#include #include #include #include #include #include #include "bam.h" #include "errmod.h" #include "kseq.h" KSTREAM_INIT(gzFile, gzread, 16384) #define MAX_VARS 256 #define FLIP_PENALTY 2 #define FLIP_THRES 4 #define MASK_THRES 3 #define FLAG_FIX_CHIMERA 0x1 #define FLAG_LIST_EXCL 0x4 #define FLAG_DROP_AMBI 0x8 typedef struct { // configurations, initialized in the main function int flag, k, min_baseQ, min_varLOD, max_depth; // other global variables int vpos_shift; bamFile fp; char *pre; bamFile out[3]; // alignment queue int n, m; bam1_t **b; } phaseg_t; typedef struct { int8_t seq[MAX_VARS]; // TODO: change to dynamic memory allocation! int vpos, beg, end; uint32_t vlen:16, single:1, flip:1, phase:1, phased:1, ambig:1; uint32_t in:16, out:16; // in-phase and out-phase } frag_t, *frag_p; #define rseq_lt(a,b) ((a)->vpos < (b)->vpos) #include "khash.h" KHASH_SET_INIT_INT64(set64) KHASH_MAP_INIT_INT64(64, frag_t) typedef khash_t(64) nseq_t; #include "ksort.h" KSORT_INIT(rseq, frag_p, rseq_lt) static char nt16_nt4_table[] = { 4, 0, 1, 4, 2, 4, 4, 4, 3, 4, 4, 4, 4, 4, 4, 4 }; static inline uint64_t X31_hash_string(const char *s) { uint64_t h = *s; if (h) for (++s ; *s; ++s) h = (h << 5) - h + *s; return h; } static void count1(int l, const uint8_t *seq, int *cnt) { int i, j, n_ambi; uint32_t z, x; if (seq[l-1] == 0) return; // do nothing is the last base is ambiguous for (i = n_ambi = 0; i < l; ++i) // collect ambiguous bases if (seq[i] == 0) ++n_ambi; if (l - n_ambi <= 1) return; // only one SNP for (x = 0; x < 1u<>j&1; ++j; } z = z<<1 | c; } ++cnt[z]; } } static int **count_all(int l, int vpos, nseq_t *hash) { khint_t k; int i, j, **cnt; uint8_t *seq; seq = calloc(l, 1); cnt = calloc(vpos, sizeof(void*)); for (i = 0; i < vpos; ++i) cnt[i] = calloc(1<vpos >= vpos || f->single) continue; // out of region; or singleton if (f->vlen == 1) { // such reads should be flagged as deleted previously if everything is right f->single = 1; continue; } for (j = 1; j < f->vlen; ++j) { for (i = 0; i < l; ++i) seq[i] = j < l - 1 - i? 0 : f->seq[j - (l - 1 - i)]; count1(l, seq, cnt[f->vpos + j]); } } } free(seq); return cnt; } // phasing static int8_t *dynaprog(int l, int vpos, int **w) { int *f[2], *curr, *prev, max, i; int8_t **b, *h = 0; uint32_t x, z = 1u<<(l-1), mask = (1u<>1; y1 = xc>>1; c0 = prev[y0] + wi[x] + wi[xc]; c1 = prev[y1] + wi[x] + wi[xc]; if (c0 > c1) bi[x] = 0, curr[x] = c0; else bi[x] = 1, curr[x] = c1; } tmp = prev; prev = curr; curr = tmp; // swap } { // backtrack uint32_t max_x = 0; int which = 0; h = calloc(vpos, 1); for (x = 0, max = 0, max_x = 0; x < z; ++x) if (prev[x] > max) max = prev[x], max_x = x; for (i = vpos - 1, x = max_x; i >= 0; --i) { h[i] = which? (~x&1) : (x&1); which = b[i][x]? !which : which; x = b[i][x]? (~x&mask)>>1 : x>>1; } } // free for (i = 0; i < vpos; ++i) free(b[i]); free(f[0]); free(f[1]); free(b); return h; } // phase each fragment static uint64_t *fragphase(int vpos, const int8_t *path, nseq_t *hash, int flip) { khint_t k; uint64_t *pcnt; uint32_t *left, *rght, max; left = rght = 0; max = 0; pcnt = calloc(vpos, 8); for (k = 0; k < kh_end(hash); ++k) { if (kh_exist(hash, k)) { int i, c[2]; frag_t *f = &kh_val(hash, k); if (f->vpos >= vpos) continue; // get the phase c[0] = c[1] = 0; for (i = 0; i < f->vlen; ++i) { if (f->seq[i] == 0) continue; ++c[f->seq[i] == path[f->vpos + i] + 1? 