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bam_plcmd.c

#include <math.h>
#include <stdio.h>
#include <unistd.h>
#include <ctype.h>
#include <string.h>
#include <errno.h>
#include "sam.h"
#include "faidx.h"
#include "bam_maqcns.h"
#include "khash.h"
#include "glf.h"
#include "kstring.h"

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
#define BAM_PLF_NOBAQ      0x400

typedef struct {
      bam_header_t *h;
      bam_maqcns_t *c;
      bam_maqindel_opt_t *ido;
      faidx_t *fai;
      khash_t(64) *hash;
      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_glf; // for glf output only
} pu_data_t;

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)
{
      char **list;
      int i, j, n, *fields, max_fields;
      khash_t(64) *hash;
      bam_init_header_hash(h);
      list = __bam_get_lines(fn, &n);
      hash = kh_init(64);
      max_fields = 0; fields = 0;
      for (i = 0; i < n; ++i) {
            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 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)
{
      pu_data_t *d = (pu_data_t*)data;
      bam_maqindel_ret_t *r = 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 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 c = bam_nt16_rev_table[bam1_seqi(bam1_seq(p->b), p->qpos)];
            if (ref) {
                  int rb = pos < ref_len? ref[pos] : 'N';
                  if (c == '=' || bam_nt16_table[c] == bam_nt16_table[rb]) c = bam1_strand(p->b)? ',' : '.';
                  else c = bam1_strand(p->b)? tolower(c) : toupper(c);
            } else {
                  if (c == '=') 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, 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 {
                  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);
            }
      }
    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;
                  }
            }
      }
      // 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');
      for (i = 0; i < n; ++i)
            pileup_seq(pu + i, pos, d->len, d->ref);
      // finalize rms_mapq
      if (d->format & BAM_PLF_CNS) {
            for (i = rms_aux = 0; i < n; ++i) {
                  const bam_pileup1_t *p = pu + i;
                  int tmp = p->b->core.qual < d->c->cap_mapQ? p->b->core.qual : d->c->cap_mapQ;
                  rms_aux += tmp * tmp;
            }
            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) {
                  int c = pu[i].b->core.qual + 33;
                  if (c > 126) c = 126;
                  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');
      // 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;
}

int bam_pileup(int argc, char *argv[])
{
      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 = 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: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); 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 (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, "        -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        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);
      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);
            { // 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;
                        if ((int)b->core.tid < 0) break;
                        // 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
      }

      // 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 *
 ***********/

#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
#define MPLP_FMT_DP 0x100
#define MPLP_FMT_SP 0x200
#define MPLP_NO_INDEL 0x400

typedef struct {
      int max_mq, min_mq, flag, min_baseQ, capQ_thres, max_depth;
      int openQ, extQ, tandemQ;
      char *reg, *fn_pos, *pl_list;
      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_core(bam1_t *b, const char *ref, int);
      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_core(b, ma->ref, 1);
            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)
{
      extern void *bcf_call_add_rg(void *rghash, const char *hdtext, const char *list);
      extern void bcf_call_del_rghash(void *rghash);
      mplp_aux_t **data;
      int i, tid, pos, *n_plp, beg0 = 0, end0 = 1u<<29, ref_len, ref_tid, max_depth;
      const bam_pileup1_t **plp;
      bam_mplp_t iter;
      bam_header_t *h = 0;
      char *ref;
      khash_t(64) *hash = 0;
      void *rghash = 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);
            rghash = bcf_call_add_rg(rghash, h_tmp->text, conf->pl_list);
            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));
            bca->rghash = rghash;
            bca->openQ = conf->openQ, bca->extQ = conf->extQ, bca->tandemQ = conf->tandemQ;
      }
      ref_tid = -1; ref = 0;
      iter = bam_mplp_init(n, mplp_func, (void**)data);
      max_depth = conf->max_depth;
      if (max_depth * sm->n > 1<<20)
            fprintf(stderr, "(%s) Max depth is above 1M. Potential memory hog!\n", __func__);
      if (max_depth * sm->n < 8000) {
            max_depth = 8000 / sm->n;
            fprintf(stderr, "<%s> Set max per-sample depth to %d\n", __func__, max_depth);
      }
      bam_mplp_set_maxcnt(iter, max_depth);
      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, (conf->flag&(MPLP_FMT_DP|MPLP_FMT_SP))? bcr : 0,
                                     (conf->flag&MPLP_FMT_SP), 0, 0);
                  bcf_write(bp, bh, b);
                  bcf_destroy(b);
                  // call indels
                  if (!(conf->flag&MPLP_NO_INDEL) && bcf_call_gap_prep(gplp.n, gplp.n_plp, gplp.plp, pos, bca, ref, rghash) >= 0) {
                        for (i = 0; i < gplp.n; ++i)
                              bcf_call_glfgen(gplp.n_plp[i], gplp.plp[i], -1, bca, bcr + i);
                        if (bcf_call_combine(gplp.n, bcr, -1, &bc) >= 0) {
                              b = calloc(1, sizeof(bcf1_t));
                              bcf_call2bcf(tid, pos, &bc, b, (conf->flag&(MPLP_FMT_DP|MPLP_FMT_SP))? bcr : 0,
                                                 (conf->flag&MPLP_FMT_SP), bca, ref);
                              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);
      bcf_call_del_rghash(rghash);
      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;
}

