#include <assert.h>
#include <string.h>
#include <ctype.h>
+#include <math.h>
#include "faidx.h"
-#include "bam.h"
+#include "sam.h"
#include "kstring.h"
+#include "kaln.h"
+#include "kprobaln.h"
-void bam_fillmd1(bam1_t *b, char *ref, int is_equal)
+#define USE_EQUAL 1
+#define DROP_TAG 2
+#define BIN_QUAL 4
+#define UPDATE_NM 8
+#define UPDATE_MD 16
+#define HASH_QNM 32
+
+char bam_nt16_nt4_table[] = { 4, 0, 1, 4, 2, 4, 4, 4, 3, 4, 4, 4, 4, 4, 4, 4 };
+
+int bam_aux_drop_other(bam1_t *b, uint8_t *s);
+
+void bam_fillmd1_core(bam1_t *b, char *ref, int flag, int max_nm)
{
uint8_t *seq = bam1_seq(b);
uint32_t *cigar = bam1_cigar(b);
bam1_core_t *c = &b->core;
int i, x, y, u = 0;
kstring_t *str;
- uint8_t *old_md;
+ int32_t old_nm_i = -1, nm = 0;
- old_md = bam_aux_get(b, "MD");
- if (c->flag & BAM_FUNMAP) return;
- if (old_md && !is_equal) return; // no need to add MD
str = (kstring_t*)calloc(1, sizeof(kstring_t));
for (i = y = 0, x = c->pos; i < c->n_cigar; ++i) {
int j, l = cigar[i]>>4, op = cigar[i]&0xf;
- if (op == BAM_CMATCH) {
+ if (op == BAM_CMATCH || op == BAM_CEQUAL || op == BAM_CDIFF) {
for (j = 0; j < l; ++j) {
int z = y + j;
int c1 = bam1_seqi(seq, z), c2 = bam_nt16_table[(int)ref[x+j]];
if (ref[x+j] == 0) break; // out of boundary
- if ((c1 == c2 && c1 != 15 && c2 != 15) || c1 == 0) {
- if (is_equal) seq[z/2] &= (z&1)? 0xf0 : 0x0f;
+ if ((c1 == c2 && c1 != 15 && c2 != 15) || c1 == 0) { // a match
+ if (flag&USE_EQUAL) seq[z/2] &= (z&1)? 0xf0 : 0x0f;
++u;
} else {
- ksprintf(str, "%d", u);
- kputc(ref[x+j], str);
- u = 0;
+ kputw(u, str); kputc(ref[x+j], str);
+ u = 0; ++nm;
}
}
if (j < l) break;
x += l; y += l;
} else if (op == BAM_CDEL) {
- ksprintf(str, "%d", u);
- kputc('^', str);
+ kputw(u, str); kputc('^', str);
for (j = 0; j < l; ++j) {
if (ref[x+j] == 0) break;
kputc(ref[x+j], str);
}
u = 0;
if (j < l) break;
- x += l;
+ x += l; nm += l;
} else if (op == BAM_CINS || op == BAM_CSOFT_CLIP) {
y += l;
+ if (op == BAM_CINS) nm += l;
} else if (op == BAM_CREF_SKIP) {
x += l;
}
}
- ksprintf(str, "%d", u);
- if (!old_md) bam_aux_append(b, "MD", 'Z', str->l + 1, (uint8_t*)str->s);
- else {
- int is_diff = 0;
- if (strlen((char*)old_md+1) == str->l) {
- for (i = 0; i < str->l; ++i)
- if (toupper(old_md[i+1]) != toupper(str->s[i]))
- break;
- if (i < str->l) is_diff = 1;
- } else is_diff = 1;
- if (is_diff)
- fprintf(stderr, "[bam_fillmd1] different MD for read '%s': '%s' != '%s'\n", bam1_qname(b), old_md+1, str->s);
+ kputw(u, str);
+ // apply max_nm
+ if (max_nm > 0 && nm >= max_nm) {
+ for (i = y = 0, x = c->pos; i < c->n_cigar; ++i) {
+ int j, l = cigar[i]>>4, op = cigar[i]&0xf;
+ if (op == BAM_CMATCH || op == BAM_CEQUAL || op == BAM_CDIFF) {
+ for (j = 0; j < l; ++j) {
+ int z = y + j;
+ int c1 = bam1_seqi(seq, z), c2 = bam_nt16_table[(int)ref[x+j]];
