diff PsiCLASS-1.0.2/samtools-0.1.19/bam2bcf_indel.c @ 0:903fc43d6227 draft default tip

Uploaded

--- /dev/null	Thu Jan 01 00:00:00 1970 +0000
+++ b/PsiCLASS-1.0.2/samtools-0.1.19/bam2bcf_indel.c	Fri Mar 26 16:52:45 2021 +0000
@@ -0,0 +1,498 @@
+#include <assert.h>
+#include <ctype.h>
+#include <string.h>
+#include "bam.h"
+#include "bam2bcf.h"
+#include "kaln.h"
+#include "kprobaln.h"
+#include "khash.h"
+KHASH_SET_INIT_STR(rg)
+
+#include "ksort.h"
+KSORT_INIT_GENERIC(uint32_t)
+
+#define MINUS_CONST 0x10000000
+#define INDEL_WINDOW_SIZE 50
+
+void *bcf_call_add_rg(void *_hash, const char *hdtext, const char *list)
+{
+	const char *s, *p, *q, *r, *t;
+	khash_t(rg) *hash;
+	if (list == 0 || hdtext == 0) return _hash;
+	if (_hash == 0) _hash = kh_init(rg);
+	hash = (khash_t(rg)*)_hash;
+	if ((s = strstr(hdtext, "@RG\t")) == 0) return hash;
+	do {
+		t = strstr(s + 4, "@RG\t"); // the next @RG
+		if ((p = strstr(s, "\tID:")) != 0) p += 4;
+		if ((q = strstr(s, "\tPL:")) != 0) q += 4;
+		if (p && q && (t == 0 || (p < t && q < t))) { // ID and PL are both present
+			int lp, lq;
+			char *x;
+			for (r = p; *r && *r != '\t' && *r != '\n'; ++r); lp = r - p;
+			for (r = q; *r && *r != '\t' && *r != '\n'; ++r); lq = r - q;
+			x = calloc((lp > lq? lp : lq) + 1, 1);
+			for (r = q; *r && *r != '\t' && *r != '\n'; ++r) x[r-q] = *r;
+			if (strstr(list, x)) { // insert ID to the hash table
+				khint_t k;
+				int ret;
+				for (r = p; *r && *r != '\t' && *r != '\n'; ++r) x[r-p] = *r;
+				x[r-p] = 0;
+				k = kh_get(rg, hash, x);
+				if (k == kh_end(hash)) k = kh_put(rg, hash, x, &ret);
+				else free(x);
+			} else free(x);
+		}
+		s = t;
+	} while (s);
+	return hash;
+}
+
+void bcf_call_del_rghash(void *_hash)
+{
+	khint_t k;
+	khash_t(rg) *hash = (khash_t(rg)*)_hash;
+	if (hash == 0) return;
+	for (k = kh_begin(hash); k < kh_end(hash); ++k)
+		if (kh_exist(hash, k))
+			free((char*)kh_key(hash, k));
+	kh_destroy(rg, hash);
+}
+
+static int tpos2qpos(const bam1_core_t *c, const uint32_t *cigar, int32_t tpos, int is_left, int32_t *_tpos)
+{
+	int k, x = c->pos, y = 0, last_y = 0;
+	*_tpos = c->pos;
+	for (k = 0; k < c->n_cigar; ++k) {
+		int op = cigar[k] & BAM_CIGAR_MASK;
+		int l = cigar[k] >> BAM_CIGAR_SHIFT;
+		if (op == BAM_CMATCH || op == BAM_CEQUAL || op == BAM_CDIFF) {
+			if (c->pos > tpos) return y;
+			if (x + l > tpos) {
+				*_tpos = tpos;
+				return y + (tpos - x);
+			}
+			x += l; y += l;
+			last_y = y;
+		} else if (op == BAM_CINS || op == BAM_CSOFT_CLIP) y += l;
+		else if (op == BAM_CDEL || op == BAM_CREF_SKIP) {
+			if (x + l > tpos) {
+				*_tpos = is_left? x : x + l;
+				return y;
+			}
+			x += l;
+		}
+	}
+	*_tpos = x;
+	return last_y;
+}
+// FIXME: check if the inserted sequence is consistent with the homopolymer run
+// l is the relative gap length and l_run is the length of the homopolymer on the reference
+static inline int est_seqQ(const bcf_callaux_t *bca, int l, int l_run)
+{
+	int q, qh;