0 : 1]; } f->phase = c[0] > c[1]? 0 : 1; f->in = c[f->phase]; f->out = c[1 - f->phase]; f->phased = f->in == f->out? 0 : 1; f->ambig = (f->in && f->out && f->out < 3 && f->in <= f->out + 1)? 1 : 0; // fix chimera f->flip = 0; if (flip && c[0] >= 3 && c[1] >= 3) { int sum[2], m, mi, md; if (f->vlen > max) { // enlarge the array max = f->vlen; kroundup32(max); left = realloc(left, max * 4); rght = realloc(rght, max * 4); } for (i = 0, sum[0] = sum[1] = 0; i < f->vlen; ++i) { // get left counts if (f->seq[i]) { int c = f->phase? 2 - f->seq[i] : f->seq[i] - 1; ++sum[c == path[f->vpos + i]? 0 : 1]; } left[i] = sum[1]<<16 | sum[0]; } for (i = f->vlen - 1, sum[0] = sum[1] = 0; i >= 0; --i) { // get right counts if (f->seq[i]) { int c = f->phase? 2 - f->seq[i] : f->seq[i] - 1; ++sum[c == path[f->vpos + i]? 0 : 1]; } rght[i] = sum[1]<<16 | sum[0]; } // find the best flip point for (i = m = 0, mi = -1, md = -1; i < f->vlen - 1; ++i) { int a[2]; a[0] = (left[i]&0xffff) + (rght[i+1]>>16&0xffff) - (rght[i+1]&0xffff) * FLIP_PENALTY; a[1] = (left[i]>>16&0xffff) + (rght[i+1]&0xffff) - (rght[i+1]>>16&0xffff) * FLIP_PENALTY; if (a[0] > a[1]) { if (a[0] > m) m = a[0], md = 0, mi = i; } else { if (a[1] > m) m = a[1], md = 1, mi = i; } } if (m - c[0] >= FLIP_THRES && m - c[1] >= FLIP_THRES) { // then flip f->flip = 1; if (md == 0) { // flip the tail for (i = mi + 1; i < f->vlen; ++i) if (f->seq[i] == 1) f->seq[i] = 2; else if (f->seq[i] == 2) f->seq[i] = 1; } else { // flip the head for (i = 0; i <= mi; ++i) if (f->seq[i] == 1) f->seq[i] = 2; else if (f->seq[i] == 2) f->seq[i] = 1; } } } // update pcnt[] if (!f->single) { for (i = 0; i < f->vlen; ++i) { int c; if (f->seq[i] == 0) continue; c = f->phase? 2 - f->seq[i] : f->seq[i] - 1; if (c == path[f->vpos + i]) { if (f->phase == 0) ++pcnt[f->vpos + i]; else pcnt[f->vpos + i] += 1ull<<32; } else { if (f->phase == 0) pcnt[f->vpos + i] += 1<<16; else pcnt[f->vpos + i] += 1ull<<48; } } } } } free(left); free(rght); return pcnt; } static uint64_t *genmask(int vpos, const uint64_t *pcnt, int *_n) { int i, max = 0, max_i = -1, m = 0, n = 0, beg = 0, score = 0; uint64_t *list = 0; for (i = 0; i < vpos; ++i) { uint64_t x = pcnt[i]; int c[4], pre = score, s; c[0] = x&0xffff; c[1] = x>>16&0xffff; c[2] = x>>32&0xffff; c[3] = x>>48&0xffff; s = (c[1] + c[3] == 0)? -(c[0] + c[2]) : (c[1] + c[3] - 1); if (c[3] > c[2]) s += c[3] - c[2]; if (c[1] > c[0]) s += c[1] - c[0]; score += s; if (score < 0) score = 0; if (pre == 0 && score > 0) beg = i; // change from zero to non-zero if ((i == vpos - 1 || score == 0) && max >= MASK_THRES) { if (n == m) { m = m? m<<1 : 4; list = realloc(list, m * 8); } list[n++] = (uint64_t)beg<<32 | max_i; i = max_i; // reset i to max_i score = 0; } else if (score > max) max = score, max_i = i; if (score == 0) max = 0; } *_n = n; return list; } // trim heading and tailing ambiguous bases; mark deleted and remove sequence static int clean_seqs(int vpos, nseq_t *hash) { khint_t k; int ret = 0; for (k = 0; k < kh_end(hash); ++k) { if (kh_exist(hash, k)) { frag_t *f = &kh_val(hash, k); int beg, end, i; if (f->vpos >= vpos) { ret = 1; continue; } for (i = 0; i < f->vlen; ++i) if (f->seq[i] != 0) break; beg = i; for (i = f->vlen - 1; i >= 0; --i) if (f->seq[i] != 0) break; end = i + 1; if (end - beg <= 0) kh_del(64, hash, k); else { if (beg != 0) memmove(f->seq, f->seq + beg, end - beg); f->vpos += beg; f->vlen = end - beg; f->single = f->vlen == 1? 1 : 0; } } } return ret; } static void dump_aln(phaseg_t *g, int min_pos, const nseq_t *hash) { int i, is_flip, drop_ambi; drop_ambi = g->flag & FLAG_DROP_AMBI; is_flip = (drand48() < 0.5); for (i = 0; i < g->n; ++i) { int end, which; uint64_t key; khint_t k; bam1_t *b = g->b[i]; key = X31_hash_string(bam1_qname(b)); end = bam_calend(&b->core, bam1_cigar(b)); if (end > min_pos) break; k = kh_get(64, hash, key); if (k == kh_end(hash)) which = 3; else { frag_t *f = &kh_val(hash, k); if (f->ambig) which = drop_ambi? 2 : 3; else if (f->phased && f->flip) which = 2; else if (f->phased == 0) which = 3; else { // phased and not flipped char c = 'Y'; which = f->phase; bam_aux_append(b, "ZP", 'A', 1, (uint8_t*)&c); } if (which < 2 && is_flip) which = 1 - which; // increase the randomness } if (which == 3) which = (drand48() < 0.5); bam_write1(g->out[which], b); bam_destroy1(b); g->b[i] = 0; } memmove(g->b, g->b + i, (g->n - i) * sizeof(void*)); g->n -= i; } static int phase(phaseg_t *g, const char *chr, int vpos, uint64_t *cns, nseq_t *hash) { int i, j, n_seqs = kh_size(hash), n_masked = 0, min_pos; khint_t k; frag_t **seqs; int8_t *path, *sitemask; uint64_t *pcnt, *regmask; if (vpos == 0) return 0; i = clean_seqs(vpos, hash); // i is true if hash has an element with its vpos >= vpos min_pos = i? cns[vpos]>>32 : 0x7fffffff; if (vpos == 1) { printf("PS\t%s\t%d\t%d\n", chr, (int)(cns[0]>>32) + 1, (int)(cns[0]>>32) + 1); printf("M0\t%s\t%d\t%d\t%c\t%c\t%d\t0\t0\t0\t0\n//\n", chr, (int)(cns[0]>>32) + 1, (int)(cns[0]>>32) + 1, "ACGTX"[cns[0]&3], "ACGTX"[cns[0]>>16&3], g->vpos_shift + 1); for (k = 0; k < kh_end(hash); ++k) { if (kh_exist(hash, k)) { frag_t *f = &kh_val(hash, k); if (f->vpos) continue; f->flip = 0; if (f->seq[0] == 0) f->phased = 0; else f->phased = 1, f->phase = f->seq[0] - 1; } } dump_aln(g, min_pos, hash); ++g->vpos_shift; return 1; } { // phase int **cnt; uint64_t *mask; printf("PS\t%s\t%d\t%d\n", chr, (int)(cns[0]>>32) + 1, (int)(cns[vpos-1]>>32) + 1); sitemask = calloc(vpos, 1); cnt = count_all(g->k, vpos, hash); path = dynaprog(g->k, vpos, cnt); for (i = 0; i < vpos; ++i) free(cnt[i]); free(cnt); pcnt = fragphase(vpos, path, hash, 0); // do not fix chimeras when masking mask = genmask(vpos, pcnt, &n_masked); regmask = calloc(n_masked, 8); for (i = 0; i < n_masked; ++i) { regmask[i] = cns[mask[i]>>32]>>32<<32 | cns[(uint32_t)mask[i]]>>32; for (j = mask[i]>>32; j <= (int32_t)mask[i]; ++j) sitemask[j] = 1; } free(mask); if (g->flag & FLAG_FIX_CHIMERA) { free(pcnt); pcnt = fragphase(vpos, path, hash, 1); } } for (i = 0; i < n_masked; ++i) printf("FL\t%s\t%d\t%d\n", chr, (int)(regmask[i]>>32) + 1, (int)regmask[i] + 1); for (i = 0; i < vpos; ++i) { uint64_t x = pcnt[i]; int8_t c[2]; c[0] = (cns[i]&0xffff)>>2 == 0? 