+ if (ref[x+j] == 0) break; // out of boundary
+ if ((c1 == c2 && c1 != 15 && c2 != 15) || c1 == 0) { // a match
+ seq[z/2] |= (z&1)? 0x0f : 0xf0;
+ bam1_qual(b)[z] = 0;
+ }
+ }
+ if (j < l) break;
+ x += l; y += l;
+ } else if (op == BAM_CDEL || op == BAM_CREF_SKIP) x += l;
+ else if (op == BAM_CINS || op == BAM_CSOFT_CLIP) y += l;
+ }
+ }
+ // update NM
+ if (flag & UPDATE_NM) {
+ uint8_t *old_nm = bam_aux_get(b, "NM");
+ if (c->flag & BAM_FUNMAP) return;
+ if (old_nm) old_nm_i = bam_aux2i(old_nm);
+ if (!old_nm) bam_aux_append(b, "NM", 'i', 4, (uint8_t*)&nm);
+ else if (nm != old_nm_i) {
+ fprintf(stderr, "[bam_fillmd1] different NM for read '%s': %d -> %d\n", bam1_qname(b), old_nm_i, nm);
+ bam_aux_del(b, old_nm);
+ bam_aux_append(b, "NM", 'i', 4, (uint8_t*)&nm);
+ }
+ }
+ // update MD
+ if (flag & UPDATE_MD) {
+ uint8_t *old_md = bam_aux_get(b, "MD");
+ if (c->flag & BAM_FUNMAP) return;
+ if (!old_md) bam_aux_append(b, "MD", 'Z', str->l + 1, (uint8_t*)str->s);
+ else {
+ int is_diff = 0;
+ if (strlen((char*)old_md+1) == str->l) {
+ for (i = 0; i < str->l; ++i)
+ if (toupper(old_md[i+1]) != toupper(str->s[i]))
+ break;
+ if (i < str->l) is_diff = 1;
+ } else is_diff = 1;
+ if (is_diff) {
+ fprintf(stderr, "[bam_fillmd1] different MD for read '%s': '%s' -> '%s'\n", bam1_qname(b), old_md+1, str->s);
+ bam_aux_del(b, old_md);
+ bam_aux_append(b, "MD", 'Z', str->l + 1, (uint8_t*)str->s);
+ }
+ }
+ }
+ // drop all tags but RG
+ if (flag&DROP_TAG) {
+ uint8_t *q = bam_aux_get(b, "RG");
+ bam_aux_drop_other(b, q);
+ }
+ // reduce the resolution of base quality
+ if (flag&BIN_QUAL) {
+ uint8_t *qual = bam1_qual(b);
+ for (i = 0; i < b->core.l_qseq; ++i)
+ if (qual[i] >= 3) qual[i] = qual[i]/10*10 + 7;
}
free(str->s); free(str);
}
+void bam_fillmd1(bam1_t *b, char *ref, int flag)
+{
+ bam_fillmd1_core(b, ref, flag, 0);
+}
+
+int bam_cap_mapQ(bam1_t *b, char *ref, int thres)
+{
+ uint8_t *seq = bam1_seq(b), *qual = bam1_qual(b);
+ uint32_t *cigar = bam1_cigar(b);
+ bam1_core_t *c = &b->core;
+ int i, x, y, mm, q, len, clip_l, clip_q;
+ double t;
+ if (thres < 0) thres = 40; // set the default
+ mm = q = len = clip_l = clip_q = 0;
+ for (i = y = 0, x = c->pos; i < c->n_cigar; ++i) {
+ int j, l = cigar[i]>>4, op = cigar[i]&0xf;
+ if (op == BAM_CMATCH || op == BAM_CEQUAL || op == BAM_CDIFF) {
+ for (j = 0; j < l; ++j) {
+ int z = y + j;
+ int c1 = bam1_seqi(seq, z), c2 = bam_nt16_table[(int)ref[x+j]];
+ if (ref[x+j] == 0) break; // out of boundary
+ if (c2 != 15 && c1 != 15 && qual[z] >= 13) { // not ambiguous
+ ++len;
+ if (c1 && c1 != c2 && qual[z] >= 13) { // mismatch
+ ++mm;
+ q += qual[z] > 33? 33 : qual[z];
+ }
+ }
+ }
+ if (j < l) break;
+ x += l; y += l; len += l;
+ } else if (op == BAM_CDEL) {
+ for (j = 0; j < l; ++j)
+ if (ref[x+j] == 0) break;
+ if (j < l) break;
+ x += l;
+ } else if (op == BAM_CSOFT_CLIP) {
