#include <math.h>

#include <stdio.h>

#include <stdlib.h>

#include <string.h>

#include "utils.h"

#include "bwt.h"

#include "bntseq.h"

#include "bwtsw2.h"

#include "kstring.h"

#ifndef _NO_SSE2

#include "ksw.h"

#include "stdaln.h"

#define MIN_RATIO 0.8

#define OUTLIER_BOUND 2.0

#define MAX_STDDEV 4.0

#define EXT_STDDEV 4.0

typedef struct {

int low, high, failed;

double avg, std;

} bsw2pestat_t;

bsw2pestat_t bsw2_stat(int n, bwtsw2_t **buf, kstring_t *msg, int max_ins)

{

extern void ks_introsort_uint64_t(size_t n, uint64_t *a);

int i, k, x, p25, p50, p75, tmp, max_len = 0;

uint64_t *isize;

bsw2pestat_t r;

memset(&r, 0, sizeof(bsw2pestat_t));

isize = xcalloc(n, 8);

for (i = k = 0; i < n; i += 2) {

bsw2hit_t *t[2];

int l;

if (buf[i] == 0 || buf[i]->n != 1 || buf[i+1]->n != 1) continue; // more than 1 hits

t[0] = &buf[i]->hits[0]; t[1] = &buf[i+1]->hits[0];

if (t[0]->G2 > 0.8 * t[0]->G) continue; // the best hit is not good enough

if (t[1]->G2 > 0.8 * t[1]->G) continue; // the best hit is not good enough

l = t[0]->k > t[1]->k? t[0]->k - t[1]->k + t[1]->len : t[1]->k - t[0]->k + t[0]->len;

if (l >= max_ins) continue; // skip pairs with excessively large insert

max_len = max_len > t[0]->end - t[0]->beg? max_len : t[0]->end - t[0]->beg;

max_len = max_len > t[1]->end - t[1]->beg? max_len : t[1]->end - t[1]->beg;

isize[k++] = l;

}

ks_introsort_uint64_t(k, isize);

p25 = isize[(int)(.25 * k + .499)];

p50 = isize[(int)(.50 * k + .499)];

p75 = isize[(int)(.75 * k + .499)];

ksprintf(msg, "[%s] infer the insert size distribution from %d high-quality pairs.\n", __func__, k);

if (k < 8) {

ksprintf(msg, "[%s] fail to infer the insert size distribution.\n", __func__);

free(isize);

r.failed = 1;

return r;

}

tmp = (int)(p25 - OUTLIER_BOUND * (p75 - p25) + .499);

r.low = tmp > max_len? tmp : max_len;

if (r.low < 1) r.low = 1;

r.high = (int)(p75 + OUTLIER_BOUND * (p75 - p25) + .499);

ksprintf(msg, "[%s] (25, 50, 75) percentile: (%d, %d, %d)\n", __func__, p25, p50, p75);

ksprintf(msg, "[%s] low and high boundaries for computing mean and std.dev: (%d, %d)\n", __func__, r.low, r.high);

for (i = x = 0, r.avg = 0; i < k; ++i)

if (isize[i] >= r.low && isize[i] <= r.high)

r.avg += isize[i], ++x;

r.avg /= x;

for (i = 0, r.std = 0; i < k; ++i)

if (isize[i] >= r.low && isize[i] <= r.high)

r.std += (isize[i] - r.avg) * (isize[i] - r.avg);

r.std = sqrt(r.std / x);

ksprintf(msg, "[%s] mean and std.dev: (%.2f, %.2f)\n", __func__, r.avg, r.std);

tmp = (int)(p25 - 3. * (p75 - p25) + .499);

r.low = tmp > max_len? tmp : max_len;

if (r.low < 1) r.low = 1;

r.high = (int)(p75 + 3. * (p75 - p25) + .499);

if (r.low > r.avg - MAX_STDDEV * 4.) r.low = (int)(r.avg - MAX_STDDEV * 4. + .499);

r.low = tmp > max_len? tmp : max_len;

if (r.high < r.avg - MAX_STDDEV * 4.) r.high = (int)(r.avg + MAX_STDDEV * 4. + .499);

ksprintf(msg, "[%s] low and high boundaries for proper pairs: (%d, %d)\n", __func__, r.low, r.high);

free(isize);

return r;

}

typedef struct {

int n_cigar, beg, end, len;

int64_t pos;

uint32_t *cigar;

} pairaux_t;

extern unsigned char nst_nt4_table[256];

void bsw2_pair1(const bsw2opt_t *opt, int64_t l_pac, const uint8_t *pac, const bsw2pestat_t *st, const bsw2hit_t *h, int l_mseq, const char *mseq, bsw2hit_t *a, int8_t g_mat[25])

{

extern void seq_reverse(int len, ubyte_t *seq, int is_comp);

int64_t k, beg, end;

uint8_t *seq, *ref;

int i;

// compute the region start and end

a->n_seeds = 1; a->flag |= BSW2_FLAG_MATESW; // before calling this routine, *a has been cleared with memset(0); the flag is set with 1<<6/7

if (h->is_rev == 0) {

beg = (int64_t)(h->k + st->avg - EXT_STDDEV * st->std - l_mseq + .499);

if (beg < h->k) beg = h->k;

end = (int64_t)(h->k + st->avg + EXT_STDDEV * st->std + .499);

a->is_rev = 1; a->flag |= 16;

