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#include <unistd.h>
#include <math.h>
#include <stdlib.h>
#include <time.h>
#include <stdio.h>
#include <string.h>
#include "bwtaln.h"
#include "kvec.h"
#include "bntseq.h"
#include "utils.h"
#include "bwase.h"
#include "bwa.h"
#include "ksw.h"
#ifdef USE_MALLOC_WRAPPERS
# include "malloc_wrap.h"
#endif
typedef struct {
int n;
bwtint_t *a;
} poslist_t;
typedef struct {
double avg, std, ap_prior;
bwtint_t low, high, high_bayesian;
} isize_info_t;
#define b128_eq(a, b) ((a).x == (b).x && (a).y == (b).y)
#define b128_hash(a) ((uint32_t)(a).x)
#include "khash.h"
KHASH_INIT(b128, pair64_t, poslist_t, 1, b128_hash, b128_eq)
typedef struct {
pair64_v arr;
pair64_v pos[2];
kvec_t(bwt_aln1_t) aln[2];
} pe_data_t;
#define MIN_HASH_WIDTH 1000
extern int g_log_n[256]; // in bwase.c
static kh_b128_t *g_hash;
void bwa_aln2seq_core(int n_aln, const bwt_aln1_t *aln, bwa_seq_t *s, int set_main, int n_multi);
void bwa_aln2seq(int n_aln, const bwt_aln1_t *aln, bwa_seq_t *s);
int bwa_approx_mapQ(const bwa_seq_t *p, int mm);
void bwa_print_sam1(const bntseq_t *bns, bwa_seq_t *p, const bwa_seq_t *mate, int mode, int max_top2);
bntseq_t *bwa_open_nt(const char *prefix);
void bwa_print_sam_SQ(const bntseq_t *bns);
pe_opt_t *bwa_init_pe_opt()
{
pe_opt_t *po;
po = (pe_opt_t*)calloc(1, sizeof(pe_opt_t));
po->max_isize = 500;
po->force_isize = 0;
po->max_occ = 100000;
po->n_multi = 3;
po->N_multi = 10;
po->type = BWA_PET_STD;
po->is_sw = 1;
po->ap_prior = 1e-5;
return po;
}
/*
static double ierfc(double x) // inverse erfc(); iphi(x) = M_SQRT2 *ierfc(2 * x);
{
const double a = 0.140012;
double b, c;
b = log(x * (2 - x));
c = 2./M_PI/a + b / 2.;
return sqrt(sqrt(c * c - b / a) - c);
}
*/
// for normal distribution, this is about 3std
#define OUTLIER_BOUND 2.0
static int infer_isize(int n_seqs, bwa_seq_t *seqs[2], isize_info_t *ii, double ap_prior, int64_t L)
{
uint64_t x, *isizes, n_ap = 0;
int n, i, tot, p25, p75, p50, max_len = 1, tmp;
double skewness = 0.0, kurtosis = 0.0, y;
ii->avg = ii->std = -1.0;
ii->low = ii->high = ii->high_bayesian = 0;
isizes = (uint64_t*)calloc(n_seqs, 8);
for (i = 0, tot = 0; i != n_seqs; ++i) {
bwa_seq_t *p[2];
p[0] = seqs[0] + i; p[1] = seqs[1] + i;
if (p[0]->mapQ >= 20 && p[1]->mapQ >= 20) {
x = (p[0]->pos < p[1]->pos)? p[1]->pos + p[1]->len - p[0]->pos : p[0]->pos + p[0]->len - p[1]->pos;
if (x < 100000) isizes[tot++] = x;
}
if (p[0]->len > max_len) max_len = p[0]->len;
if (p[1]->len > max_len) max_len = p[1]->len;
}
if (tot < 20) {
fprintf(stderr, "[infer_isize] fail to infer insert size: too few good pairs\n");
free(isizes);
return -1;
}
ks_introsort_64(tot, isizes);
p25 = isizes[(int)(tot*0.25 + 0.5)];
p50 = isizes[(int)(tot*0.50 + 0.5)];
p75 = isizes[(int)(tot*0.75 + 0.5)];
tmp = (int)(p25 - OUTLIER_BOUND * (p75 - p25) + .499);
ii->low = tmp > max_len? tmp : max_len; // ii->low is unsigned
ii->high = (int)(p75 + OUTLIER_BOUND * (p75 - p25) + .499);
if (ii->low > ii->high) {
fprintf(stderr, "[infer_isize] fail to infer insert size: upper bound is smaller than read length\n");
free(isizes);
return -1;
}
for (i = 0, x = n = 0; i < tot; ++i)
if (isizes[i] >= ii->low && isizes[i] <= ii->high)
++n, x += isizes[i];
ii->avg = (double)x / n;
for (i = 0; i < tot; ++i) {
if (isizes[i] >= ii->low && isizes[i] <= ii->high) {
double tmp = (isizes[i] - ii->avg) * (isizes[i] - ii->avg);