4 : (cns[i]&3); c[1] = (cns[i]>>16&0xffff)>>2 == 0? 4 : (cns[i]>>16&3); printf("M%d\t%s\t%d\t%d\t%c\t%c\t%d\t%d\t%d\t%d\t%d\n", sitemask[i]+1, chr, (int)(cns[0]>>32) + 1, (int)(cns[i]>>32) + 1, "ACGTX"[c[path[i]]], "ACGTX"[c[1-path[i]]], i + g->vpos_shift + 1, (int)(x&0xffff), (int)(x>>16&0xffff), (int)(x>>32&0xffff), (int)(x>>48&0xffff)); } free(path); free(pcnt); free(regmask); free(sitemask); seqs = calloc(n_seqs, sizeof(void*)); for (k = 0, i = 0; k < kh_end(hash); ++k) if (kh_exist(hash, k) && kh_val(hash, k).vpos < vpos && !kh_val(hash, k).single) seqs[i++] = &kh_val(hash, k); n_seqs = i; ks_introsort_rseq(n_seqs, seqs); for (i = 0; i < n_seqs; ++i) { frag_t *f = seqs[i]; printf("EV\t0\t%s\t%d\t40\t%dM\t*\t0\t0\t", chr, f->vpos + 1 + g->vpos_shift, f->vlen); for (j = 0; j < f->vlen; ++j) { uint32_t c = cns[f->vpos + j]; if (f->seq[j] == 0) putchar('N'); else putchar("ACGT"[f->seq[j] == 1? (c&3) : (c>>16&3)]); } printf("\t*\tYP:i:%d\tYF:i:%d\tYI:i:%d\tYO:i:%d\tYS:i:%d\n", f->phase, f->flip, f->in, f->out, f->beg+1); } free(seqs); printf("//\n"); fflush(stdout); g->vpos_shift += vpos; dump_aln(g, min_pos, hash); return vpos; } static void update_vpos(int vpos, nseq_t *hash) { khint_t k; for (k = 0; k < kh_end(hash); ++k) { if (kh_exist(hash, k)) { frag_t *f = &kh_val(hash, k); if (f->vpos < vpos) kh_del(64, hash, k); // TODO: if frag_t::seq is allocated dynamically, free it else f->vpos -= vpos; } } } static nseq_t *shrink_hash(nseq_t *hash) // TODO: to implement { return hash; } static int readaln(void *data, bam1_t *b) { phaseg_t *g = (phaseg_t*)data; int ret; ret = bam_read1(g->fp, b); if (ret < 0) return ret; if (!(b->core.flag & (BAM_FUNMAP|BAM_FSECONDARY|BAM_FQCFAIL|BAM_FDUP)) && g->pre) { if (g->n == g->m) { g->m = g->m? g->m<<1 : 16; g->b = realloc(g->b, g->m * sizeof(void*)); } g->b[g->n++] = bam_dup1(b); } return ret; } static khash_t(set64) *loadpos(const char *fn, bam_header_t *h) { gzFile fp; kstream_t *ks; int ret, dret; kstring_t *str; khash_t(set64) *hash; hash = kh_init(set64); str = calloc(1, sizeof(kstring_t)); fp = strcmp(fn, "-")? gzopen(fn, "r") : gzdopen(fileno(stdin), "r"); ks = ks_init(fp); while (ks_getuntil(ks, 0, str, &dret) >= 0) { int tid = bam_get_tid(h, str->s); if (tid >= 0 && dret != '\n') { if (ks_getuntil(ks, 0, str, &dret) >= 0) { uint64_t x = (uint64_t)tid<<32 | (atoi(str->s) - 1); kh_put(set64, hash, x, &ret); } else break; } if (dret != '\n') while ((dret = ks_getc(ks)) > 0 && dret != '\n'); if (dret < 0) break; } ks_destroy(ks); gzclose(fp); free(str->s); free(str); return hash; } static int gl2cns(float q[16]) { int i, j, min_ij; float min, min2; min = min2 = 1e30; min_ij = -1; for (i = 0; i < 4; ++i) { for (j = i; j < 4; ++j) { if (q[i<<2|j] < min) min_ij = i<<2|j, min2 = min, min = q[i<<2|j]; else if (q[i<<2|j] < min2) min2 = q[i<<2|j]; } } return (min_ij>>2&3) == (min_ij&3)? 