} else {

beg = (int64_t)(h->k + h->end - h->beg - st->avg - EXT_STDDEV * st->std + .499);

end = (int64_t)(h->k + h->end - h->beg - st->avg + EXT_STDDEV * st->std + l_mseq + .499);

if (end > h->k + (h->end - h->beg)) end = h->k + (h->end - h->beg);

a->is_rev = 0;

}

if (beg < 1) beg = 1;

if (end > l_pac) end = l_pac;

if (end - beg < l_mseq) return;

// generate the sequence

seq = xmalloc(l_mseq + (end - beg));

ref = seq + l_mseq;

for (k = beg; k < end; ++k)

ref[k - beg] = pac[k>>2] >> ((~k&3)<<1) & 0x3;

if (h->is_rev == 0) {

for (i = 0; i < l_mseq; ++i) { // on the reverse strand

int c = nst_nt4_table[(int)mseq[i]];

seq[l_mseq - 1 - i] = c > 3? 4 : 3 - c;

}

} else {

for (i = 0; i < l_mseq; ++i) // on the forward strand

seq[i] = nst_nt4_table[(int)mseq[i]];

}

#ifndef _NO_SSE2

{

ksw_query_t *q;

ksw_aux_t aux[2];

// forward Smith-Waterman

aux[0].T = opt->t; aux[0].gapo = opt->q; aux[0].gape = opt->r; aux[1] = aux[0];

q = ksw_qinit(l_mseq * g_mat[0] < 250? 1 : 2, l_mseq, seq, 5, g_mat);

ksw_sse2(q, end - beg, ref, &aux[0]);

free(q);

if (aux[0].score < opt->t) {

free(seq);

return;

}

++aux[0].qe; ++aux[0].te;

// reverse Smith-Waterman

seq_reverse(aux[0].qe, seq, 0);

seq_reverse(aux[0].te, ref, 0);

q = ksw_qinit(aux[0].qe * g_mat[0] < 250? 1 : 2, aux[0].qe, seq, 5, g_mat);

ksw_sse2(q, aux[0].te, ref, &aux[1]);

free(q);

++aux[1].qe; ++aux[1].te;

// write output

a->G = aux[0].score;

a->G2 = aux[0].score2 > aux[1].score2? aux[0].score2 : aux[1].score2;

if (a->G2 < opt->t) a->G2 = 0;

if (a->G2) a->flag |= BSW2_FLAG_TANDEM;

a->k = beg + (aux[0].te - aux[1].te);

a->len = aux[1].te;

a->beg = aux[0].qe - aux[1].qe;

a->end = aux[0].qe;

}

{

AlnParam ap;

path_t path[2];

int matrix[25];

for (i = 0; i < 25; ++i) matrix[i] = g_mat[i];

ap.gap_open = opt->q; ap.gap_ext = opt->r; ap.gap_end = opt->r;

ap.matrix = matrix; ap.row = 5; ap.band_width = 50;

a->G = aln_local_core(ref, end - beg, seq, l_mseq, &ap, path, 0, opt->t, &a->G2);

if (a->G < opt->t) a->G = 0;

if (a->G2 < opt->t) a->G2 = 0;

a->k = beg + path[0].i - 1;

a->len = path[1].i - path[0].i + 1;

a->beg = path[0].j - 1;

a->end = path[1].j;

}

if (a->is_rev) i = a->beg, a->beg = l_mseq - a->end, a->end = l_mseq - i;

free(seq);

}

void bsw2_pair(const bsw2opt_t *opt, int64_t l_pac, const uint8_t *pac, int n, bsw2seq1_t *seq, bwtsw2_t **hits)

{

extern int bsw2_resolve_duphits(const bntseq_t *bns, const bwt_t *bwt, bwtsw2_t *b, int IS);

bsw2pestat_t pes;

int i, j, k, n_rescued = 0, n_moved = 0, n_fixed = 0;

int8_t g_mat[25];

kstring_t msg;

memset(&msg, 0, sizeof(kstring_t));

pes = bsw2_stat(n, hits, &msg, opt->max_ins);

for (i = k = 0; i < 5; ++i) {

for (j = 0; j < 4; ++j)

g_mat[k++] = i == j? opt->a : -opt->b;

g_mat[k++] = 0;

}

for (i = 0; i < n; i += 2) {

bsw2hit_t a[2];

memset(&a, 0, sizeof(bsw2hit_t) * 2);

a[0].flag = 1<<6; a[1].flag = 1<<7;

for (j = 0; j < 2; ++j) { // set the read1/2 flag

if (hits[i+j] == 0) continue;

for (k = 0; k < hits[i+j]->n; ++k) {

bsw2hit_t *p = &hits[i+j]->hits[k];

p->flag |= 1<<(6+j);