+ for (j = 0; j < l; ++j) clip_q += qual[y+j];
+ clip_l += l;
+ y += l;
+ } else if (op == BAM_CHARD_CLIP) {
+ clip_q += 13 * l;
+ clip_l += l;
+ } else if (op == BAM_CINS) y += l;
+ else if (op == BAM_CREF_SKIP) x += l;
+ }
+ for (i = 0, t = 1; i < mm; ++i)
+ t *= (double)len / (i+1);
+ t = q - 4.343 * log(t) + clip_q / 5.;
+ if (t > thres) return -1;
+ if (t < 0) t = 0;
+ t = sqrt((thres - t) / thres) * thres;
+// fprintf(stderr, "%s %lf %d\n", bam1_qname(b), t, q);
+ return (int)(t + .499);
+}
+
+int bam_prob_realn_core(bam1_t *b, const char *ref, int flag)
+{
+ int k, i, bw, x, y, yb, ye, xb, xe, apply_baq = flag&1, extend_baq = flag>>1&1;
+ uint32_t *cigar = bam1_cigar(b);
+ bam1_core_t *c = &b->core;
+ kpa_par_t conf = kpa_par_def;
+ uint8_t *bq = 0, *zq = 0, *qual = bam1_qual(b);
+ if ((c->flag & BAM_FUNMAP) || b->core.l_qseq == 0) return -1; // do nothing
+ // test if BQ or ZQ is present
+ if ((bq = bam_aux_get(b, "BQ")) != 0) ++bq;
+ if ((zq = bam_aux_get(b, "ZQ")) != 0 && *zq == 'Z') ++zq;
+ if (bq && zq) { // remove the ZQ tag
+ bam_aux_del(b, zq-1);
+ zq = 0;
+ }
+ if (bq || zq) {
+ if ((apply_baq && zq) || (!apply_baq && bq)) return -3; // in both cases, do nothing
+ if (bq && apply_baq) { // then convert BQ to ZQ
+ for (i = 0; i < c->l_qseq; ++i)
+ qual[i] = qual[i] + 64 < bq[i]? 0 : qual[i] - ((int)bq[i] - 64);
+ *(bq - 3) = 'Z';
+ } else if (zq && !apply_baq) { // then convert ZQ to BQ
+ for (i = 0; i < c->l_qseq; ++i)
+ qual[i] += (int)zq[i] - 64;
+ *(zq - 3) = 'B';
+ }
+ return 0;
+ }
+ // find the start and end of the alignment
+ x = c->pos, y = 0, yb = ye = xb = xe = -1;
+ for (k = 0; k < c->n_cigar; ++k) {
+ int op, l;
+ op = cigar[k]&0xf; l = cigar[k]>>4;
+ if (op == BAM_CMATCH || op == BAM_CEQUAL || op == BAM_CDIFF) {
+ if (yb < 0) yb = y;
+ if (xb < 0) xb = x;
+ ye = y + l; xe = x + l;
+ x += l; y += l;
+ } else if (op == BAM_CSOFT_CLIP || op == BAM_CINS) y += l;
+ else if (op == BAM_CDEL) x += l;
+ else if (op == BAM_CREF_SKIP) return -1; // do nothing if there is a reference skip
+ }
+ // set bandwidth and the start and the end
+ bw = 7;
+ if (abs((xe - xb) - (ye - yb)) > bw)
+ bw = abs((xe - xb) - (ye - yb)) + 3;
+ conf.bw = bw;
+ xb -= yb + bw/2; if (xb < 0) xb = 0;
+ xe += c->l_qseq - ye + bw/2;
+ if (xe - xb - c->l_qseq > bw)
+ xb += (xe - xb - c->l_qseq - bw) / 2, xe -= (xe - xb - c->l_qseq - bw) / 2;
+ { // glocal
+ uint8_t *s, *r, *q, *seq = bam1_seq(b), *bq;
+ int *state;
+ bq = calloc(c->l_qseq + 1, 1);
+ memcpy(bq, qual, c->l_qseq);
+ s = calloc(c->l_qseq, 1);
+ for (i = 0; i < c->l_qseq; ++i) s[i] = bam_nt16_nt4_table[bam1_seqi(seq, i)];
+ r = calloc(xe - xb, 1);
+ for (i = xb; i < xe; ++i) {
+ if (ref[i] == 0) { xe = i; break; }
+ r[i-xb] = bam_nt16_nt4_table[bam_nt16_table[(int)ref[i]]];
+ }
+ state = calloc(c->l_qseq, sizeof(int));