+	q = bca->openQ + bca->extQ * (abs(l) - 1);
+	qh = l_run >= 3? (int)(bca->tandemQ * (double)abs(l) / l_run + .499) : 1000;
+	return q < qh? q : qh;
+}
+
+static inline int est_indelreg(int pos, const char *ref, int l, char *ins4)
+{
+	int i, j, max = 0, max_i = pos, score = 0;
+	l = abs(l);
+	for (i = pos + 1, j = 0; ref[i]; ++i, ++j) {
+		if (ins4) score += (toupper(ref[i]) != "ACGTN"[(int)ins4[j%l]])? -10 : 1;
+		else score += (toupper(ref[i]) != toupper(ref[pos+1+j%l]))? -10 : 1;
+		if (score < 0) break;
+		if (max < score) max = score, max_i = i;
+	}
+	return max_i - pos;
+}
+
+/*
+ *  @n:  number of samples
+ */
+int bcf_call_gap_prep(int n, int *n_plp, bam_pileup1_t **plp, int pos, bcf_callaux_t *bca, const char *ref,
+					  const void *rghash)
+{
+	int i, s, j, k, t, n_types, *types, max_rd_len, left, right, max_ins, *score1, *score2, max_ref2;
+	int N, K, l_run, ref_type, n_alt;
+	char *inscns = 0, *ref2, *query, **ref_sample;
+	khash_t(rg) *hash = (khash_t(rg)*)rghash;
+	if (ref == 0 || bca == 0) return -1;
+	// mark filtered reads
+	if (rghash) {
+		N = 0;
+		for (s = N = 0; s < n; ++s) {
+			for (i = 0; i < n_plp[s]; ++i) {
+				bam_pileup1_t *p = plp[s] + i;
+				const uint8_t *rg = bam_aux_get(p->b, "RG");
+				p->aux = 1; // filtered by default
+				if (rg) {
+					khint_t k = kh_get(rg, hash, (const char*)(rg + 1));
+					if (k != kh_end(hash)) p->aux = 0, ++N; // not filtered
+				}
+			}
+		}
+		if (N == 0) return -1; // no reads left
+	}
+	// determine if there is a gap
+	for (s = N = 0; s < n; ++s) {
+		for (i = 0; i < n_plp[s]; ++i)
+			if (plp[s][i].indel != 0) break;
+		if (i < n_plp[s]) break;
+	}
+	if (s == n) return -1; // there is no indel at this position.
+	for (s = N = 0; s < n; ++s) N += n_plp[s]; // N is the total number of reads
+	{ // find out how many types of indels are present
+        bca->max_support = bca->max_frac = 0;
+		int m, n_alt = 0, n_tot = 0, indel_support_ok = 0;
+		uint32_t *aux;
+		aux = calloc(N + 1, 4);
+		m = max_rd_len = 0;
+		aux[m++] = MINUS_CONST; // zero indel is always a type
+		for (s = 0; s < n; ++s) {
+            int na = 0, nt = 0; 
+			for (i = 0; i < n_plp[s]; ++i) {
+				const bam_pileup1_t *p = plp[s] + i;
+				if (rghash == 0 || p->aux == 0) {
+					++nt;
+					if (p->indel != 0) {
+						++na;
+						aux[m++] = MINUS_CONST + p->indel;
+					}
+				}
+				j = bam_cigar2qlen(&p->b->core, bam1_cigar(p->b));
+				if (j > max_rd_len) max_rd_len = j;
+			}
+            float frac = (float)na/nt;
+            if ( !indel_support_ok && na >= bca->min_support && frac >= bca->min_frac )
+                indel_support_ok = 1;
+            if ( na > bca->max_support && frac > 0 ) bca->max_support = na, bca->max_frac = frac;
+            n_alt += na; 
+            n_tot += nt;
+		}
+        // To prevent long stretches of N's to be mistaken for indels (sometimes thousands of bases),
+        //  check the number of N's in the sequence and skip places where half or more reference bases are Ns.