#define MAX_PATH_LEN 1024
int read_file_list(const char *file_list,int *n,char **argv[])
{
    char buf[MAX_PATH_LEN];
    int len, nfiles;
    char **files;

    FILE *fh = fopen(file_list,"r");
    if ( !fh )
    {
        fprintf(stderr,"%s: %s\n", file_list,strerror(errno));
        return 1;
    }

    // Speed is not an issue here, determine the number of files by reading the file twice
    nfiles = 0;
    while ( fgets(buf,MAX_PATH_LEN,fh) ) nfiles++;

    if ( fseek(fh, 0L, SEEK_SET) )
    {
        fprintf(stderr,"%s: %s\n", file_list,strerror(errno));
        return 1;
    }

    files = calloc(nfiles,sizeof(char*));
    nfiles = 0;
    while ( fgets(buf,MAX_PATH_LEN,fh) ) 
    {
        len = strlen(buf);
        while ( len>0 && isspace(buf[len-1]) ) len--;
        if ( !len ) continue;

        files[nfiles] = malloc(sizeof(char)*(len+1)); 
        strncpy(files[nfiles],buf,len);
        files[nfiles][len] = 0;
        nfiles++;
    }
    fclose(fh);
    if ( !nfiles )
    {
        fprintf(stderr,"No files read from %s\n", file_list);
        return 1;
    }
    *argv = files;
    *n    = nfiles;
    return 0;
}
#undef MAX_PATH_LEN

int bam_mpileup(int argc, char *argv[])
{
      int c;
    const char *file_list = NULL;
    char **fn = NULL;
    int nfiles = 0;
      mplp_conf_t mplp;
      memset(&mplp, 0, sizeof(mplp_conf_t));
      mplp.max_mq = 60;
      mplp.min_baseQ = 13;
      mplp.capQ_thres = 0;
      mplp.max_depth = 250;
      mplp.openQ = 40; mplp.extQ = 20; mplp.tandemQ = 100;
      mplp.flag = MPLP_NO_ORPHAN | MPLP_REALN;
      while ((c = getopt(argc, argv, "gf:r:l:M:q:Q:uaORC:BDSd:b:P:o:e:h:I")) >= 0) {
            switch (c) {
            case 'f':
                  mplp.fai = fai_load(optarg);
                  if (mplp.fai == 0) return 1;
                  break;
            case 'd': mplp.max_depth = atoi(optarg); break;
            case 'r': mplp.reg = strdup(optarg); break;
            case 'l': mplp.fn_pos = strdup(optarg); break;
            case 'P': mplp.pl_list = 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 'B': mplp.flag &= ~MPLP_REALN & ~MPLP_NO_ORPHAN; break;
            case 'O': mplp.flag |= MPLP_NO_ORPHAN; break;
            case 'R': mplp.flag |= MPLP_REALN; break;
            case 'D': mplp.flag |= MPLP_FMT_DP; break;
            case 'S': mplp.flag |= MPLP_FMT_SP; break;
            case 'I': mplp.flag |= MPLP_NO_INDEL; 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;
        case 'b': file_list = optarg; break;
            case 'o': mplp.openQ = atoi(optarg); break;
            case 'e': mplp.extQ = atoi(optarg); break;
            case 'h': mplp.tandemQ = 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, "         -b FILE     list of input BAM files [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, "         -d INT      max per-sample depth [%d]\n", mplp.max_depth);
            fprintf(stderr, "         -P STR      comma separated list of platforms for indels [all]\n");
            fprintf(stderr, "         -o INT      Phred-scaled gap open sequencing error probability [%d]\n", mplp.openQ);
            fprintf(stderr, "         -e INT      Phred-scaled gap extension seq error probability [%d]\n", mplp.extQ);
            fprintf(stderr, "         -h INT      coefficient for homopolyer errors [%d]\n", mplp.tandemQ);
            fprintf(stderr, "         -g          generate BCF output\n");
            fprintf(stderr, "         -u          do not compress BCF output\n");
            fprintf(stderr, "         -B          disable BAQ computation\n");
            fprintf(stderr, "         -D          output per-sample DP\n");
            fprintf(stderr, "         -S          output per-sample SP (strand bias P-value, slow)\n");
            fprintf(stderr, "         -I          do not perform indel calling\n");
            fprintf(stderr, "\n");
            fprintf(stderr, "Notes: Assuming diploid individuals.\n\n");
            return 1;
      }
    if ( file_list )
    {
        if ( read_file_list(file_list,&nfiles,&fn) ) return 1;
        mpileup(&mplp,nfiles,fn);
        for (c=0; c<nfiles; c++) free(fn[c]);
        free(fn);
    }
    else
          mpileup(&mplp, argc - optind, argv + optind);
      free(mplp.reg); free(mplp.pl_list);
      if (mplp.fai) fai_destroy(mplp.fai);
      return 0;
}

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