ii->std += tmp;
skewness += tmp * (isizes[i] - ii->avg);
kurtosis += tmp * tmp;
}
}
kurtosis = kurtosis/n / (ii->std / n * ii->std / n) - 3;
ii->std = sqrt(ii->std / n); // it would be better as n-1, but n is usually very large
skewness = skewness / n / (ii->std * ii->std * ii->std);
for (y = 1.0; y < 10.0; y += 0.01)
if (.5 * erfc(y / M_SQRT2) < ap_prior / L * (y * ii->std + ii->avg)) break;
ii->high_bayesian = (bwtint_t)(y * ii->std + ii->avg + .499);
for (i = 0; i < tot; ++i)
if (isizes[i] > ii->high_bayesian) ++n_ap;
ii->ap_prior = .01 * (n_ap + .01) / tot;
if (ii->ap_prior < ap_prior) ii->ap_prior = ap_prior;
free(isizes);
fprintf(stderr, "[infer_isize] (25, 50, 75) percentile: (%d, %d, %d)\n", p25, p50, p75);
if (isnan(ii->std) || p75 > 100000) {
ii->low = ii->high = ii->high_bayesian = 0; ii->avg = ii->std = -1.0;
fprintf(stderr, "[infer_isize] fail to infer insert size: weird pairing\n");
return -1;
}
for (y = 1.0; y < 10.0; y += 0.01)
if (.5 * erfc(y / M_SQRT2) < ap_prior / L * (y * ii->std + ii->avg)) break;
ii->high_bayesian = (bwtint_t)(y * ii->std + ii->avg + .499);
fprintf(stderr, "[infer_isize] low and high boundaries: %ld and %ld for estimating avg and std\n", (long)ii->low, (long)ii->high);
fprintf(stderr, "[infer_isize] inferred external isize from %d pairs: %.3lf +/- %.3lf\n", n, ii->avg, ii->std);
fprintf(stderr, "[infer_isize] skewness: %.3lf; kurtosis: %.3lf; ap_prior: %.2e\n", skewness, kurtosis, ii->ap_prior);
fprintf(stderr, "[infer_isize] inferred maximum insert size: %ld (%.2lf sigma)\n", (long)ii->high_bayesian, y);
return 0;
}
static int pairing(bwa_seq_t *p[2], pe_data_t *d, const pe_opt_t *opt, int s_mm, const isize_info_t *ii)
{
int i, j, o_n, subo_n, cnt_chg = 0, low_bound = ii->low, max_len;
uint64_t o_score, subo_score;
pair64_t last_pos[2][2], o_pos[2];
max_len = p[0]->full_len;
if (max_len < p[1]->full_len) max_len = p[1]->full_len;
if (low_bound < max_len) low_bound = max_len;
// here v>=u. When ii is set, we check insert size with ii; otherwise with opt->max_isize
#define __pairing_aux(u,v) do { \
bwtint_t l = (v).x + p[(v).y&1]->len - ((u).x); \
if ((u).x != (uint64_t)-1 && (v).x > (u).x && l >= max_len \
&& ((ii->high && l <= ii->high_bayesian) || (ii->high == 0 && l <= opt->max_isize))) \
{ \
uint64_t s = d->aln[(v).y&1].a[(v).y>>2].score + d->aln[(u).y&1].a[(u).y>>2].score; \
s *= 10; \
if (ii->high) s += (int)(-4.343 * log(.5 * erfc(M_SQRT1_2 * fabs(l - ii->avg) / ii->std)) + .499); \
s = s<<32 | (uint32_t)hash_64((u).x<<32 | (v).x); \
if (s>>32 == o_score>>32) ++o_n; \
else if (s>>32 < o_score>>32) { subo_n += o_n; o_n = 1; } \
else ++subo_n; \
if (s < o_score) subo_score = o_score, o_score = s, o_pos[(u).y&1] = (u), o_pos[(v).y&1] = (v); \
else if (s < subo_score) subo_score = s; \
} \
} while (0)
#define __pairing_aux2(q, w) do { \
const bwt_aln1_t *r = d->aln[(w).y&1].a + ((w).y>>2); \
(q)->extra_flag |= SAM_FPP; \
if ((q)->pos != (w).x || (q)->strand != ((w).y>>1&1)) { \
(q)->n_mm = r->n_mm; (q)->n_gapo = r->n_gapo; (q)->n_gape = r->n_gape; (q)->strand = (w).y>>1&1; \
(q)->score = r->score; \
(q)->pos = (w).x; \
if ((q)->mapQ > 0) ++cnt_chg; \
} \
} while (0)
o_score = subo_score = (uint64_t)-1;
o_n = subo_n = 0;
ks_introsort_128(d->arr.n, d->arr.a);
for (j = 0; j < 2; ++j) last_pos[j][0].x = last_pos[j][0].y = last_pos[j][1].x = last_pos[j][1].y = (uint64_t)-1;
if (opt->type == BWA_PET_STD) {
