#include <unistd.h>
#include <assert.h>
#include <string.h>
#include <ctype.h>
#include <math.h>
#include "faidx.h"
#include "sam.h"
#include "kstring.h"
#include "kaln.h"
#include "kprobaln.h"
#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;
int32_t old_nm_i = -1, nm = 0;
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 || 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
if (flag&USE_EQUAL) seq[z/2] &= (z&1)? 0xf0 : 0x0f;
++u;
} else {
kputw(u, str); kputc(ref[x+j], str);
u = 0; ++nm;
}
}
if (j < l) break;
x += l; y += l;
} else if (op == BAM_CDEL) {
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; 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;
}
}
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, redo_baq = flag&4;
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 && redo_baq)
{
bam_aux_del(b, bq-1);
bq = 0;
}
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, 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, mode_w[8], mode_r[8];
bam1_t *b;
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 '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, "\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 = 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 = samread(fp, b)) >= 0) {
if (b->core.tid >= 0) {
if (tid != b->core.tid) {
free(ref);
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;
}
if (ref) bam_fillmd1_core(b, ref, flt_flag, max_nm);
}
samwrite(fpout, b);
}
bam_destroy1(b);
free(ref);
fai_destroy(fai);
samclose(fp); samclose(fpout);
return 0;
}
