#include #include #include #include #include #include #include #include "bwase.h" #include "bwtaln.h" #include "bntseq.h" #include "utils.h" #include "kstring.h" #include "bwa.h" #include "ksw.h" #ifdef USE_MALLOC_WRAPPERS # include "malloc_wrap.h" #endif int g_log_n[256]; void bwa_aln2seq_core(int n_aln, const bwt_aln1_t *aln, bwa_seq_t *s, int set_main, int n_multi) { int i, cnt, best; if (n_aln == 0) { s->type = BWA_TYPE_NO_MATCH; s->c1 = s->c2 = 0; return; } if (set_main) { best = aln[0].score; for (i = cnt = 0; i < n_aln; ++i) { const bwt_aln1_t *p = aln + i; if (p->score > best) break; if (drand48() * (p->l - p->k + 1 + cnt) > (double)cnt) { s->n_mm = p->n_mm; s->n_gapo = p->n_gapo; s->n_gape = p->n_gape; s->ref_shift = (int)p->n_del - (int)p->n_ins; s->score = p->score; s->sa = p->k + (bwtint_t)((p->l - p->k + 1) * drand48()); } cnt += p->l - p->k + 1; } s->c1 = cnt; for (; i < n_aln; ++i) cnt += aln[i].l - aln[i].k + 1; s->c2 = cnt - s->c1; s->type = s->c1 > 1? BWA_TYPE_REPEAT : BWA_TYPE_UNIQUE; } if (n_multi) { int k, rest, n_occ, z = 0; for (k = n_occ = 0; k < n_aln; ++k) { const bwt_aln1_t *q = aln + k; n_occ += q->l - q->k + 1; } if (s->multi) free(s->multi); if (n_occ > n_multi + 1) { // if there are too many hits, generate none of them s->multi = 0; s->n_multi = 0; return; } /* The following code is more flexible than what is required * here. In principle, due to the requirement above, we can * simply output all hits, but the following samples "rest" * number of random hits. */ rest = n_occ > n_multi + 1? n_multi + 1 : n_occ; // find one additional for ->sa s->multi = calloc(rest, sizeof(bwt_multi1_t)); for (k = 0; k < n_aln; ++k) { const bwt_aln1_t *q = aln + k; if (q->l - q->k + 1 <= rest) { bwtint_t l; for (l = q->k; l <= q->l; ++l) { s->multi[z].pos = l; s->multi[z].gap = q->n_gapo + q->n_gape; s->multi[z].ref_shift = (int)q->n_del - (int)q->n_ins; s->multi[z++].mm = q->n_mm; } rest -= q->l - q->k + 1; } else { // Random sampling (http://code.activestate.com/recipes/272884/). In fact, we never come here. int j, i; for (j = rest, i = q->l - q->k + 1; j > 0; --j) { double p = 1.0, x = drand48(); while (x < p) p -= p * j / (i--); s->multi[z].pos = q->l - i; s->multi[z].gap = q->n_gapo + q->n_gape; s->multi[z].ref_shift = (int)q->n_del - (int)q->n_ins; s->multi[z++].mm = q->n_mm; } rest = 0; break; } } s->n_multi = z; } } void bwa_aln2seq(int n_aln, const bwt_aln1_t *aln, bwa_seq_t *s) { bwa_aln2seq_core(n_aln, aln, s, 1, 0); } int bwa_approx_mapQ(const bwa_seq_t *p, int mm) { int n; if (p->c1 == 0) return 23; if (p->c1 > 1) return 0; if (p->n_mm == mm) return 25; if (p->c2 == 0) return 37; n = (p->c2 >= 255)? 255 : p->c2; return (23 < g_log_n[n])? 