+        int nN=0; for (i=pos; i-pos<max_rd_len && ref[i]; i++) if ( ref[i]=='N' ) nN++;
+        if ( nN*2>i ) { free(aux); return -1; }
+
+		ks_introsort(uint32_t, m, aux);
+		// squeeze out identical types
+		for (i = 1, n_types = 1; i < m; ++i)
+			if (aux[i] != aux[i-1]) ++n_types;
+        // Taking totals makes it hard to call rare indels
+        if ( !bca->per_sample_flt )
+            indel_support_ok = ( (float)n_alt / n_tot < bca->min_frac || n_alt < bca->min_support ) ? 0 : 1;
+        if ( n_types == 1 || !indel_support_ok ) { // then skip
+            free(aux); return -1;
+        }
+		if (n_types >= 64) {
+			free(aux);
+			if (bam_verbose >= 2) 
+				fprintf(stderr, "[%s] excessive INDEL alleles at position %d. Skip the position.\n", __func__, pos + 1);
+			return -1;
+		}
+		types = (int*)calloc(n_types, sizeof(int));
+		t = 0;
+		types[t++] = aux[0] - MINUS_CONST; 
+		for (i = 1; i < m; ++i)
+			if (aux[i] != aux[i-1])
+				types[t++] = aux[i] - MINUS_CONST;
+		free(aux);
+		for (t = 0; t < n_types; ++t)
+			if (types[t] == 0) break;
+		ref_type = t; // the index of the reference type (0)
+	}
+	{ // calculate left and right boundary
+		left = pos > INDEL_WINDOW_SIZE? pos - INDEL_WINDOW_SIZE : 0;
+		right = pos + INDEL_WINDOW_SIZE;
+		if (types[0] < 0) right -= types[0];
+		// in case the alignments stand out the reference
+		for (i = pos; i < right; ++i)
+			if (ref[i] == 0) break;
+		right = i;
+	}
+	/* The following block fixes a long-existing flaw in the INDEL
+	 * calling model: the interference of nearby SNPs. However, it also
+	 * reduces the power because sometimes, substitutions caused by
+	 * indels are not distinguishable from true mutations. Multiple
+	 * sequence realignment helps to increase the power.
+     *
+     * Masks mismatches present in at least 70% of the reads with 'N'.
+	 */
+	{ // construct per-sample consensus
+		int L = right - left + 1, max_i, max2_i;
+		uint32_t *cns, max, max2;
+		char *ref0, *r;
+		ref_sample = calloc(n, sizeof(void*));
+		cns = calloc(L, 4);
+		ref0 = calloc(L, 1);
+		for (i = 0; i < right - left; ++i)
+			ref0[i] = bam_nt16_table[(int)ref[i+left]];
+		for (s = 0; s < n; ++s) {
+			r = ref_sample[s] = calloc(L, 1);
+			memset(cns, 0, sizeof(int) * L);
+			// collect ref and non-ref counts
+			for (i = 0; i < n_plp[s]; ++i) {
+				bam_pileup1_t *p = plp[s] + i;
+				bam1_t *b = p->b;
+				uint32_t *cigar = bam1_cigar(b);
+				uint8_t *seq = bam1_seq(b);
+				int x = b->core.pos, y = 0;
+				for (k = 0; k < b->core.n_cigar; ++k) {
+					int op = cigar[k]&0xf;
+					int j, l = cigar[k]>>4;
+					if (op == BAM_CMATCH || op == BAM_CEQUAL || op == BAM_CDIFF) {
+						for (j = 0; j < l; ++j)
+							if (x + j >= left && x + j < right)
+								cns[x+j-left] += (bam1_seqi(seq, y+j) == ref0[x+j-left])? 1 : 0x10000;
+						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;
+				}
+			}
+			// determine the consensus
+			for (i = 0; i < right - left; ++i) r[i] = ref0[i];
+			max = max2 = 0; max_i = max2_i = -1;
+			for (i = 0; i < right - left; ++i) {
+				if (cns[i]>>16 >= max>>16) max2 = max, max2_i = max_i, max = cns[i], max_i = i;
+				else if (cns[i]>>16 >= max2>>16) max2 = cns[i], max2_i = i;
+			}
+			if ((double)(max&0xffff) / ((max&0xffff) + (max>>16)) >= 0.7) max_i = -1;
+			if ((double)(max2&0xffff) / ((max2&0xffff) + (max2>>16)) >= 0.7) max2_i = -1;
+			if (max_i >= 0) r[max_i] = 15;
+			if (max2_i >= 0) r[max2_i] = 15;
