]> git.donarmstrong.com Git - samtools.git/blobdiff - bam_plcmd.c
* samtools-0.1.8-1 (r615)
[samtools.git] / bam_plcmd.c
index 79d0e63b8e75a8c6d55c19f6433d9d4c42d159cc..c665a923a8a2ec1eaab957444012689ffd8fab16 100644 (file)
@@ -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 +16,12 @@ 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
 
 typedef struct {
        bam_header_t *h;
@@ -25,8 +32,9 @@ typedef struct {
        uint32_t format;
        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; // for glf output only
+       glfFile fp_glf; // for glf output only
 } pu_data_t;
 
 char **__bam_get_lines(const char *fn, int *_n);
@@ -103,9 +111,9 @@ 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 +125,13 @@ 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 +150,7 @@ 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 +158,146 @@ static int glt3_func(uint32_t tid, uint32_t pos, int n, const bam_pileup1_t *pu,
        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, 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;
        }
+       // 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);
+               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);
        }
-       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);
-       }
-       // 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 (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 +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
+       // 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\t%d\n", r->cnt1, r->cnt2, r->cnt_ambi, 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;
@@ -246,41 +335,64 @@ 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->max_depth = 0;
        d->tid = -1; d->mask = BAM_DEF_MASK;
        d->c = bam_maqcns_init();
+       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:im:gI:G:")) >= 0) {
+       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 '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 (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, "        -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, "        -t FILE   list of reference sequences (assume the input is in SAM)\n");
+               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, "        -g        output in the extended GLT format (suppressing -c/-i/-s)\n");
+               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);
@@ -290,53 +402,184 @@ int bam_pileup(int argc, char *argv[])
                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);
-       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)
+       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 (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);
+               sampileup(fp, d->mask, pileup_func, d);
+               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 *
+ ***********/
+
+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;
+}