for (i = 0; i < d->arr.n; ++i) {
pair64_t x = d->arr.a[i];
int strand = x.y>>1&1;
if (strand == 1) { // reverse strand, then check
int y = 1 - (x.y&1);
__pairing_aux(last_pos[y][1], x);
__pairing_aux(last_pos[y][0], x);
} else { // forward strand, then push
last_pos[x.y&1][0] = last_pos[x.y&1][1];
last_pos[x.y&1][1] = x;
}
}
} else {
fprintf(stderr, "[paring] not implemented yet!\n");
exit(1);
}
// set pairing
//fprintf(stderr, "[%ld, %d, %d, %d]\n", d->arr.n, (int)(o_score>>32), (int)(subo_score>>32), o_n);
if (o_score != (uint64_t)-1) {
int mapQ_p = 0; // this is the maximum mapping quality when one end is moved
//fprintf(stderr, "%d, %d\n", o_n, subo_n);
if (o_n == 1) {
if (subo_score == (uint64_t)-1) mapQ_p = 29; // no sub-optimal pair
else if ((subo_score>>32) - (o_score>>32) > s_mm * 10) mapQ_p = 23; // poor sub-optimal pair
else {
int n = subo_n > 255? 255 : subo_n;
mapQ_p = ((subo_score>>32) - (o_score>>32)) / 2 - g_log_n[n];
if (mapQ_p < 0) mapQ_p = 0;
}
}
if ((p[0]->pos == o_pos[0].x && p[0]->strand == (o_pos[0].y>>1&1)) && (p[1]->pos == o_pos[1].x && p[1]->strand == (o_pos[1].y>>1&1))) { // both ends not moved
if (p[0]->mapQ > 0 && p[1]->mapQ > 0) {
int mapQ = p[0]->mapQ + p[1]->mapQ;
if (mapQ > 60) mapQ = 60;
p[0]->mapQ = p[1]->mapQ = mapQ;
} else {
if (p[0]->mapQ == 0) p[0]->mapQ = (mapQ_p + 7 < p[1]->mapQ)? mapQ_p + 7 : p[1]->mapQ;
if (p[1]->mapQ == 0) p[1]->mapQ = (mapQ_p + 7 < p[0]->mapQ)? mapQ_p + 7 : p[0]->mapQ;
}
} else if (p[0]->pos == o_pos[0].x && p[0]->strand == (o_pos[0].y>>1&1)) { // [1] moved
p[1]->seQ = 0; p[1]->mapQ = p[0]->mapQ;
if (p[1]->mapQ > mapQ_p) p[1]->mapQ = mapQ_p;
} else if (p[1]->pos == o_pos[1].x && p[1]->strand == (o_pos[1].y>>1&1)) { // [0] moved
p[0]->seQ = 0; p[0]->mapQ = p[1]->mapQ;
if (p[0]->mapQ > mapQ_p) p[0]->mapQ = mapQ_p;
} else { // both ends moved
p[0]->seQ = p[1]->seQ = 0;
mapQ_p -= 20;
if (mapQ_p < 0) mapQ_p = 0;
p[0]->mapQ = p[1]->mapQ = mapQ_p;
}
__pairing_aux2(p[0], o_pos[0]);
__pairing_aux2(p[1], o_pos[1]);
}
return cnt_chg;
}
typedef struct {
kvec_t(bwt_aln1_t) aln;
} aln_buf_t;
int bwa_cal_pac_pos_pe(const bntseq_t *bns, const char *prefix, bwt_t *const _bwt, int n_seqs, bwa_seq_t *seqs[2], FILE *fp_sa[2], isize_info_t *ii,
const pe_opt_t *opt, const gap_opt_t *gopt, const isize_info_t *last_ii)
{
int i, j, cnt_chg = 0;
char str[1024];
bwt_t *bwt;
pe_data_t *d;
aln_buf_t *buf[2];
d = (pe_data_t*)calloc(1, sizeof(pe_data_t));
buf[0] = (aln_buf_t*)calloc(n_seqs, sizeof(aln_buf_t));
buf[1] = (aln_buf_t*)calloc(n_seqs, sizeof(aln_buf_t));
if (_bwt == 0) { // 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);
} else bwt = _bwt;
// SE
for (i = 0; i != n_seqs; ++i) {
bwa_seq_t *p[2];
for (j = 0; j < 2; ++j) {
int n_aln;
p[j] = seqs[j] + i;
p[j]->n_multi = 0;
p[j]->extra_flag |= SAM_FPD | (j == 0? SAM_FR1 : SAM_FR2);
err_fread_noeof(&n_aln, 4, 1, fp_sa[j]);
if (n_aln > kv_max(d->aln[j]))
kv_resize(bwt_aln1_t, d->aln[j], n_aln);
d->aln[j].n = n_aln;
err_fread_noeof(d->aln[j].a, sizeof(bwt_aln1_t), n_aln, fp_sa[j]);
kv_copy(bwt_aln1_t, buf[j][i].aln, d->aln[j]); // backup d->aln[j]
// generate SE alignment and mapping quality
bwa_aln2seq(n_aln, d->aln[j].a, p[j]);
if (p[j]->type == BWA_TYPE_UNIQUE || p[j]->type == BWA_TYPE_REPEAT) {
int strand;
int max_diff = gopt->fnr > 0.0? bwa_cal_maxdiff(p[j]->len, BWA_AVG_ERR, gopt->fnr) : gopt->max_diff;