0 : 1<<18 | (min_ij>>2&3)<<16 | (min_ij&3) | (int)(min2 - min + .499) << 2; } int main_phase(int argc, char *argv[]) { extern void bam_init_header_hash(bam_header_t *header); int c, tid, pos, vpos = 0, n, lasttid = -1, max_vpos = 0; const bam_pileup1_t *plp; bam_plp_t iter; bam_header_t *h; nseq_t *seqs; uint64_t *cns = 0; phaseg_t g; char *fn_list = 0; khash_t(set64) *set = 0; errmod_t *em; uint16_t *bases; memset(&g, 0, sizeof(phaseg_t)); g.flag = FLAG_FIX_CHIMERA; g.min_varLOD = 37; g.k = 13; g.min_baseQ = 13; g.max_depth = 256; while ((c = getopt(argc, argv, "Q:eFq:k:b:l:D:A:")) >= 0) { switch (c) { case 'D': g.max_depth = atoi(optarg); break; case 'q': g.min_varLOD = atoi(optarg); break; case 'Q': g.min_baseQ = atoi(optarg); break; case 'k': g.k = atoi(optarg); break; case 'F': g.flag &= ~FLAG_FIX_CHIMERA; break; case 'e': g.flag |= FLAG_LIST_EXCL; break; case 'A': g.flag |= FLAG_DROP_AMBI; break; case 'b': g.pre = strdup(optarg); break; case 'l': fn_list = strdup(optarg); break; } } if (argc == optind) { fprintf(stderr, "\n"); fprintf(stderr, "Usage: samtools phase [options] \n\n"); fprintf(stderr, "Options: -k INT block length [%d]\n", g.k); fprintf(stderr, " -b STR prefix of BAMs to output [null]\n"); fprintf(stderr, " -q INT min het phred-LOD [%d]\n", g.min_varLOD); fprintf(stderr, " -Q INT min base quality in het calling [%d]\n", g.min_baseQ); fprintf(stderr, " -D INT max read depth [%d]\n", g.max_depth); // fprintf(stderr, " -l FILE list of sites to phase [null]\n"); fprintf(stderr, " -F do not attempt to fix chimeras\n"); fprintf(stderr, " -A drop reads with ambiguous phase\n"); // fprintf(stderr, " -e do not discover SNPs (effective with -l)\n"); fprintf(stderr, "\n"); return 1; } g.fp = strcmp(argv[optind], "-")? bam_open(argv[optind], "r") : bam_dopen(fileno(stdin), "r"); h = bam_header_read(g.fp); if (fn_list) { // read the list of sites to phase bam_init_header_hash(h); set = loadpos(fn_list, h); free(fn_list); } else g.flag &= ~FLAG_LIST_EXCL; if (g.pre) { // open BAMs to write char *s = malloc(strlen(g.pre) + 20); strcpy(s, g.pre); strcat(s, ".0.bam"); g.out[0] = bam_open(s, "w"); strcpy(s, g.pre); strcat(s, ".1.bam"); g.out[1] = bam_open(s, "w"); strcpy(s, g.pre); strcat(s, ".chimera.bam"); g.out[2] = bam_open(s, "w"); for (c = 0; c <= 2; ++c) bam_header_write(g.out[c], h); free(s); } iter = bam_plp_init(readaln, &g); g.vpos_shift = 0; seqs = kh_init(64); em = errmod_init(1. - 0.83); bases = calloc(g.max_depth, 2); printf("CC\n"); printf("CC\tDescriptions:\nCC\n"); printf("CC\t CC comments\n"); printf("CC\t PS start of a phase set\n"); printf("CC\t FL filtered region\n"); printf("CC\t M[012] markers; 0 for singletons, 1 for phased and 2 for filtered\n"); printf("CC\t EV supporting reads; SAM format\n"); printf("CC\t // end of a phase set\nCC\n"); printf("CC\tFormats of PS, FL and M[012] lines (1-based