plp[id] = m->m_plp[id]? m->m_plp[id]<<1 : 8;
+				m->plp[id] = realloc(m->plp[id], sizeof(bam_pileup1_t) * m->m_plp[id]);
+			}
+			m->plp[id][m->n_plp[id]++] = *p;
+		}
+	}
+}
+
+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;
+
+	bcf_callaux_t *bca = 0;
+	bcf_callret1_t *bcr = 0;
+	bcf_call_t bc;
+	bcf_t *bp = 0;
+	bcf_hdr_t *bh = 0;
+
+	bam_sample_t *sm = 0;
+	kstring_t buf;
+	mplp_pileup_t gplp;
+
+	memset(&gplp, 0, sizeof(mplp_pileup_t));
+	memset(&buf, 0, sizeof(kstring_t));
+	memset(&bc, 0, sizeof(bcf_call_t));
+	data = calloc(n, sizeof(void*));
+	plp = calloc(n, sizeof(void*));
+	n_plp = calloc(n, sizeof(int*));
+	sm = bam_smpl_init();
+
+	// 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]->min_mq = conf->min_mq;
+		data[i]->flag = conf->flag;
+		data[i]->capQ_thres = conf->capQ_thres;
+		data[i]->fp = strcmp(fn[i], "-") == 0? bam_dopen(fileno(stdin), "r") : bam_open(fn[i], "r");
+		h_tmp = bam_header_read(data[i]->fp);
+		bam_smpl_add(sm, fn[i], h_tmp->text);
+		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);
+		}
+	}
+	gplp.n = sm->n;
+	gplp.n_plp = calloc(sm->n, sizeof(int));
+	gplp.m_plp = calloc(sm->n, sizeof(int));
+	gplp.plp = calloc(sm->n, sizeof(void*));
+
+	fprintf(stderr, "[%s] %d samples in %d input files\n", __func__, sm->n, n);
+	if (conf->fn_pos) hash = load_pos(conf->fn_pos, h);
+	// write the VCF header
+	if (conf->flag & MPLP_GLF) {
+		kstring_t s;
+		bh = calloc(1, sizeof(bcf_hdr_t));
+		s.l = s.m = 0; s.s = 0;
+		bp = bcf_open("-", (conf->flag&MPLP_NO_COMP)? "wu" : "w");
+		for (i = 0; i < h->n_targets; ++i) {
+			kputs(h->target_name[i], &s);
+			kputc('\0', &s);
+		}
+		bh->l_nm = s.l;
+		bh->name = malloc(s.l);
+		memcpy(bh->name, s.s, s.l);
+		s.l = 0;
+		for (i = 0; i < sm->n; ++i) {
+			kputs(sm->smpl[i], &s); kputc('\0', &s);
+		}
+		bh->l_smpl = s.l;
+		bh->sname = malloc(s.l);
+		memcpy(bh->sname, s.s, s.l);
+		bh->l_txt = 0;
+		free(s.s);
+		bcf_hdr_sync(bh);
+		bcf_hdr_write(bp, bh);
+		bca = bcf_call_init(-1., conf->min_baseQ);
+		bcr = calloc(sm->n, sizeof(bcf_callret1_t));
+	}
+	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); ref = 0;
+			if (conf->fai) ref = fai_fetch(conf->fai, h->target_name[tid], &ref_len);
+			for (i = 0; i < n; ++i) data[i]->ref = ref, data[i]->ref_id = tid;
+			ref_tid = tid;
+		}
+		if (conf->flag & MPLP_GLF) {
+			int _ref0, ref16;
+			bcf1_t *b = calloc(1, sizeof(bcf1_t));
+			group_smpl(&gplp, sm, &buf, n, fn, n_plp, plp);
+			_ref0 = (ref && pos < ref_len)? ref[pos] : 'N';
+			ref16 = bam_nt16_table[_ref0];
+			for (i = 0; i < gplp.n; ++i)
+				bcf_call_glfgen(gplp.n_plp[i], gplp.plp[i], ref16, bca, bcr + i);
+			bcf_call_combine(gplp.n, bcr, ref16, &bc);
+			bcf_call2bcf(tid, pos, &bc, b);
+			bcf_write(bp, bh, b);
+			bcf_destroy(b);
+		} else {
+			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');
+		}
+	}
+
+	bcf_close(bp);
+	bam_smpl_destroy(sm); free(buf.s);
+	for (i = 0; i < gplp.n; ++i) free(gplp.plp[i]);
+	free(gplp.plp); free(gplp.n_plp); free(gplp.m_plp);
+	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);
+	}
+	bcf_hdr_destroy(bh); bcf_call_destroy(bca); free(bc.PL); free(bcr);
+	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));
+	mplp.max_mq = 60;
+	mplp.min_baseQ = 13;
+	mplp.capQ_thres = 0;
+	while ((c = getopt(argc, argv, "gf:r:l:M:q:Q:uaORC:")) >= 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;
+		case 'g': mplp.flag |= MPLP_GLF; break;
+		case 'u': mplp.flag |= MPLP_NO_COMP | MPLP_GLF; break;
+		case 'a': mplp.flag |= MPLP_NO_ORPHAN | MPLP_REALN; break;
+		case 'O': mplp.flag |= MPLP_NO_ORPHAN; break;
+		case 'R': mplp.flag |= MPLP_REALN; break;
+		case 'C': mplp.capQ_thres = atoi(optarg); break;
+		case 'M': mplp.max_mq = atoi(optarg); break;
+		case 'q': mplp.min_mq = atoi(optarg); break;
+		case 'Q': mplp.min_baseQ = atoi(optarg); break;
+		}
+	}
+	if (argc == 1) {
+		fprintf(stderr, "\n");
+		fprintf(stderr, "Usage:   samtools mpileup [options] in1.bam [in2.bam [...]]\n\n");
+		fprintf(stderr, "Options: -f FILE     reference sequence file [null]\n");
+		fprintf(stderr, "         -r STR      region in which pileup is generated [null]\n");
+		fprintf(stderr, "         -l FILE     list of positions (format: chr pos) [null]\n");
+		fprintf(stderr, "         -M INT      cap mapping quality at INT [%d]\n", mplp.max_mq);
+		fprintf(stderr, "         -Q INT      min base quality [%d]\n", mplp.min_baseQ);
+		fprintf(stderr, "         -q INT      filter out alignment with MQ smaller than INT [%d]\n", mplp.min_mq);
+		fprintf(stderr, "         -g          generate BCF output\n");
+		fprintf(stderr, "         -u          do not compress BCF output\n");
+		fprintf(stderr, "\n");
+		fprintf(stderr, "Notes: Assuming diploid individuals.\n\n");
+		return 1;
+	}
+	mpileup(&mplp, argc - optind, argv + optind);
+	free(mplp.reg);
+	if (mplp.fai) fai_destroy(mplp.fai);
+	return 0;
+}