p[j]->seQ = p[j]->mapQ = bwa_approx_mapQ(p[j], max_diff);
p[j]->pos = bwa_sa2pos(bns, bwt, p[j]->sa, p[j]->len + p[j]->ref_shift, &strand);
p[j]->strand = strand;
if (p[j]->pos == (bwtint_t)-1) p[j]->type = BWA_TYPE_NO_MATCH;
}
}
}
// infer isize
infer_isize(n_seqs, seqs, ii, opt->ap_prior, bwt->seq_len/2);
if (ii->avg < 0.0 && last_ii->avg > 0.0) *ii = *last_ii;
if (opt->force_isize) {
fprintf(stderr, "[%s] discard insert size estimate as user's request.\n", __func__);
ii->low = ii->high = 0; ii->avg = ii->std = -1.0;
}
// PE
for (i = 0; i != n_seqs; ++i) {
bwa_seq_t *p[2];
for (j = 0; j < 2; ++j) {
p[j] = seqs[j] + i;
kv_copy(bwt_aln1_t, d->aln[j], buf[j][i].aln);
}
if ((p[0]->type == BWA_TYPE_UNIQUE || p[0]->type == BWA_TYPE_REPEAT)
&& (p[1]->type == BWA_TYPE_UNIQUE || p[1]->type == BWA_TYPE_REPEAT))
{ // only when both ends mapped
pair64_t x;
int j, k;
long long n_occ[2];
for (j = 0; j < 2; ++j) {
n_occ[j] = 0;
for (k = 0; k < d->aln[j].n; ++k)
n_occ[j] += d->aln[j].a[k].l - d->aln[j].a[k].k + 1;
}
if (n_occ[0] > opt->max_occ || n_occ[1] > opt->max_occ) continue;
d->arr.n = 0;
for (j = 0; j < 2; ++j) {
for (k = 0; k < d->aln[j].n; ++k) {
bwt_aln1_t *r = d->aln[j].a + k;
bwtint_t l;
if (0 && r->l - r->k + 1 >= MIN_HASH_WIDTH) { // then check hash table
pair64_t key;
int ret;
key.x = r->k; key.y = r->l;
khint_t iter = kh_put(b128, g_hash, key, &ret);
if (ret) { // not in the hash table; ret must equal 1 as we never remove elements
poslist_t *z = &kh_val(g_hash, iter);
z->n = r->l - r->k + 1;
z->a = (bwtint_t*)malloc(sizeof(bwtint_t) * z->n);
for (l = r->k; l <= r->l; ++l) {
int strand;
z->a[l - r->k] = bwa_sa2pos(bns, bwt, l, p[j]->len + p[j]->ref_shift, &strand)<<1;
z->a[l - r->k] |= strand;
}
}
for (l = 0; l < kh_val(g_hash, iter).n; ++l) {
x.x = kh_val(g_hash, iter).a[l]>>1;
x.y = k<<2 | (kh_val(g_hash, iter).a[l]&1)<<1 | j;
kv_push(pair64_t, d->arr, x);
}
} else { // then calculate on the fly
for (l = r->k; l <= r->l; ++l) {
int strand;
x.x = bwa_sa2pos(bns, bwt, l, p[j]->len + p[j]->ref_shift, &strand);
x.y = k<<2 | strand<<1 | j;
kv_push(pair64_t, d->arr, x);
}
}
}
}
cnt_chg += pairing(p, d, opt, gopt->s_mm, ii);
}
if (opt->N_multi || opt->n_multi) {
for (j = 0; j < 2; ++j) {
if (p[j]->type != BWA_TYPE_NO_MATCH) {
int k, n_multi;
if (!(p[j]->extra_flag&SAM_FPP) && p[1-j]->type != BWA_TYPE_NO_MATCH) {
bwa_aln2seq_core(d->aln[j].n, d->aln[j].a, p[j], 0, p[j]->c1+p[j]->c2-1 > opt->N_multi? opt->n_multi : opt->N_multi);
} else bwa_aln2seq_core(d->aln[j].n, d->aln[j].a, p[j], 0, opt->n_multi);
for (k = 0, n_multi = 0; k < p[j]->n_multi; ++k) {
int strand;
bwt_multi1_t *q = p[j]->multi + k;
q->pos = bwa_sa2pos(bns, bwt, q->pos, p[j]->len + q->ref_shift, &strand);
q->strand = strand;
if (q->pos != p[j]->pos && q->pos != (bwtint_t)-1)
p[j]->multi[n_multi++] = *q;
}
p[j]->n_multi = n_multi;
}
}
}
}
// free
for (i = 0; i < n_seqs; ++i) {
kv_destroy(buf[0][i].aln);
kv_destroy(buf[1][i].aln);
}
free(buf[0]); free(buf[1]);
if (_bwt == 0) bwt_destroy(bwt);
kv_destroy(d->arr);
kv_destroy(d->pos[0]); kv_destroy(d->pos[1]);
kv_destroy(d->aln[0]); kv_destroy(d->aln[1]);
free(d);
return cnt_chg;
}
#define SW_MIN_MATCH_LEN 20
#define SW_MIN_MAPQ 17
// cnt = n_mm<<16 | n_gapo<<8 | n_gape
bwa_cigar_t *bwa_sw_core(bwtint_t l_pac, const ubyte_t *pacseq, int len, const ubyte_t *seq, int64_t *beg, int reglen, int *n_cigar, uint32_t *_cnt)
{
kswr_t r;
uint32_t *cigar32 = 0;
