bio-bwa 0.2.0

data/ext/bwape.c

@@ -0,0 +1,807 @@
+#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 "stdaln.h"
+
+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;
+
+#include "khash.h"
+KHASH_MAP_INIT_INT64(64, poslist_t)
+
+#include "ksort.h"
+KSORT_INIT_GENERIC(uint64_t)
+
+typedef struct {
+	kvec_t(uint64_t) arr;
+	kvec_t(uint64_t) 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_64_t *g_hash;
+
+void bwase_initialize();
+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);
+void bwa_refine_gapped(const bntseq_t *bns, int n_seqs, bwa_seq_t *seqs, ubyte_t *_pacseq, bntseq_t *ntbns);
+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);
+void bwa_print_sam_PG();
+
+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 inline uint64_t hash_64(uint64_t key)
+{
+	key += ~(key << 32);
+	key ^= (key >> 22);
+	key += ~(key << 13);
+	key ^= (key >> 8);
+	key += (key << 3);
+	key ^= (key >> 15);
+	key += ~(key << 27);
+	key ^= (key >> 31);
+	return key;
+}
+/*
+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(uint64_t, 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);
+	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: %d and %d for estimating avg and std\n", ii->low, 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: %d (%.2lf sigma)\n", 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 last_pos[2][2], o_pos[2], subo_score, o_score;
+	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)>>32) + p[(v)&1]->len - ((u)>>32);				\
+		if ((u) != (uint64_t)-1 && (v)>>32 > (u)>>32 && l >= max_len	\
+			&& ((ii->high && l <= ii->high_bayesian) || (ii->high == 0 && l <= opt->max_isize))) \
+		{																\
+			uint64_t s = d->aln[(v)&1].a[(uint32_t)(v)>>1].score + d->aln[(u)&1].a[(uint32_t)(u)>>1].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)>>32<<32 | (v)>>32);		\
+			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)&1] = (u), o_pos[(v)&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)&1].a + ((uint32_t)(w)>>1);		\
+		(q)->extra_flag |= SAM_FPP;										\
+		if ((q)->pos != (w)>>32 || (q)->strand != r->a) {				\
+			(q)->n_mm = r->n_mm; (q)->n_gapo = r->n_gapo; (q)->n_gape = r->n_gape; (q)->strand = r->a; \
+			(q)->score = r->score;										\
+			(q)->pos = (w)>>32;											\
+			if ((q)->mapQ > 0) ++cnt_chg;								\
+		}																\
+	} while (0)
+
+	o_score = subo_score = (uint64_t)-1;
+	o_n = subo_n = 0;
+	ks_introsort(uint64_t, d->arr.n, d->arr.a);
+	for (j = 0; j < 2; ++j) last_pos[j][0] = last_pos[j][1] = (uint64_t)-1;
+	if (opt->type == BWA_PET_STD) {
+		for (i = 0; i < d->arr.n; ++i) {
+			uint64_t x = d->arr.a[i];
+			int strand = d->aln[x&1].a[(uint32_t)x>>1].a;
+			if (strand == 1) { // reverse strand, then check
+				int y = 1 - (x&1);
+				__pairing_aux(last_pos[y][1], x);
+				__pairing_aux(last_pos[y][0], x);
+			} else { // forward strand, then push
+				last_pos[x&1][0] = last_pos[x&1][1];
+				last_pos[x&1][1] = x;
+			}
+		}
+	} else if (opt->type == BWA_PET_SOLID) {
+		for (i = 0; i < d->arr.n; ++i) {
+			uint64_t x = d->arr.a[i];
+			int strand = d->aln[x&1].a[(uint32_t)x>>1].a;
+			if ((strand^x)&1) { // push
+				int y = 1 - (x&1);
+				__pairing_aux(last_pos[y][1], x);
+				__pairing_aux(last_pos[y][0], x);
+			} else { // check
+				last_pos[x&1][0] = last_pos[x&1][1];
+				last_pos[x&1][1] = x;
+			}
+		}
+	} else {
+		fprintf(stderr, "[paring] not implemented yet!\n");
+		exit(1);
+	}
+	// set pairing
+	//fprintf(stderr, "[%d, %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
+		int rr[2];
+		//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;
+			}
+		}
+		rr[0] = d->aln[o_pos[0]&1].a[(uint32_t)o_pos[0]>>1].a;