+ q = calloc(c->l_qseq, 1);
+ kpa_glocal(r, xe-xb, s, c->l_qseq, qual, &conf, state, q);
+ if (!extend_baq) { // in this block, bq[] is capped by base quality qual[]
+ for (k = 0, x = c->pos, y = 0; k < c->n_cigar; ++k) {
+ int op = cigar[k]&0xf, l = cigar[k]>>4;
+ if (op == BAM_CMATCH || op == BAM_CEQUAL || op == BAM_CDIFF) {
+ for (i = y; i < y + l; ++i) {
+ if ((state[i]&3) != 0 || state[i]>>2 != x - xb + (i - y)) bq[i] = 0;
+ else bq[i] = bq[i] < q[i]? bq[i] : q[i];
+ }
+ x += l; y += l;
+ } else if (op == BAM_CSOFT_CLIP || op == BAM_CINS) y += l;
+ else if (op == BAM_CDEL) x += l;
+ }
+ for (i = 0; i < c->l_qseq; ++i) bq[i] = qual[i] - bq[i] + 64; // finalize BQ
+ } else { // in this block, bq[] is BAQ that can be larger than qual[] (different from the above!)
+ uint8_t *left, *rght;
+ left = calloc(c->l_qseq, 1); rght = calloc(c->l_qseq, 1);
+ for (k = 0, x = c->pos, y = 0; k < c->n_cigar; ++k) {
+ int op = cigar[k]&0xf, l = cigar[k]>>4;
+ if (op == BAM_CMATCH || op == BAM_CEQUAL || op == BAM_CDIFF) {
+ for (i = y; i < y + l; ++i)
+ bq[i] = ((state[i]&3) != 0 || state[i]>>2 != x - xb + (i - y))? 0 : q[i];
+ for (left[y] = bq[y], i = y + 1; i < y + l; ++i)
+ left[i] = bq[i] > left[i-1]? bq[i] : left[i-1];
+ for (rght[y+l-1] = bq[y+l-1], i = y + l - 2; i >= y; --i)
+ rght[i] = bq[i] > rght[i+1]? bq[i] : rght[i+1];
+ for (i = y; i < y + l; ++i)
+ bq[i] = left[i] < rght[i]? left[i] : rght[i];
+ x += l; y += l;
+ } else if (op == BAM_CSOFT_CLIP || op == BAM_CINS) y += l;
+ else if (op == BAM_CDEL) x += l;
+ }
+ for (i = 0; i < c->l_qseq; ++i) bq[i] = 64 + (qual[i] <= bq[i]? 0 : qual[i] - bq[i]); // finalize BQ
+ free(left); free(rght);
+ }
+ if (apply_baq) {
+ for (i = 0; i < c->l_qseq; ++i) qual[i] -= bq[i] - 64; // modify qual
+ bam_aux_append(b, "ZQ", 'Z', c->l_qseq + 1, bq);
+ } else bam_aux_append(b, "BQ", 'Z', c->l_qseq + 1, bq);
+ free(bq); free(s); free(r); free(q); free(state);
+ }
+ return 0;
+}
+
+int bam_prob_realn(bam1_t *b, const char *ref)
+{
+ return bam_prob_realn_core(b, ref, 1);
+}
+
int bam_fillmd(int argc, char *argv[])
{
- int c, is_equal = 0, tid = -2, ret, len;
- bamFile fp, fpout = 0;
- bam_header_t *header;
+ int c, flt_flag, tid = -2, ret, len, is_bam_out, is_sam_in, is_uncompressed, max_nm, is_realn, capQ, baq_flag;
+ samfile_t *fp, *fpout = 0;
faidx_t *fai;
- char *ref = 0;
+ char *ref = 0, mode_w[8], mode_r[8];
bam1_t *b;
- while ((c = getopt(argc, argv, "e")) >= 0) {
+ flt_flag = UPDATE_NM | UPDATE_MD;
+ is_bam_out = is_sam_in = is_uncompressed = is_realn = max_nm = capQ = baq_flag = 0;
+ mode_w[0] = mode_r[0] = 0;
+ strcpy(mode_r, "r"); strcpy(mode_w, "w");
+ while ((c = getopt(argc, argv, "EqreuNhbSC:n:Ad")) >= 0) {
switch (c) {
- case 'e': is_equal = 1; break;
+ case 'r': is_realn = 1; break;
+ case 'e': flt_flag |= USE_EQUAL; break;