bwa_cigar_t *cigar = 0;
ubyte_t *ref_seq;
bwtint_t k, x, y, l;
int xtra, gscore;
int8_t mat[25];
bwa_fill_scmat(1, 3, mat);
// check whether there are too many N's
if (reglen < SW_MIN_MATCH_LEN || (int64_t)l_pac - *beg < len) return 0;
for (k = 0, x = 0; k < len; ++k)
if (seq[k] >= 4) ++x;
if ((float)x/len >= 0.25 || len - x < SW_MIN_MATCH_LEN) return 0;
// get reference subsequence
ref_seq = (ubyte_t*)calloc(reglen, 1);
for (k = *beg, l = 0; l < reglen && k < l_pac; ++k)
ref_seq[l++] = pacseq[k>>2] >> ((~k&3)<<1) & 3;
// do alignment
xtra = KSW_XSUBO | KSW_XSTART | (len < 250? KSW_XBYTE : 0);
r = ksw_align(len, (uint8_t*)seq, l, ref_seq, 5, mat, 5, 1, xtra, 0);
gscore = ksw_global(r.qe - r.qb + 1, &seq[r.qb], r.te - r.tb + 1, &ref_seq[r.tb], 5, mat, 5, 1, 50, n_cigar, &cigar32);
cigar = (bwa_cigar_t*)cigar32;
for (k = 0; k < *n_cigar; ++k)
cigar[k] = __cigar_create((cigar32[k]&0xf), (cigar32[k]>>4));
if (r.score < SW_MIN_MATCH_LEN || r.score2 == r.score || gscore != r.score) { // poor hit or tandem hits or weird alignment
free(cigar); free(ref_seq); *n_cigar = 0;
return 0;
}
// check whether the alignment is good enough
for (k = 0, x = y = 0; k < *n_cigar; ++k) {
bwa_cigar_t c = cigar[k];
if (__cigar_op(c) == FROM_M) x += __cigar_len(c), y += __cigar_len(c);
else if (__cigar_op(c) == FROM_D) x += __cigar_len(c);
else y += __cigar_len(c);
}
if (x < SW_MIN_MATCH_LEN || y < SW_MIN_MATCH_LEN) { // not good enough
free(cigar); free(ref_seq);
*n_cigar = 0;
return 0;
}
{ // update cigar and coordinate;
int start = r.qb, end = r.qe + 1;
*beg += r.tb;
cigar = (bwa_cigar_t*)realloc(cigar, sizeof(bwa_cigar_t) * (*n_cigar + 2));
if (start) {
memmove(cigar + 1, cigar, sizeof(bwa_cigar_t) * (*n_cigar));
cigar[0] = __cigar_create(3, start);
++(*n_cigar);
}
if (end < len) {
/*cigar[*n_cigar] = 3<<14 | (len - end);*/
cigar[*n_cigar] = __cigar_create(3, (len - end));
++(*n_cigar);
}
}
{ // set *cnt
int n_mm, n_gapo, n_gape;
n_mm = n_gapo = n_gape = 0;
x = r.tb; y = r.qb;
for (k = 0; k < *n_cigar; ++k) {
bwa_cigar_t c = cigar[k];
if (__cigar_op(c) == FROM_M) {
for (l = 0; l < (__cigar_len(c)); ++l)
if (ref_seq[x+l] < 4 && seq[y+l] < 4 && ref_seq[x+l] != seq[y+l]) ++n_mm;
x += __cigar_len(c), y += __cigar_len(c);
} else if (__cigar_op(c) == FROM_D) {
x += __cigar_len(c), ++n_gapo, n_gape += (__cigar_len(c)) - 1;
} else if (__cigar_op(c) == FROM_I) {
y += __cigar_len(c), ++n_gapo, n_gape += (__cigar_len(c)) - 1;
}
}
*_cnt = (uint32_t)n_mm<<16 | n_gapo<<8 | n_gape;
}
free(ref_seq);
return cigar;
}
ubyte_t *bwa_paired_sw(const bntseq_t *bns, const ubyte_t *_pacseq, int n_seqs, bwa_seq_t *seqs[2], const pe_opt_t *popt, const isize_info_t *ii)
{
ubyte_t *pacseq;
int i;
uint64_t n_tot[2], n_mapped[2];
// load reference sequence
if (_pacseq == 0) {
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 = (ubyte_t*)_pacseq;
if (!popt->is_sw || ii->avg < 0.0) return pacseq;
// perform mate alignment
n_tot[0] = n_tot[1] = n_mapped[0] = n_mapped[1] = 0;
for (i = 0; i != n_seqs; ++i) {
bwa_seq_t *p[2];
p[0] = seqs[0] + i; p[1] = seqs[1] + i;
if ((p[0]->mapQ >= SW_MIN_MAPQ || p[1]->mapQ >= SW_MIN_MAPQ) && (p[0]->extra_flag&SAM_FPP) == 0) { // unpaired and one read has high mapQ
int k, n_cigar[2], is_singleton, mapQ = 0, mq_adjust[2];
int64_t beg[2], end[2];
bwa_cigar_t *cigar[2];
uint32_t cnt[2];
/* In the following, _pref points to the reference read