+		rr[1] = d->aln[o_pos[1]&1].a[(uint32_t)o_pos[1]>>1].a;
+		if ((p[0]->pos == o_pos[0]>>32 && p[0]->strand == rr[0]) && (p[1]->pos == o_pos[1]>>32 && p[1]->strand == rr[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]>>32 && p[0]->strand == rr[0]) { // [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]>>32 && p[1]->strand == rr[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 char *prefix, bwt_t *const _bwt[2], 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[2];
+	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] == 0) { // load forward SA
+		strcpy(str, prefix); strcat(str, ".bwt");  bwt[0] = bwt_restore_bwt(str);
+		strcpy(str, prefix); strcat(str, ".sa"); bwt_restore_sa(str, bwt[0]);
+		strcpy(str, prefix); strcat(str, ".rbwt"); bwt[1] = bwt_restore_bwt(str);
+		strcpy(str, prefix); strcat(str, ".rsa"); bwt_restore_sa(str, bwt[1]);
+	} else bwt[0] = _bwt[0], bwt[1] = _bwt[1];
+
+	// 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);
+			fread(&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;
+			fread(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 max_diff = gopt->fnr > 0.0? bwa_cal_maxdiff(p[j]->len, BWA_AVG_ERR, gopt->fnr) : gopt->max_diff;
+				p[j]->pos = p[j]->strand? bwt_sa(bwt[0], p[j]->sa)
+					: bwt[1]->seq_len - (bwt_sa(bwt[1], p[j]->sa) + p[j]->len);
+				p[j]->seQ = p[j]->mapQ = bwa_approx_mapQ(p[j], max_diff);
+			}
+		}
+	}
+
+	// infer isize
+	infer_isize(n_seqs, seqs, ii, opt->ap_prior, bwt[0]->seq_len);
+	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
+			uint64_t x;
+			int j, k, 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 (r->l - r->k + 1 >= MIN_HASH_WIDTH) { // then check hash table
+						uint64_t key = (uint64_t)r->k<<32 | r->l;
+						int ret;
+						khint_t iter = kh_put(64, 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)
+								z->a[l - r->k] = r->a? bwt_sa(bwt[0], l) : bwt[1]->seq_len - (bwt_sa(bwt[1], l) + p[j]->len);
+						}
+						for (l = 0; l < kh_val(g_hash, iter).n; ++l) {
+							x = kh_val(g_hash, iter).a[l];
+							x = x<<32 | k<<1 | j;
+							kv_push(uint64_t, d->arr, x);
+						}
+					} else { // then calculate on the fly
+						for (l = r->k; l <= r->l; ++l) {
+							x = r->a? bwt_sa(bwt[0], l) : bwt[1]->seq_len - (bwt_sa(bwt[1], l) + p[j]->len);
+							x = x<<32 | k<<1 | j;
+							kv_push(uint64_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;
+					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; k < p[j]->n_multi; ++k) {
+						bwt_multi1_t *q = p[j]->multi + k;
+						q->pos = q->strand? bwt_sa(bwt[0], q->pos) : bwt[1]->seq_len - (bwt_sa(bwt[1], q->pos) + p[j]->len);
+					}
+				}
+			}
+		}
+	}
+
+	// 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] == 0) {
+		bwt_destroy(bwt[0]); bwt_destroy(bwt[1]);
+	}
+	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)
+{
+	bwa_cigar_t *cigar = 0;
+	ubyte_t *ref_seq;
+	bwtint_t k, x, y, l;
+	int path_len, ret;
+	AlnParam ap = aln_param_bwa;
+	path_t *path, *p;
+
+	// 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;
+	path = (path_t*)calloc(l+len, sizeof(path_t));
+
+	// do alignment
+	ret = aln_local_core(ref_seq, l, (ubyte_t*)seq, len, &ap, path, &path_len, 1, 0);
+	if (ret < 0) {
+		free(path); free(cigar); free(ref_seq); *n_cigar = 0;
+		return 0;
+	}
+	cigar = bwa_aln_path2cigar(path, path_len, n_cigar);
+
+	// 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(path); free(cigar); free(ref_seq);
+		*n_cigar = 0;
+		return 0;
+	}
+
+	{ // update cigar and coordinate;
+		int start, end;
+		p = path + path_len - 1;
+		*beg += (p->i? p->i : 1) - 1;
+		start = (p->j? p->j : 1) - 1;
+		end = path->j;
+		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;