0 : 23 - g_log_n[n]; } bwtint_t bwa_sa2pos(const bntseq_t *bns, const bwt_t *bwt, bwtint_t sapos, int ref_len, int *strand) { bwtint_t pos_f; int is_rev; *strand = 0; // initialise strand to 0 otherwise we could return without setting it pos_f = bwt_sa(bwt, sapos); // position on the forward-reverse coordinate if (pos_f < bns->l_pac && bns->l_pac < pos_f + ref_len) return (bwtint_t)-1; pos_f = bns_depos(bns, pos_f, &is_rev); // position on the forward strand; this may be the first base or the last base *strand = !is_rev; if (is_rev) pos_f = pos_f + 1 < ref_len? 0 : pos_f - ref_len + 1; // position of the first base return pos_f; // FIXME: it is possible that pos_f < bns->anns[ref_id].offset } /** * Derive the actual position in the read from the given suffix array * coordinates. Note that the position will be approximate based on * whether indels appear in the read and whether calculations are * performed from the start or end of the read. */ void bwa_cal_pac_pos_core(const bntseq_t *bns, const bwt_t *bwt, bwa_seq_t *seq, const int max_mm, const float fnr) { int max_diff, strand; if (seq->type != BWA_TYPE_UNIQUE && seq->type != BWA_TYPE_REPEAT) return; max_diff = fnr > 0.0? bwa_cal_maxdiff(seq->len, BWA_AVG_ERR, fnr) : max_mm; seq->seQ = seq->mapQ = bwa_approx_mapQ(seq, max_diff); //fprintf(stderr, "%d\n", seq->ref_shift); seq->pos = bwa_sa2pos(bns, bwt, seq->sa, seq->len + seq->ref_shift, &strand); seq->strand = strand; seq->seQ = seq->mapQ = bwa_approx_mapQ(seq, max_diff); if (seq->pos == (bwtint_t)-1) seq->type = BWA_TYPE_NO_MATCH; } void bwa_cal_pac_pos(const bntseq_t *bns, const char *prefix, int n_seqs, bwa_seq_t *seqs, int max_mm, float fnr) { int i, j, strand, n_multi; char str[1024]; bwt_t *bwt; // load forward SA strcpy(str, prefix); strcat(str, ".bwt"); bwt = bwt_restore_bwt(str); strcpy(str, prefix); strcat(str, ".sa"); bwt_restore_sa(str, bwt); for (i = 0; i != n_seqs; ++i) { bwa_seq_t *p = &seqs[i]; bwa_cal_pac_pos_core(bns, bwt, p, max_mm, fnr); for (j = n_multi = 0; j < p->n_multi; ++j) { bwt_multi1_t *q = p->multi + j; q->pos = bwa_sa2pos(bns, bwt, q->pos, p->len + q->ref_shift, &strand); q->strand = strand; if (q->pos != p->pos && q->pos != (bwtint_t)-1) p->multi[n_multi++] = *q; } p->n_multi = n_multi; } bwt_destroy(bwt); } #define SW_BW 50 bwa_cigar_t *bwa_refine_gapped_core(bwtint_t l_pac, const ubyte_t *pacseq, int len, ubyte_t *seq, int ref_shift, bwtint_t *_rb, int *n_cigar) { bwa_cigar_t *cigar = 0; uint32_t *cigar32 = 0; ubyte_t *rseq; int64_t k, rb, re, rlen; int8_t mat[25]; bwa_fill_scmat(1, 3, mat); rb = *_rb; re = rb + len + ref_shift; assert(re <= l_pac); rseq = bns_get_seq(l_pac, pacseq, rb, re, &rlen); assert(re - rb == rlen); ksw_global(len, seq, rlen, rseq, 5, mat, 5, 1, SW_BW > abs(rlen - len) * 1.5? SW_BW : abs(rlen - len) * 1.5, n_cigar, &cigar32); assert(*n_cigar > 0); if ((cigar32[*n_cigar - 1]&0xf) == 1) cigar32[*n_cigar - 1] = (cigar32[*n_cigar - 1]>>4<<4) | 3; // change endding ins to soft clipping if ((cigar32[0]&0xf) == 1) cigar32[0] = (cigar32[0]>>4<<4) | 3; // change beginning ins to soft clipping if ((cigar32[*n_cigar - 1]&0xf) == 2) --*n_cigar; // delete endding del if ((cigar32[0]&0xf) == 2) { // delete beginning del *_rb += cigar32[0]>>4; --*n_cigar; memmove(cigar32, cigar32+1, (*n_cigar) * 4); } cigar = (bwa_cigar_t*)cigar32; for (k = 0; k < *n_cigar; ++k) cigar[k] = __cigar_create((cigar32[k]&0xf), (cigar32[k]>>4)); free(rseq); return cigar; } char *bwa_cal_md1(int n_cigar, bwa_cigar_t *cigar, int len, bwtint_t pos, ubyte_t *seq, bwtint_t l_pac, ubyte_t *pacseq, kstring_t *str, int *_nm) { bwtint_t x, y; int z, u, c, nm = 0; str->l = 0; // reset x = pos; y = 0; if (cigar) { int k, l; for (k = u = 0; k < n_cigar; ++k) { l = __cigar_len(cigar[k]); if (__cigar_op(cigar[k]) == FROM_M) { for (z = 0; z < l && x+z < l_pac; ++z) { c = pacseq[(x+z)>>2] >> ((~(x+z)&3)<<1) & 3; if (c > 3 || seq[y+z] > 3 || c != seq[y+z]) { ksprintf(str, "%d", u); kputc("ACGTN"[c], str); ++nm; u = 0; } else ++u; } x += l; y += l; } else if (__cigar_op(cigar[k]) == FROM_I || __cigar_op(cigar[k]) == FROM_S) { y += l; if (__cigar_op(cigar[k]) == FROM_I) nm += l; } else if (__cigar_op(cigar[k]) == FROM_D) { ksprintf(str, "%d", u); kputc('^', str); for (z = 0; z < l && x+z < l_pac; ++z) kputc("ACGT"[pacseq[(x+z)>>2] >> ((~(x+z)&3)<<1) & 3], str); u = 0; x += l; nm += l; } } } else { // no gaps for (z = u = 0; z < (bwtint_t)len && x+z < l_pac; ++z) { c = pacseq[(x+z)>>2] >> ((~(x+z)&3)<<1) & 3; if (c > 3 || seq[y+z] > 3 || c != seq[y+z]) { ksprintf(str, "%d", u); kputc("ACGTN"[c], str); ++nm; u = 0; } else ++u; } } ksprintf(str, "%d", u); *_nm = nm; return strdup(str->s); } void bwa_correct_trimmed(bwa_seq_t *s) { if (s->len == s->full_len) return; if (s->strand == 0) { // forward if (s->cigar && __cigar_op(s->cigar[s->n_cigar-1]) == FROM_S) { // the last is S s->cigar[s->n_cigar-1] += s->full_len - s->len; } else { if (s->cigar == 0) { s->n_cigar = 2; s->cigar = calloc(s->n_cigar, sizeof(bwa_cigar_t)); s->cigar[0] = __cigar_create(0, s->len); } else { ++s->n_cigar; s->cigar = realloc(s->cigar, s->n_cigar * sizeof(bwa_cigar_t)); } s->cigar[s->n_cigar-1] = __cigar_create(3, (s->full_len - s->len)); } } else { // reverse if (s->cigar && __cigar_op(s->cigar[0]) == FROM_S) { // the first is S s->cigar[0] += s->full_len - s->len; } else { if (s->cigar == 0) { s->n_cigar = 2; s->cigar = calloc(s->n_cigar, sizeof(bwa_cigar_t)); s->cigar[1] = __cigar_create(0, s->len); } else { ++s->n_cigar; s->cigar = realloc(s->cigar, s->n_cigar * sizeof(bwa_cigar_t)); memmove(s->cigar + 1, s->cigar, (s->n_cigar-1) * sizeof(bwa_cigar_t)); } s->cigar[0] = __cigar_create(3, (s->full_len - s->len)); } } s->len = s->full_len; } void bwa_refine_gapped(const bntseq_t *bns, int n_seqs, bwa_seq_t *seqs, ubyte_t *_pacseq) { ubyte_t *pacseq; int i, j, k; kstring_t *str; if (!_pacseq) { pacseq = (ubyte_t*)calloc(bns->l_pac/4+1, 1); err_rewind(bns->fp_pac); err_fread_noeof(pacseq, 1, bns->l_pac/4+1, bns->fp_pac); } else pacseq = _pacseq; for (i = 0; i != n_seqs; ++i) { bwa_seq_t *s = seqs + i; seq_reverse(s->len, s->seq, 0); // IMPORTANT: s->seq is reversed here!!! for (j = k = 0; j < s->n_multi; ++j) { bwt_multi1_t *q = s->multi + j; int n_cigar; if (q->gap) { // gapped alignment q->cigar = bwa_refine_gapped_core(bns->l_pac, pacseq, s->len, q->strand? s->rseq : s->seq, q->ref_shift, &q->pos, &n_cigar); q->n_cigar = n_cigar; if (q->cigar) s->multi[k++] = *q; } else s->multi[k++] = *q; } s->n_multi = k; // this squeezes out gapped alignments which failed the CIGAR generation if (s->type == BWA_TYPE_NO_MATCH || s->type == BWA_TYPE_MATESW || s->n_gapo == 0) continue; s->cigar = bwa_refine_gapped_core(bns->l_pac, pacseq, s->len, s->strand? s->rseq : s->seq, s->ref_shift, &s->pos, &s->n_cigar); if (s->cigar == 0) s->type = BWA_TYPE_NO_MATCH; } // generate MD tag str = (kstring_t*)calloc(1, sizeof(kstring_t)); for (i = 0; i != n_seqs; ++i) { bwa_seq_t *s = seqs + i; if (s->type != BWA_TYPE_NO_MATCH) { int nm; s->md = bwa_cal_md1(s->n_cigar, s->cigar, s->len, s->pos, s->strand? s->rseq : s->seq, bns->l_pac, pacseq, str, &nm); s->nm = nm; } } free(str->s); free(str); // correct for trimmed reads for (i = 0; i < n_seqs; ++i) bwa_correct_trimmed(seqs + i); if (!_pacseq) free(pacseq); } int64_t pos_end(const bwa_seq_t *p) { if (p->cigar) { int j; int64_t x = p->pos; for (j = 0; j != p->n_cigar; ++j) { int op = __cigar_op(p->cigar[j]); if (op == 0 || op == 2) x += __cigar_len(p->cigar[j]); } return x; } else return p->pos + p->len; } int64_t pos_end_multi(const bwt_multi1_t *p, int len) // analogy to pos_end() { if (p->cigar) { int j; int64_t x = p->pos; for (j = 0; j != p->n_cigar; ++j) { int op = __cigar_op(p->cigar[j]); if (op == 0 || op == 2) x += __cigar_len(p->cigar[j]); } return x; } else return p->pos + len; } static int64_t pos_5(const bwa_seq_t *p) { if (p->type != BWA_TYPE_NO_MATCH) return p->strand? pos_end(p) : p->pos; return -1; } void bwa_print_seq(FILE *stream, bwa_seq_t *seq) { char buffer[4096]; const int bsz = sizeof(buffer); int i, j, l; if (seq->strand == 0) { for (i = 0; i < seq->full_len; i += bsz) { l = seq->full_len - i > bsz ? bsz : seq->full_len - i; for (j = 0; j < l; j++) buffer[j] = "ACGTN"[seq->seq[i + j]]; err_fwrite(buffer, 1, l, stream); } } else { for (i = seq->full_len - 1; i >= 0; i -= bsz) { l = i + 1 > bsz ? bsz : i + 1; for (j = 0; j < l; j++) buffer[j] = "TGCAN"[seq->seq[i - j]]; err_fwrite(buffer, 1, l, stream); } } } void bwa_print_sam1(const bntseq_t *bns, bwa_seq_t *p, const bwa_seq_t *mate, int mode, int max_top2) { int j; if (p->type != BWA_TYPE_NO_MATCH || (mate && mate->type != BWA_TYPE_NO_MATCH)) { int seqid, nn, am = 0, flag = p->extra_flag; char XT; if (p->type == BWA_TYPE_NO_MATCH) { p->pos = mate->pos; p->strand = mate->strand; flag |= SAM_FSU; j = 1; } else j = pos_end(p) - p->pos; // j is the length of the reference in the alignment // get seqid nn = bns_cnt_ambi(bns, p->pos, j, &seqid); if (p->type != BWA_TYPE_NO_MATCH && p->pos + j - bns->anns[seqid].offset > bns->anns[seqid].len) flag |= SAM_FSU; // flag UNMAP as this alignment bridges two adjacent reference sequences // update flag and print it if (p->strand) flag |= SAM_FSR; if (mate) { if (mate->type != BWA_TYPE_NO_MATCH) { if (mate->strand) flag |= SAM_FMR; } else flag |= SAM_FMU; } err_printf("%s\t%d\t%s\t", p->name, flag, bns->anns[seqid].name); err_printf("%d\t%d\t", (int)(p->pos - bns->anns[seqid].offset + 