* which must be aligned; _pmate points to its mate which is
* considered to be modified. */
#define __set_rght_coor(_a, _b, _pref, _pmate) do { \
(_a) = (int64_t)_pref->pos + ii->avg - 3 * ii->std - _pmate->len * 1.5; \
(_b) = (_a) + 6 * ii->std + 2 * _pmate->len; \
if ((_a) < (int64_t)_pref->pos + _pref->len) (_a) = _pref->pos + _pref->len; \
if ((_b) > bns->l_pac) (_b) = bns->l_pac; \
} while (0)
#define __set_left_coor(_a, _b, _pref, _pmate) do { \
(_a) = (int64_t)_pref->pos + _pref->len - ii->avg - 3 * ii->std - _pmate->len * 0.5; \
(_b) = (_a) + 6 * ii->std + 2 * _pmate->len; \
if ((_a) < 0) (_a) = 0; \
if ((_b) > _pref->pos) (_b) = _pref->pos; \
} while (0)
#define __set_fixed(_pref, _pmate, _beg, _cnt) do { \
_pmate->type = BWA_TYPE_MATESW; \
_pmate->pos = _beg; \
_pmate->seQ = _pref->seQ; \
_pmate->strand = (popt->type == BWA_PET_STD)? 1 - _pref->strand : _pref->strand; \
_pmate->n_mm = _cnt>>16; _pmate->n_gapo = _cnt>>8&0xff; _pmate->n_gape = _cnt&0xff; \
_pmate->extra_flag |= SAM_FPP; \
_pref->extra_flag |= SAM_FPP; \
} while (0)
mq_adjust[0] = mq_adjust[1] = 255; // not effective
is_singleton = (p[0]->type == BWA_TYPE_NO_MATCH || p[1]->type == BWA_TYPE_NO_MATCH)? 1 : 0;
++n_tot[is_singleton];
cigar[0] = cigar[1] = 0;
n_cigar[0] = n_cigar[1] = 0;
if (popt->type != BWA_PET_STD) continue; // other types of pairing is not considered
for (k = 0; k < 2; ++k) { // p[1-k] is the reference read and p[k] is the read considered to be modified
ubyte_t *seq;
if (p[1-k]->type == BWA_TYPE_NO_MATCH) continue; // if p[1-k] is unmapped, skip
{ // note that popt->type == BWA_PET_STD always true; in older versions, there was a branch for color-space FF/RR reads
if (p[1-k]->strand == 0) { // then the mate is on the reverse strand and has larger coordinate
__set_rght_coor(beg[k], end[k], p[1-k], p[k]);
seq = p[k]->rseq;
} else { // then the mate is on forward stand and has smaller coordinate
__set_left_coor(beg[k], end[k], p[1-k], p[k]);
seq = p[k]->seq;
seq_reverse(p[k]->len, seq, 0); // because ->seq is reversed; this will reversed back shortly
}
}
// perform SW alignment
cigar[k] = bwa_sw_core(bns->l_pac, pacseq, p[k]->len, seq, &beg[k], end[k] - beg[k], &n_cigar[k], &cnt[k]);
if (cigar[k] && p[k]->type != BWA_TYPE_NO_MATCH) { // re-evaluate cigar[k]
int s_old, clip = 0, s_new;
if (__cigar_op(cigar[k][0]) == 3) clip += __cigar_len(cigar[k][0]);
if (__cigar_op(cigar[k][n_cigar[k]-1]) == 3) clip += __cigar_len(cigar[k][n_cigar[k]-1]);
s_old = (int)((p[k]->n_mm * 9 + p[k]->n_gapo * 13 + p[k]->n_gape * 2) / 3. * 8. + .499);
s_new = (int)(((cnt[k]>>16) * 9 + (cnt[k]>>8&0xff) * 13 + (cnt[k]&0xff) * 2 + clip * 3) / 3. * 8. + .499);
s_old += -4.343 * log(ii->ap_prior / bns->l_pac);
s_new += (int)(-4.343 * log(.5 * erfc(M_SQRT1_2 * 1.5) + .499)); // assume the mapped isize is 1.5\sigma
if (s_old < s_new) { // reject SW alignment
mq_adjust[k] = s_new - s_old;
free(cigar[k]); cigar[k] = 0; n_cigar[k] = 0;
} else mq_adjust[k] = s_old - s_new;
}
// now revserse sequence back such that p[*]->seq looks untouched
if (popt->type == BWA_PET_STD) {
if (p[1-k]->strand == 1) seq_reverse(p[k]->len, seq, 0);
} else {
if (p[1-k]->strand == 0) seq_reverse(p[k]->len, seq, 0);
}
}
k = -1; // no read to be changed
if (cigar[0] && cigar[1]) {
k = p[0]->mapQ < p[1]->mapQ? 0 : 1; // p[k] to be fixed
mapQ = abs(p[1]->mapQ - p[0]->mapQ);
} else if (cigar[0]) k = 0, mapQ = p[1]->mapQ;