+ case 'd': flt_flag |= DROP_TAG; break;
+ case 'q': flt_flag |= BIN_QUAL; break;
+ case 'h': flt_flag |= HASH_QNM; break;
+ case 'N': flt_flag &= ~(UPDATE_MD|UPDATE_NM); break;
+ case 'b': is_bam_out = 1; break;
+ case 'u': is_uncompressed = is_bam_out = 1; break;
+ case 'S': is_sam_in = 1; break;
+ case 'n': max_nm = atoi(optarg); break;
+ case 'C': capQ = atoi(optarg); break;
+ case 'A': baq_flag |= 1; break;
+ case 'E': baq_flag |= 2; break;
default: fprintf(stderr, "[bam_fillmd] unrecognized option '-%c'\n", c); return 1;
}
}
+ if (!is_sam_in) strcat(mode_r, "b");
+ if (is_bam_out) strcat(mode_w, "b");
+ else strcat(mode_w, "h");
+ if (is_uncompressed) strcat(mode_w, "u");
if (optind + 1 >= argc) {
- fprintf(stderr, "Usage: bam fillmd [-e] <aln.bam> <ref.fasta>\n");
+ fprintf(stderr, "\n");
+ fprintf(stderr, "Usage: samtools fillmd [-eubrS] <aln.bam> <ref.fasta>\n\n");
+ fprintf(stderr, "Options: -e change identical bases to '='\n");
+ fprintf(stderr, " -u uncompressed BAM output (for piping)\n");
+ fprintf(stderr, " -b compressed BAM output\n");
+ fprintf(stderr, " -S the input is SAM with header\n");
+ fprintf(stderr, " -A modify the quality string\n");
+ fprintf(stderr, " -r compute the BQ tag (without -A) or cap baseQ by BAQ (with -A)\n");
+ fprintf(stderr, " -E extended BAQ for better sensitivity but lower specificity\n\n");
return 1;
}
- fp = strcmp(argv[optind], "-")? bam_open(argv[optind], "r") : bam_dopen(fileno(stdin), "r");
- assert(fp);
- header = bam_header_read(fp);
- fpout = bam_dopen(fileno(stdout), "w");
- bam_header_write(fpout, header);
+ fp = samopen(argv[optind], mode_r, 0);
+ if (fp == 0) return 1;
+ if (is_sam_in && (fp->header == 0 || fp->header->n_targets == 0)) {
+ fprintf(stderr, "[bam_fillmd] input SAM does not have header. Abort!\n");
+ return 1;
+ }
+ fpout = samopen("-", mode_w, fp->header);
fai = fai_load(argv[optind+1]);
b = bam_init1();
- while ((ret = bam_read1(fp, b)) >= 0) {
+ while ((ret = samread(fp, b)) >= 0) {
if (b->core.tid >= 0) {
if (tid != b->core.tid) {
free(ref);
- ref = fai_fetch(fai, header->target_name[b->core.tid], &len);
+ ref = fai_fetch(fai, fp->header->target_name[b->core.tid], &len);
tid = b->core.tid;
+ if (ref == 0)
+ fprintf(stderr, "[bam_fillmd] fail to find sequence '%s' in the reference.\n",
+ fp->header->target_name[tid]);
+ }
+ if (is_realn) bam_prob_realn_core(b, ref, baq_flag);
+ if (capQ > 10) {
+ int q = bam_cap_mapQ(b, ref, capQ);
+ if (b->core.qual > q) b->core.qual = q;
}
- bam_fillmd1(b, ref, is_equal);
+ if (ref) bam_fillmd1_core(b, ref, flt_flag, max_nm);
}
- bam_write1(fpout, b);
+ samwrite(fpout, b);
}
bam_destroy1(b);
free(ref);
fai_destroy(fai);
- bam_header_destroy(header);
- bam_close(fp); bam_close(fpout);
+ samclose(fp); samclose(fpout);
return 0;
}
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