+			//for (i = 0; i < right - left; ++i) fputc("=ACMGRSVTWYHKDBN"[(int)r[i]], stderr); fputc('\n', stderr);
+		}
+		free(ref0); free(cns);
+	}
+	{ // the length of the homopolymer run around the current position
+		int c = bam_nt16_table[(int)ref[pos + 1]];
+		if (c == 15) l_run = 1;
+		else {
+			for (i = pos + 2; ref[i]; ++i)
+				if (bam_nt16_table[(int)ref[i]] != c) break;
+			l_run = i;
+			for (i = pos; i >= 0; --i)
+				if (bam_nt16_table[(int)ref[i]] != c) break;
+			l_run -= i + 1;
+		}
+	}
+	// construct the consensus sequence
+	max_ins = types[n_types - 1];   // max_ins is at least 0
+	if (max_ins > 0) {
+		int *inscns_aux = calloc(5 * n_types * max_ins, sizeof(int));
+		// count the number of occurrences of each base at each position for each type of insertion
+		for (t = 0; t < n_types; ++t) {
+			if (types[t] > 0) {
+				for (s = 0; s < n; ++s) {
+					for (i = 0; i < n_plp[s]; ++i) {
+						bam_pileup1_t *p = plp[s] + i;
+						if (p->indel == types[t]) {
+							uint8_t *seq = bam1_seq(p->b);
+							for (k = 1; k <= p->indel; ++k) {
+								int c = bam_nt16_nt4_table[bam1_seqi(seq, p->qpos + k)];
+                                assert(c<5);
+								++inscns_aux[(t*max_ins+(k-1))*5 + c];
+							}
+						}
+					}
+				}
+			}
+		}
+		// use the majority rule to construct the consensus
+		inscns = calloc(n_types * max_ins, 1);
+		for (t = 0; t < n_types; ++t) {
+			for (j = 0; j < types[t]; ++j) {
+				int max = 0, max_k = -1, *ia = &inscns_aux[(t*max_ins+j)*5];
+				for (k = 0; k < 5; ++k)
+					if (ia[k] > max)
+						max = ia[k], max_k = k;
+				inscns[t*max_ins + j] = max? max_k : 4;
+                if ( max_k==4 ) { types[t] = 0; break; } // discard insertions which contain N's
+			}
+		}
+		free(inscns_aux);
+	}
+	// compute the likelihood given each type of indel for each read
+	max_ref2 = right - left + 2 + 2 * (max_ins > -types[0]? max_ins : -types[0]);
+	ref2  = calloc(max_ref2, 1);
+	query = calloc(right - left + max_rd_len + max_ins + 2, 1);
+	score1 = calloc(N * n_types, sizeof(int));
+	score2 = calloc(N * n_types, sizeof(int));
+	bca->indelreg = 0;
+	for (t = 0; t < n_types; ++t) {
+		int l, ir;
+		kpa_par_t apf1 = { 1e-4, 1e-2, 10 }, apf2 = { 1e-6, 1e-3, 10 };
+		apf1.bw = apf2.bw = abs(types[t]) + 3;
+		// compute indelreg
+		if (types[t] == 0) ir = 0;
+		else if (types[t] > 0) ir = est_indelreg(pos, ref, types[t], &inscns[t*max_ins]);
+		else ir = est_indelreg(pos, ref, -types[t], 0);
+		if (ir > bca->indelreg) bca->indelreg = ir;
+//		fprintf(stderr, "%d, %d, %d\n", pos, types[t], ir);
+		// realignment
+		for (s = K = 0; s < n; ++s) {
+			// write ref2
+			for (k = 0, j = left; j <= pos; ++j)
+				ref2[k++] = bam_nt16_nt4_table[(int)ref_sample[s][j-left]];
+			if (types[t] <= 0) j += -types[t];
+			else for (l = 0; l < types[t]; ++l)
+					 ref2[k++] = inscns[t*max_ins + l];
+			for (; j < right && ref[j]; ++j)
+				ref2[k++] = bam_nt16_nt4_table[(int)ref_sample[s][j-left]];
+			for (; k < max_ref2; ++k) ref2[k] = 4;
+			if (j < right) right = j;
+			// align each read to ref2
+			for (i = 0; i < n_plp[s]; ++i, ++K) {
+				bam_pileup1_t *p = plp[s] + i;
+				int qbeg, qend, tbeg, tend, sc, kk;
+				uint8_t *seq = bam1_seq(p->b);
+				uint32_t *cigar = bam1_cigar(p->b);
+				if (p->b->core.flag&4) continue; // unmapped reads
+				// FIXME: the following loop should be better moved outside; nonetheless, realignment should be much slower anyway.