else if (cigar[1]) k = 1, mapQ = p[0]->mapQ;
if (k >= 0 && p[k]->pos != beg[k]) {
++n_mapped[is_singleton];
{ // recalculate mapping quality
int tmp = (int)p[1-k]->mapQ - p[k]->mapQ/2 - 8;
if (tmp <= 0) tmp = 1;
if (mapQ > tmp) mapQ = tmp;
p[k]->mapQ = p[1-k]->mapQ = mapQ;
p[k]->seQ = p[1-k]->seQ = p[1-k]->seQ < mapQ? p[1-k]->seQ : mapQ;
if (p[k]->mapQ > mq_adjust[k]) p[k]->mapQ = mq_adjust[k];
if (p[k]->seQ > mq_adjust[k]) p[k]->seQ = mq_adjust[k];
}
// update CIGAR
free(p[k]->cigar); p[k]->cigar = cigar[k]; cigar[k] = 0;
p[k]->n_cigar = n_cigar[k];
// update the rest of information
__set_fixed(p[1-k], p[k], beg[k], cnt[k]);
}
free(cigar[0]); free(cigar[1]);
}
}
fprintf(stderr, "[bwa_paired_sw] %lld out of %lld Q%d singletons are mated.\n",
(long long)n_mapped[1], (long long)n_tot[1], SW_MIN_MAPQ);
fprintf(stderr, "[bwa_paired_sw] %lld out of %lld Q%d discordant pairs are fixed.\n",
(long long)n_mapped[0], (long long)n_tot[0], SW_MIN_MAPQ);
return pacseq;
}
void bwa_sai2sam_pe_core(const char *prefix, char *const fn_sa[2], char *const fn_fa[2], pe_opt_t *popt, const char *rg_line)
{
extern bwa_seqio_t *bwa_open_reads(int mode, const char *fn_fa);
int i, j, n_seqs;
long long tot_seqs = 0;
bwa_seq_t *seqs[2];
bwa_seqio_t *ks[2];
clock_t t;
bntseq_t *bns;
FILE *fp_sa[2];
gap_opt_t opt, opt0;
khint_t iter;
isize_info_t last_ii; // this is for the last batch of reads
char str[1024], magic[2][4];
bwt_t *bwt;
uint8_t *pac;
// initialization
bwase_initialize(); // initialize g_log_n[] in bwase.c
pac = 0; bwt = 0;
for (i = 1; i != 256; ++i) g_log_n[i] = (int)(4.343 * log(i) + 0.5);
bns = bns_restore(prefix);
srand48(bns->seed);
fp_sa[0] = xopen(fn_sa[0], "r");
fp_sa[1] = xopen(fn_sa[1], "r");
g_hash = kh_init(b128);
last_ii.avg = -1.0;
err_fread_noeof(magic[0], 1, 4, fp_sa[0]);
err_fread_noeof(magic[1], 1, 4, fp_sa[1]);
if (strncmp(magic[0], SAI_MAGIC, 4) != 0 || strncmp(magic[1], 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[0]);
ks[0] = bwa_open_reads(opt.mode, fn_fa[0]);
opt0 = opt;
err_fread_noeof(&opt, sizeof(gap_opt_t), 1, fp_sa[1]); // overwritten!
ks[1] = bwa_open_reads(opt.mode, fn_fa[1]);
{ // for Illumina alignment only
if (popt->is_preload) {
strcpy(str, prefix); strcat(str, ".bwt"); bwt = bwt_restore_bwt(str);
strcpy(str, prefix); strcat(str, ".sa"); bwt_restore_sa(str, bwt);
pac = (ubyte_t*)calloc(bns->l_pac/4+1, 1);
err_rewind(bns->fp_pac);
err_fread_noeof(pac, 1, bns->l_pac/4+1, bns->fp_pac);
}
}
// core loop
bwa_print_sam_hdr(bns, rg_line);
while ((seqs[0] = bwa_read_seq(ks[0], 0x40000, &n_seqs, opt0.mode, opt0.trim_qual)) != 0) {
int cnt_chg;
isize_info_t ii;
ubyte_t *pacseq;
seqs[1] = bwa_read_seq(ks[1], 0x40000, &n_seqs, opt.mode, opt.trim_qual);
tot_seqs += n_seqs;
t = clock();
fprintf(stderr, "[bwa_sai2sam_pe_core] convert to sequence coordinate... \n");
cnt_chg = bwa_cal_pac_pos_pe(bns, prefix, bwt, n_seqs, seqs, fp_sa, &ii, popt, &opt, &last_ii);
fprintf(stderr, "[bwa_sai2sam_pe_core] time elapses: %.2f sec\n", (float)(clock() - t) / CLOCKS_PER_SEC); t = clock();
fprintf(stderr, "[bwa_sai2sam_pe_core] changing coordinates of %d alignments.\n", cnt_chg);
fprintf(stderr, "[bwa_sai2sam_pe_core] align unmapped mate...\n");
pacseq = bwa_paired_sw(bns, pac, n_seqs, seqs, popt, &ii);
fprintf(stderr, "[bwa_sai2sam_pe_core] time elapses: %.2f sec\n", (float)(clock() - t) / CLOCKS_PER_SEC); t = clock();