+		p = path + path_len - 1;
+		x = p->i? p->i - 1 : 0; y = p->j? p->j - 1 : 0;
+		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); free(path);
+	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);
+		rewind(bns->fp_pac);
+		fread(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 && popt->type != BWA_PET_SOLID) 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
+				if (popt->type == BWA_PET_STD) {
+					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
+					}
+				} else { // BWA_PET_SOLID
+					if (p[1-k]->strand == 0) { // R3-F3 pairing
+						if (k == 0) __set_left_coor(beg[k], end[k], p[1-k], p[k]); // p[k] is R3
+						else __set_rght_coor(beg[k], end[k], p[1-k], p[k]); // p[k] is F3
+						seq = p[k]->rseq;
+						seq_reverse(p[k]->len, seq, 0); // because ->seq is reversed
+					} else { // F3-R3 pairing
+						if (k == 0) __set_rght_coor(beg[k], end[k], p[1-k], p[k]); // p[k] is R3
+						else __set_left_coor(beg[k], end[k], p[1-k], p[k]); // p[k] is F3
+						seq = p[k]->seq;
+					}
+				}
+				// 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)
+{
+	extern bwa_seqio_t *bwa_open_reads(int mode, const char *fn_fa);
+	int i, j, n_seqs, tot_seqs = 0;
+	bwa_seq_t *seqs[2];
+	bwa_seqio_t *ks[2];
+	clock_t t;
+	bntseq_t *bns, *ntbns = 0;
+	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];
+	bwt_t *bwt[2];
+	uint8_t *pac;
+
+	// initialization
+	bwase_initialize(); // initialize g_log_n[] in bwase.c
+	pac = 0; bwt[0] = bwt[1] = 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(64);
+	last_ii.avg = -1.0;
+
+	fread(&opt, sizeof(gap_opt_t), 1, fp_sa[0]);
+	ks[0] = bwa_open_reads(opt.mode, fn_fa[0]);
+	opt0 = opt;
+	fread(&opt, sizeof(gap_opt_t), 1, fp_sa[1]); // overwritten!
+	ks[1] = bwa_open_reads(opt.mode, fn_fa[1]);
+	if (!(opt.mode & BWA_MODE_COMPREAD)) {
+		popt->type = BWA_PET_SOLID;
+		ntbns = bwa_open_nt(prefix);
+	} else { // for Illumina alignment only
+		if (popt->is_preload) {
+			strcpy(str, prefix); strcat(str, ".bwt");  bwt[0] = bwt_restore_bwt(str);
+			strcpy(str, prefix); strcat(str, ".sa"); bwt_restore_sa(str, bwt[0]);
+			strcpy(str, prefix); strcat(str, ".rbwt"); bwt[1] = bwt_restore_bwt(str);
+			strcpy(str, prefix); strcat(str, ".rsa"); bwt_restore_sa(str, bwt[1]);
+			pac = (ubyte_t*)calloc(bns->l_pac/4+1, 1);
+			rewind(bns->fp_pac);
+			fread(pac, 1, bns->l_pac/4+1, bns->fp_pac);
+		}
+	}
+
+	// core loop
+	bwa_print_sam_SQ(bns);
+	bwa_print_sam_PG();
+	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(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, ntbns);
+		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);
+		}
+		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] %d sequences have been processed.\n", tot_seqs);
+		last_ii = ii;
+	}
+
+	// destroy
+	bns_destroy(bns);
+	if (ntbns) bns_destroy(ntbns);
+	for (i = 0; i < 2; ++i) {
+		bwa_seq_close(ks[i]);
+		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(64, g_hash);
+	if (pac) {
+		free(pac); bwt_destroy(bwt[0]); bwt_destroy(bwt[1]);
+	}
+}
+
+int bwa_sai2sam_pe(int argc, char *argv[])
+{
+	extern char *bwa_rg_line, *bwa_rg_id;
+	extern int bwa_set_rg(const char *s);
+	int c;
+	pe_opt_t *popt;
+	popt = bwa_init_pe_opt();
+	optind = 1;
+	while ((c = getopt(argc, argv, "a:o:sPn:N:c:f:Ar:")) >= 0) {
+		switch (c) {
+		case 'r':
+			if (bwa_set_rg(optarg) < 0) {
+				fprintf(stderr, "[%s] malformated @RG line\n", __func__);
+				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;
+	}
+	bwa_sai2sam_pe_core(argv[optind], argv + optind + 1, argv + optind+3, popt);
+	free(bwa_rg_line); free(bwa_rg_id);
+	free(popt);
+	fflush(stdout);
+	xreopen("/dev/tty","w",stdout);
+	return 0;
+}