+				for (kk = 0; kk < p->b->core.n_cigar; ++kk)
+					if ((cigar[kk]&BAM_CIGAR_MASK) == BAM_CREF_SKIP) break;
+				if (kk < p->b->core.n_cigar) continue;
+				// FIXME: the following skips soft clips, but using them may be more sensitive.
+				// determine the start and end of sequences for alignment
+				qbeg = tpos2qpos(&p->b->core, bam1_cigar(p->b), left,  0, &tbeg);
+				qend = tpos2qpos(&p->b->core, bam1_cigar(p->b), right, 1, &tend);
+				if (types[t] < 0) {
+					int l = -types[t];
+					tbeg = tbeg - l > left?  tbeg - l : left;
+				}
+				// write the query sequence
+				for (l = qbeg; l < qend; ++l)
+					query[l - qbeg] = bam_nt16_nt4_table[bam1_seqi(seq, l)];
+				{ // do realignment; this is the bottleneck
+					const uint8_t *qual = bam1_qual(p->b), *bq;
+					uint8_t *qq;
+					qq = calloc(qend - qbeg, 1);
+					bq = (uint8_t*)bam_aux_get(p->b, "ZQ");
+					if (bq) ++bq; // skip type
+					for (l = qbeg; l < qend; ++l) {
+						qq[l - qbeg] = bq? qual[l] + (bq[l] - 64) : qual[l];
+						if (qq[l - qbeg] > 30) qq[l - qbeg] = 30;
+						if (qq[l - qbeg] < 7) qq[l - qbeg] = 7;
+					}
+					sc = kpa_glocal((uint8_t*)ref2 + tbeg - left, tend - tbeg + abs(types[t]),
+									(uint8_t*)query, qend - qbeg, qq, &apf1, 0, 0);
+					l = (int)(100. * sc / (qend - qbeg) + .499); // used for adjusting indelQ below
+					if (l > 255) l = 255;
+					score1[K*n_types + t] = score2[K*n_types + t] = sc<<8 | l;
+					if (sc > 5) {
+						sc = kpa_glocal((uint8_t*)ref2 + tbeg - left, tend - tbeg + abs(types[t]),
+										(uint8_t*)query, qend - qbeg, qq, &apf2, 0, 0);
+						l = (int)(100. * sc / (qend - qbeg) + .499);
+						if (l > 255) l = 255;
+						score2[K*n_types + t] = sc<<8 | l;
+					}
+					free(qq);
+				}
+/*
+				for (l = 0; l < tend - tbeg + abs(types[t]); ++l)
+					fputc("ACGTN"[(int)ref2[tbeg-left+l]], stderr);
+				fputc('\n', stderr);
+				for (l = 0; l < qend - qbeg; ++l) fputc("ACGTN"[(int)query[l]], stderr);
+				fputc('\n', stderr);
+				fprintf(stderr, "pos=%d type=%d read=%d:%d name=%s qbeg=%d tbeg=%d score=%d\n", pos, types[t], s, i, bam1_qname(p->b), qbeg, tbeg, sc);
+*/
+			}
+		}
+	}
+	free(ref2); free(query);
+	{ // compute indelQ
+		int *sc, tmp, *sumq;
+		sc   = alloca(n_types * sizeof(int));
+		sumq = alloca(n_types * sizeof(int));
+		memset(sumq, 0, sizeof(int) * n_types);
+		for (s = K = 0; s < n; ++s) {
+			for (i = 0; i < n_plp[s]; ++i, ++K) {
+				bam_pileup1_t *p = plp[s] + i;
+				int *sct = &score1[K*n_types], indelQ1, indelQ2, seqQ, indelQ;
+				for (t = 0; t < n_types; ++t) sc[t] = sct[t]<<6 | t;
+				for (t = 1; t < n_types; ++t) // insertion sort
+					for (j = t; j > 0 && sc[j] < sc[j-1]; --j)
+						tmp = sc[j], sc[j] = sc[j-1], sc[j-1] = tmp;
+				/* errmod_cal() assumes that if the call is wrong, the
+				 * likelihoods of other events are equal. This is about
+				 * right for substitutions, but is not desired for
+				 * indels. To reuse errmod_cal(), I have to make
+				 * compromise for multi-allelic indels.