fprintf(stderr, "[bwa_sai2sam_pe_core] refine gapped alignments... ");
for (j = 0; j < 2; ++j)
bwa_refine_gapped(bns, n_seqs, seqs[j], pacseq);
fprintf(stderr, "%.2f sec\n", (float)(clock() - t) / CLOCKS_PER_SEC); t = clock();
if (pac == 0) free(pacseq);
fprintf(stderr, "[bwa_sai2sam_pe_core] print alignments... ");
for (i = 0; i < n_seqs; ++i) {
bwa_seq_t *p[2];
p[0] = seqs[0] + i; p[1] = seqs[1] + i;
if (p[0]->bc[0] || p[1]->bc[0]) {
strcat(p[0]->bc, p[1]->bc);
strcpy(p[1]->bc, p[0]->bc);
}
bwa_print_sam1(bns, p[0], p[1], opt.mode, opt.max_top2);
bwa_print_sam1(bns, p[1], p[0], opt.mode, opt.max_top2);
if (strcmp(p[0]->name, p[1]->name) != 0) err_fatal(__func__, "paired reads have different names: \"%s\", \"%s\"\n", p[0]->name, p[1]->name);
}
fprintf(stderr, "%.2f sec\n", (float)(clock() - t) / CLOCKS_PER_SEC); t = clock();
for (j = 0; j < 2; ++j)
bwa_free_read_seq(n_seqs, seqs[j]);
fprintf(stderr, "[bwa_sai2sam_pe_core] %lld sequences have been processed.\n", tot_seqs);
last_ii = ii;
}
// destroy
bns_destroy(bns);
for (i = 0; i < 2; ++i) {
bwa_seq_close(ks[i]);
err_fclose(fp_sa[i]);
}
for (iter = kh_begin(g_hash); iter != kh_end(g_hash); ++iter)
if (kh_exist(g_hash, iter)) free(kh_val(g_hash, iter).a);
kh_destroy(b128, g_hash);
if (pac) {
free(pac); bwt_destroy(bwt);
}
}
int bwa_sai2sam_pe(int argc, char *argv[])
{
int c;
pe_opt_t *popt;
char *prefix, *rg_line = 0;
popt = bwa_init_pe_opt();
while ((c = getopt(argc, argv, "a:o:sPn:N:c:f:Ar:")) >= 0) {
switch (c) {
case 'r':
if ((rg_line = bwa_set_rg(optarg)) == 0) return 1;
break;
case 'a': popt->max_isize = atoi(optarg); break;
case 'o': popt->max_occ = atoi(optarg); break;
case 's': popt->is_sw = 0; break;
case 'P': popt->is_preload = 1; break;
case 'n': popt->n_multi = atoi(optarg); break;
case 'N': popt->N_multi = atoi(optarg); break;
case 'c': popt->ap_prior = atof(optarg); break;
case 'f': xreopen(optarg, "w", stdout); break;
case 'A': popt->force_isize = 1; break;
default: return 1;
}
}
if (optind + 5 > argc) {
fprintf(stderr, "\n");
fprintf(stderr, "Usage: bwa sampe [options] <prefix> <in1.sai> <in2.sai> <in1.fq> <in2.fq>\n\n");
fprintf(stderr, "Options: -a INT maximum insert size [%d]\n", popt->max_isize);
fprintf(stderr, " -o INT maximum occurrences for one end [%d]\n", popt->max_occ);
fprintf(stderr, " -n INT maximum hits to output for paired reads [%d]\n", popt->n_multi);
fprintf(stderr, " -N INT maximum hits to output for discordant pairs [%d]\n", popt->N_multi);
fprintf(stderr, " -c FLOAT prior of chimeric rate (lower bound) [%.1le]\n", popt->ap_prior);
fprintf(stderr, " -f FILE sam file to output results to [stdout]\n");
fprintf(stderr, " -r STR read group header line such as `@RG\\tID:foo\\tSM:bar' [null]\n");
fprintf(stderr, " -P preload index into memory (for base-space reads only)\n");
fprintf(stderr, " -s disable Smith-Waterman for the unmapped mate\n");
fprintf(stderr, " -A disable insert size estimate (force -s)\n\n");
fprintf(stderr, "Notes: 1. For SOLiD reads, <in1.fq> corresponds R3 reads and <in2.fq> to F3.\n");
fprintf(stderr, " 2. For reads shorter than 30bp, applying a smaller -o is recommended to\n");
fprintf(stderr, " to get a sensible speed at the cost of pairing accuracy.\n");
fprintf(stderr, "\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_pe_core(prefix, argv + optind + 1, argv + optind+3, popt, rg_line);
free(prefix); free(popt);
return 0;
}