coordinates):\nCC\n"); printf("CC\t PS chr phaseSetStart phaseSetEnd\n"); printf("CC\t FL chr filterStart filterEnd\n"); printf("CC\t M? chr PS pos allele0 allele1 hetIndex #supports0 #errors0 #supp1 #err1\n"); printf("CC\nCC\n"); fflush(stdout); while ((plp = bam_plp_auto(iter, &tid, &pos, &n)) != 0) { int i, k, c, tmp, dophase = 1, in_set = 0; float q[16]; if (tid < 0) break; if (tid != lasttid) { // change of chromosome g.vpos_shift = 0; if (lasttid >= 0) { seqs = shrink_hash(seqs); phase(&g, h->target_name[lasttid], vpos, cns, seqs); update_vpos(0x7fffffff, seqs); } lasttid = tid; vpos = 0; } if (set && kh_get(set64, set, (uint64_t)tid<<32 | pos) != kh_end(set)) in_set = 1; if (n > g.max_depth) continue; // do not proceed if the depth is too high // fill the bases array and check if there is a variant for (i = k = 0; i < n; ++i) { const bam_pileup1_t *p = plp + i; uint8_t *seq; int q, baseQ, b; if (p->is_del || p->is_refskip) continue; baseQ = bam1_qual(p->b)[p->qpos]; if (baseQ < g.min_baseQ) continue; seq = bam1_seq(p->b); b = bam_nt16_nt4_table[bam1_seqi(seq, p->qpos)]; if (b > 3) continue; q = baseQ < p->b->core.qual? baseQ : p->b->core.qual; if (q < 4) q = 4; if (q > 63) q = 63; bases[k++] = q<<5 | (int)bam1_strand(p->b)<<4 | b; } if (k == 0) continue; errmod_cal(em, k, 4, bases, q); // compute genotype likelihood c = gl2cns(q); // get the consensus // tell if to proceed if (set && (g.flag&FLAG_LIST_EXCL) && !in_set) continue; // not in the list if (!in_set && (c&0xffff)>>2 < g.min_varLOD) continue; // not a variant // add the variant if (vpos == max_vpos) { max_vpos = max_vpos? max_vpos<<1 : 128; cns = realloc(cns, max_vpos * 8); } cns[vpos] = (uint64_t)pos<<32 | c; for (i = 0; i < n; ++i) { const bam_pileup1_t *p = plp + i; uint64_t key; khint_t k; uint8_t *seq = bam1_seq(p->b); frag_t *f; if (p->is_del || p->is_refskip) continue; if (p->b->core.qual == 0) continue; // get the base code c = nt16_nt4_table[(int)bam1_seqi(seq, p->qpos)]; if (c == (cns[vpos]&3)) c = 1; else if (c == (cns[vpos]>>16&3)) c = 2; else c = 0; // write to seqs key = X31_hash_string(bam1_qname(p->b)); k = kh_put(64, seqs, key, &tmp); f = &kh_val(seqs, k); if (tmp == 0) { // present in the hash table if (vpos - f->vpos + 1 < MAX_VARS) { f->vlen = vpos - f->vpos + 1; f->seq[f->vlen-1] = c; f->end = bam_calend(&p->b->core, bam1_cigar(p->b)); } dophase = 0; } else { // absent memset(f->seq, 0, MAX_VARS); f->beg = p->b->core.pos; f->end = bam_calend(&p->b->core, bam1_cigar(p->b)); f->vpos = vpos, f->vlen = 1, f->seq[0] = c, f->single = f->phased = f->flip = f->ambig = 0; } } if (dophase) { seqs = shrink_hash(seqs); phase(&g, h->target_name[tid], vpos, cns, seqs); update_vpos(vpos, seqs); cns[0] = cns[vpos]; vpos = 0; } ++vpos; } if (tid >= 0) phase(&g, h->target_name[tid], vpos, cns, seqs); bam_header_destroy(h); bam_plp_destroy(iter); bam_close(g.fp); kh_destroy(64, seqs); kh_destroy(set64, set); free(cns); errmod_destroy(em); free(bases); if (g.pre) { for (c = 0; c <= 2; ++c) bam_close(g.out[c]); free(g.pre); free(g.b); } return 0; }