+				 */
+				if ((sc[0]&0x3f) == ref_type) {
+					indelQ1 = (sc[1]>>14) - (sc[0]>>14);
+					seqQ = est_seqQ(bca, types[sc[1]&0x3f], l_run);
+				} else {
+					for (t = 0; t < n_types; ++t) // look for the reference type
+						if ((sc[t]&0x3f) == ref_type) break;
+					indelQ1 = (sc[t]>>14) - (sc[0]>>14);
+					seqQ = est_seqQ(bca, types[sc[0]&0x3f], l_run);
+				}
+				tmp = sc[0]>>6 & 0xff;
+				indelQ1 = tmp > 111? 0 : (int)((1. - tmp/111.) * indelQ1 + .499); // reduce indelQ
+				sct = &score2[K*n_types];
+				for (t = 0; t < n_types; ++t) sc[t] = sct[t]<<6 | t;
+				for (t = 1; t < n_types; ++t) // insertion sort
+					for (j = t; j > 0 && sc[j] < sc[j-1]; --j)
+						tmp = sc[j], sc[j] = sc[j-1], sc[j-1] = tmp;
+				if ((sc[0]&0x3f) == ref_type) {
+					indelQ2 = (sc[1]>>14) - (sc[0]>>14);
+				} else {
+					for (t = 0; t < n_types; ++t) // look for the reference type
+						if ((sc[t]&0x3f) == ref_type) break;
+					indelQ2 = (sc[t]>>14) - (sc[0]>>14);
+				}
+				tmp = sc[0]>>6 & 0xff;
+				indelQ2 = tmp > 111? 0 : (int)((1. - tmp/111.) * indelQ2 + .499);
+				// pick the smaller between indelQ1 and indelQ2
+				indelQ = indelQ1 < indelQ2? indelQ1 : indelQ2;
+				if (indelQ > 255) indelQ = 255;
+				if (seqQ > 255) seqQ = 255;
+				p->aux = (sc[0]&0x3f)<<16 | seqQ<<8 | indelQ; // use 22 bits in total
+				sumq[sc[0]&0x3f] += indelQ < seqQ? indelQ : seqQ;
+//				fprintf(stderr, "pos=%d read=%d:%d name=%s call=%d indelQ=%d seqQ=%d\n", pos, s, i, bam1_qname(p->b), types[sc[0]&0x3f], indelQ, seqQ);
+			}
+		}
+		// determine bca->indel_types[] and bca->inscns
+		bca->maxins = max_ins;
+		bca->inscns = realloc(bca->inscns, bca->maxins * 4);
+		for (t = 0; t < n_types; ++t)
+			sumq[t] = sumq[t]<<6 | t;
+		for (t = 1; t < n_types; ++t) // insertion sort
+			for (j = t; j > 0 && sumq[j] > sumq[j-1]; --j)
+				tmp = sumq[j], sumq[j] = sumq[j-1], sumq[j-1] = tmp;
+		for (t = 0; t < n_types; ++t) // look for the reference type
+			if ((sumq[t]&0x3f) == ref_type) break;
+		if (t) { // then move the reference type to the first
+			tmp = sumq[t];
+			for (; t > 0; --t) sumq[t] = sumq[t-1];
+			sumq[0] = tmp;
+		}
+		for (t = 0; t < 4; ++t) bca->indel_types[t] = B2B_INDEL_NULL;
+		for (t = 0; t < 4 && t < n_types; ++t) {
+			bca->indel_types[t] = types[sumq[t]&0x3f];
+			memcpy(&bca->inscns[t * bca->maxins], &inscns[(sumq[t]&0x3f) * max_ins], bca->maxins);
+		}
+		// update p->aux
+		for (s = n_alt = 0; s < n; ++s) {
+			for (i = 0; i < n_plp[s]; ++i) {
+				bam_pileup1_t *p = plp[s] + i;
+				int x = types[p->aux>>16&0x3f];
+				for (j = 0; j < 4; ++j)
+					if (x == bca->indel_types[j]) break;
+				p->aux = j<<16 | (j == 4? 0 : (p->aux&0xffff));
+				if ((p->aux>>16&0x3f) > 0) ++n_alt;
+//				fprintf(stderr, "X pos=%d read=%d:%d name=%s call=%d type=%d q=%d seqQ=%d\n", pos, s, i, bam1_qname(p->b), p->aux>>16&63, bca->indel_types[p->aux>>16&63], p->aux&0xff, p->aux>>8&0xff);
+			}
+		}		
+	}
+	free(score1); free(score2);
+	// free
+	for (i = 0; i < n; ++i) free(ref_sample[i]);
+	free(ref_sample);
+	free(types); free(inscns);
+	return n_alt > 0? 0 : -1;
+}