1), p->mapQ); // print CIGAR if (p->cigar) { for (j = 0; j != p->n_cigar; ++j) err_printf("%d%c", __cigar_len(p->cigar[j]), "MIDS"[__cigar_op(p->cigar[j])]); } else if (p->type == BWA_TYPE_NO_MATCH) err_printf("*"); else err_printf("%dM", p->len); // print mate coordinate if (mate && mate->type != BWA_TYPE_NO_MATCH) { int m_seqid; long long isize; am = mate->seQ < p->seQ? mate->seQ : p->seQ; // smaller single-end mapping quality // redundant calculation here, but should not matter too much bns_cnt_ambi(bns, mate->pos, mate->len, &m_seqid); err_printf("\t%s\t", (seqid == m_seqid)? "=" : bns->anns[m_seqid].name); isize = (seqid == m_seqid)? pos_5(mate) - pos_5(p) : 0; if (p->type == BWA_TYPE_NO_MATCH) isize = 0; err_printf("%d\t%lld\t", (int)(mate->pos - bns->anns[m_seqid].offset + 1), isize); } else if (mate) err_printf("\t=\t%d\t0\t", (int)(p->pos - bns->anns[seqid].offset + 1)); else err_printf("\t*\t0\t0\t"); // print sequence and quality bwa_print_seq(stdout, p); err_putchar('\t'); if (p->qual) { if (p->strand) seq_reverse(p->len, p->qual, 0); // reverse quality err_printf("%s", p->qual); } else err_printf("*"); if (bwa_rg_id[0]) err_printf("\tRG:Z:%s", bwa_rg_id); if (p->bc[0]) err_printf("\tBC:Z:%s", p->bc); if (p->clip_len < p->full_len) err_printf("\tXC:i:%d", p->clip_len); if (p->type != BWA_TYPE_NO_MATCH) { int i; // calculate XT tag XT = "NURM"[p->type]; if (nn > 10) XT = 'N'; // print tags err_printf("\tXT:A:%c\t%s:i:%d", XT, (mode & BWA_MODE_COMPREAD)? "NM" : "CM", p->nm); if (nn) err_printf("\tXN:i:%d", nn); if (mate) err_printf("\tSM:i:%d\tAM:i:%d", p->seQ, am); if (p->type != BWA_TYPE_MATESW) { // X0 and X1 are not available for this type of alignment err_printf("\tX0:i:%d", p->c1); if (p->c1 <= max_top2) err_printf("\tX1:i:%d", p->c2); } err_printf("\tXM:i:%d\tXO:i:%d\tXG:i:%d", p->n_mm, p->n_gapo, p->n_gapo+p->n_gape); if (p->md) err_printf("\tMD:Z:%s", p->md); // print multiple hits if (p->n_multi) { err_printf("\tXA:Z:"); for (i = 0; i < p->n_multi; ++i) { bwt_multi1_t *q = p->multi + i; int k; j = pos_end_multi(q, p->len) - q->pos; nn = bns_cnt_ambi(bns, q->pos, j, &seqid); err_printf("%s,%c%d,", bns->anns[seqid].name, q->strand? '-' : '+', (int)(q->pos - bns->anns[seqid].offset + 1)); if (q->cigar) { for (k = 0; k < q->n_cigar; ++k) err_printf("%d%c", __cigar_len(q->cigar[k]), "MIDS"[__cigar_op(q->cigar[k])]); } else err_printf("%dM", p->len); err_printf(",%d;", q->gap + q->mm); } } } err_putchar('\n'); } else { // this read has no match //ubyte_t *s = p->strand? p->rseq : p->seq; int flag = p->extra_flag | SAM_FSU; if (mate && mate->type == BWA_TYPE_NO_MATCH) flag |= SAM_FMU; err_printf("%s\t%d\t*\t0\t0\t*\t*\t0\t0\t", p->name, flag); //Why did this work differently to the version above?? //for (j = 0; j != p->len; ++j) putchar("ACGTN"[(int)s[j]]); bwa_print_seq(stdout, p); err_putchar('\t'); if (p->qual) { if (p->strand) seq_reverse(p->len, p->qual, 0); // reverse quality err_printf("%s", p->qual); } else err_printf("*"); if (bwa_rg_id[0]) err_printf("\tRG:Z:%s", bwa_rg_id); if (p->bc[0]) err_printf("\tBC:Z:%s", p->bc); if (p->clip_len < p->full_len) err_printf("\tXC:i:%d", p->clip_len); err_putchar('\n'); } } void bwase_initialize() { int i; for (i = 1; i != 256; ++i) g_log_n[i] = (int)(4.343 * log(i) + 0.5); } void bwa_sai2sam_se_core(const char *prefix, const char *fn_sa, const char *fn_fa, int n_occ, const char *rg_line) { extern bwa_seqio_t *bwa_open_reads(int mode, const char *fn_fa); int i, n_seqs, tot_seqs = 0, m_aln; bwt_aln1_t *aln = 0; bwa_seq_t *seqs; bwa_seqio_t *ks; clock_t t; bntseq_t *bns; FILE *fp_sa; gap_opt_t opt; char magic[4]; // initialization bwase_initialize(); bns = bns_restore(prefix); srand48(bns->seed); fp_sa = xopen(fn_sa, "r"); m_aln = 0; err_fread_noeof(magic, 1, 4, fp_sa); if (strncmp(magic, SAI_MAGIC, 4) != 0) { fprintf(stderr, "[E::%s] Unmatched SAI magic. Please re-run `aln' with the same version of bwa.\n", __func__); exit(1); } err_fread_noeof(&opt, sizeof(gap_opt_t), 1, fp_sa); bwa_print_sam_hdr(bns, rg_line); // set ks ks = bwa_open_reads(opt.mode, fn_fa); // core loop while ((seqs = bwa_read_seq(ks, 0x40000, &n_seqs, opt.mode, opt.trim_qual)) != 0) { tot_seqs += n_seqs; t = clock(); // read alignment for (i = 0; i < n_seqs; ++i) { bwa_seq_t *p = seqs + i; int n_aln; err_fread_noeof(&n_aln, 4, 1, fp_sa); if (n_aln > m_aln) { m_aln = n_aln; aln = (bwt_aln1_t*)realloc(aln, sizeof(bwt_aln1_t) * m_aln); } err_fread_noeof(aln, sizeof(bwt_aln1_t), n_aln, fp_sa); bwa_aln2seq_core(n_aln, aln, p, 1, n_occ); } fprintf(stderr, "[bwa_aln_core] convert to sequence coordinate... "); bwa_cal_pac_pos(bns, prefix, n_seqs, seqs, opt.max_diff, opt.fnr); // forward bwt will be destroyed here fprintf(stderr, "%.2f sec\n", (float)(clock() - t) / CLOCKS_PER_SEC); t = clock(); fprintf(stderr, "[bwa_aln_core] refine gapped alignments... "); bwa_refine_gapped(bns, n_seqs, seqs, 0); fprintf(stderr, "%.2f sec\n", (float)(clock() - t) / CLOCKS_PER_SEC); t = clock(); fprintf(stderr, "[bwa_aln_core] print alignments... "); for (i = 0; i < n_seqs; ++i) bwa_print_sam1(bns, seqs + i, 0, opt.mode, opt.max_top2); fprintf(stderr, "%.2f sec\n", (float)(clock() - t) / CLOCKS_PER_SEC); bwa_free_read_seq(n_seqs, seqs); fprintf(stderr, "[bwa_aln_core] %d sequences have been processed.\n", tot_seqs); } // destroy bwa_seq_close(ks); bns_destroy(bns); err_fclose(fp_sa); free(aln); } int bwa_sai2sam_se(int argc, char *argv[]) { int c, n_occ = 3; char *prefix, *rg_line = 0; while ((c = getopt(argc, argv, "hn:f:r:")) >= 0) { switch (c) { case 'h': break; case 'r': if ((rg_line = bwa_set_rg(optarg)) == 0) return 1; break; case 'n': n_occ = atoi(optarg); break; case 'f': xreopen(optarg, "w", stdout); break; default: return 1; } } if (optind + 3 > argc) { fprintf(stderr, "Usage: bwa samse [-n max_occ] [-f out.sam] [-r RG_line] \n"); return 1; } if ((prefix = bwa_idx_infer_prefix(argv[optind])) == 0) { fprintf(stderr, "[%s] fail to locate the index\n", __func__); return 1; } bwa_sai2sam_se_core(prefix, argv[optind+1], argv[optind+2], n_occ, rg_line); free(prefix); return 0; }