}

}

if (pes.failed) continue;

if (hits[i] == 0 || hits[i+1] == 0) continue; // one end has excessive N

if (hits[i]->n != 1 && hits[i+1]->n != 1) continue; // no end has exactly one hit

if (hits[i]->n > 1 || hits[i+1]->n > 1) continue; // one read has more than one hit

if (!opt->skip_sw) {

if (hits[i+0]->n == 1) bsw2_pair1(opt, l_pac, pac, &pes, &hits[i+0]->hits[0], seq[i+1].l, seq[i+1].seq, &a[1], g_mat);

if (hits[i+1]->n == 1) bsw2_pair1(opt, l_pac, pac, &pes, &hits[i+1]->hits[0], seq[i+0].l, seq[i+0].seq, &a[0], g_mat);

} // else a[0].G == a[1].G == a[0].G2 == a[1].G2 == 0

// the following enumerate all possibilities. It is tedious but necessary...

if (hits[i]->n + hits[i+1]->n == 1) { // one end mapped; the other not;

bwtsw2_t *p[2];

int which;

if (hits[i]->n == 1) p[0] = hits[i], p[1] = hits[i+1], which = 1;

else p[0] = hits[i+1], p[1] = hits[i], which = 0;

if (a[which].G == 0) continue;

a[which].flag |= BSW2_FLAG_RESCUED;

if (p[1]->max == 0) {

p[1]->max = 1;

p[1]->hits = xmalloc(sizeof(bsw2hit_t));

}

p[1]->hits[0] = a[which];

p[1]->n = 1;

p[0]->hits[0].flag |= 2;

p[1]->hits[0].flag |= 2;

++n_rescued;

} else { // then both ends mapped

int is_fixed = 0;

//fprintf(stderr, "%d; %lld,%lld; %d,%d\n", a[0].is_rev, hits[i]->hits[0].k, a[0].k, hits[i]->hits[0].end, a[0].end);

for (j = 0; j < 2; ++j) { // fix wrong mappings and wrong suboptimal alignment score

bsw2hit_t *p = &hits[i+j]->hits[0];

if (p->G < a[j].G) { // the orginal mapping is suboptimal

a[j].G2 = a[j].G2 > p->G? a[j].G2 : p->G; // FIXME: reset BSW2_FLAG_TANDEM?

*p = a[j];

++n_fixed;

is_fixed = 1;

} else if (p->k != a[j].k && p->G2 < a[j].G) {

p->G2 = a[j].G;

} else if (p->k == a[j].k && p->G2 < a[j].G2) {

p->G2 = a[j].G2;

}

}

if (hits[i]->hits[0].k == a[0].k && hits[i+1]->hits[0].k == a[1].k) { // properly paired and no ends need to be moved

for (j = 0; j < 2; ++j)

hits[i+j]->hits[0].flag |= 2 | (a[j].flag & BSW2_FLAG_TANDEM);

} else if (hits[i]->hits[0].k == a[0].k || hits[i+1]->hits[0].k == a[1].k) { // a tandem match

for (j = 0; j < 2; ++j) {

hits[i+j]->hits[0].flag |= 2;

if (hits[i+j]->hits[0].k != a[j].k)

hits[i+j]->hits[0].flag |= BSW2_FLAG_TANDEM;

}

} else if (!is_fixed && (a[0].G || a[1].G)) { // it is possible to move one end

if (a[0].G && a[1].G) { // now we have two "proper pairs"

int G[2];

double diff;

G[0] = hits[i]->hits[0].G + a[1].G;

G[1] = hits[i+1]->hits[0].G + a[0].G;

diff = fabs(G[0] - G[1]) / (opt->a + opt->b) / ((hits[i]->hits[0].len + a[1].len + hits[i+1]->hits[0].len + a[0].len) / 2.);

if (diff > 0.05) a[G[0] > G[1]? 0 : 1].G = 0;

}

if (a[0].G == 0 || a[1].G == 0) { // one proper pair only

bsw2hit_t *p[2]; // p[0] points the unchanged hit; p[1] to the hit to be moved

int which, isize;

double dev, diff;

if (a[0].G) p[0] = &hits[i+1]->hits[0], p[1] = &hits[i]->hits[0], which = 0;

else p[0] = &hits[i]->hits[0], p[1] = &hits[i+1]->hits[0], which = 1;

isize = p[0]->is_rev? p[0]->k + p[0]->len - a[which].k : a[which].k + a[which].len - p[0]->k;

dev = fabs(isize - pes.avg) / pes.std;

diff = (double)(p[1]->G - a[which].G) / (opt->a + opt->b) / (p[1]->end - p[1]->beg) * 100.0;

if (diff < dev * 2.) { // then move (heuristic)

a[which].G2 = a[which].G;

p[1][0] = a[which];

p[1]->flag |= BSW2_FLAG_MOVED | 2;

p[0]->flag |= 2;

++n_moved;

}

}

} else if (is_fixed) {

hits[i+0]->hits[0].flag |= 2;

hits[i+1]->hits[0].flag |= 2;

}

}

}

ksprintf(&msg, "[%s] #fixed=%d, #rescued=%d, #moved=%d\n", __func__, n_fixed, n_rescued, n_moved);

fputs(msg.s, stderr);

free(msg.s);

}