#include <assert.h>
+#include <ctype.h>
+#include <string.h>
#include "bam.h"
#include "bam2bcf.h"
#include "ksort.h"
#include "kaln.h"
-
-#define INDEL_DEBUG
+#include "kprobaln.h"
+#include "khash.h"
+KHASH_SET_INIT_STR(rg)
#define MINUS_CONST 0x10000000
#define INDEL_WINDOW_SIZE 50
-#define INDEL_BAD_SCORE 10000
+#define MIN_SUPPORT_COEF 500
+
+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)
{
*_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)l / l_run + .499) : 1000;
+ qh = l_run >= 3? (int)(bca->tandemQ * (double)abs(l) / l_run + .499) : 1000;
return q < qh? q : qh;
}
-int bcf_call_gap_prep(int n, int *n_plp, bam_pileup1_t **plp, int pos, bcf_callaux_t *bca, const char *ref)
+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;
+}
+
+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)
{
extern void ks_introsort_uint32_t(int, uint32_t*);
- int i, s, j, k, t, n_types, *types, max_rd_len, left, right, max_ins, *score, N, K, l_run, ref_type;
+ int i, s, j, k, t, n_types, *types, max_rd_len, left, right, max_ins, *score1, *score2;
+ int N, K, l_run, ref_type, n_alt;
char *inscns = 0, *ref2, *query;
+ 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 = 0; s < n; ++s) {
+ 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
- int m;
+ int m, n_alt = 0, n_tot = 0;
uint32_t *aux;
- aux = calloc(n + 1, 4);
+ aux = calloc(N + 1, 4);
m = max_rd_len = 0;
aux[m++] = MINUS_CONST; // zero indel is always a type
- for (s = N = 0; s < n; ++s) {
- N += n_plp[s]; // N is the total number of reads
+ for (s = 0; s < n; ++s) {
for (i = 0; i < n_plp[s]; ++i) {
const bam_pileup1_t *p = plp[s] + i;
- if (p->indel != 0)
- aux[m++] = MINUS_CONST + p->indel;
+ if (rghash == 0 || p->aux == 0) {
+ ++n_tot;
+ if (p->indel != 0) {
+ ++n_alt;
+ 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;
}
// squeeze out identical types
for (i = 1, n_types = 1; i < m; ++i)
if (aux[i] != aux[i-1]) ++n_types;
- assert(n_types > 1); // there must at least one type of non-reference indel
+ if (n_types == 1 || n_alt * MIN_SUPPORT_COEF < n_tot) { // no indels or too few supporting reads
+ free(aux); return -1;
+ }
types = (int*)calloc(n_types, sizeof(int));
t = 0;
types[t++] = aux[0] - MINUS_CONST;
// compute the likelihood given each type of indel for each read
ref2 = calloc(right - left + max_ins + 2, 1);
query = calloc(right - left + max_rd_len + max_ins + 2, 1);
- score = calloc(N * n_types, sizeof(int));
+ 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;
- ka_param2_t ap = ka_param2_qual;
- ap.band_width = abs(types[t]) + 3;
+ 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);
// write ref2
for (k = 0, j = left; j <= pos; ++j)
ref2[k++] = bam_nt16_nt4_table[bam_nt16_table[(int)ref[j]]];
bam_pileup1_t *p = plp[s] + i;
int qbeg, qend, tbeg, tend, sc;
uint8_t *seq = bam1_seq(p->b);
+ // 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);
- assert(tbeg >= left);
+ 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 alignment; this takes most of computing time for indel calling
-#ifdef INDEL_DEBUG
- for (sc = 0; sc < tend - tbeg + types[t]; ++sc)
- fputc("ACGTN"[(int)ref2[tbeg-left+sc]], stderr);
+ { // 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 (sc = 0; sc < qend - qbeg; ++sc) fputc("ACGTN"[(int)query[qbeg + sc]], stderr);
- fputc('\n', stderr);
-#endif
- sc = ka_global_score((uint8_t*)ref2 + tbeg - left, tend - tbeg + abs(types[t]),
- (uint8_t*)query + qbeg, qend - qbeg, &ap);
-#ifdef INDEL_DEBUG
- fprintf(stderr, "pos=%d type=%d read=%d:%d name=%s score=%d\n", pos, types[t], s, i, bam1_qname(p->b), sc);
-#endif
- score[K*n_types + t] = -sc;
+ 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);
- { // choose the top 4 indel types; reference must be included
- }
{ // compute indelQ
int *sc, tmp, *sumq;
sc = alloca(n_types * sizeof(int));
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 = &score[K*n_types], indelQ;
+ 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)
* compromise for multi-allelic indels.
*/
if ((sc[0]&0x3f) == ref_type) {
- indelQ = (sc[1]>>6) - (sc[0]>>6);
- tmp = est_seqQ(bca, types[sc[1]&0x3f], l_run);
+ 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;
- indelQ = (sc[t]>>6) - (sc[0]>>6);
- tmp = est_seqQ(bca, types[sc[0]&0x3f], l_run);
+ indelQ1 = (sc[t]>>14) - (sc[0]>>14);
+ seqQ = est_seqQ(bca, types[sc[0]&0x3f], l_run);
}
- if (indelQ > tmp) indelQ = tmp;
- if (indelQ > p->b->core.qual) indelQ = p->b->core.qual;
- if (indelQ > bca->capQ) indelQ = bca->capQ;
- p->aux = (sc[0]&0x3f)<<8 | indelQ;
- sumq[sc[0]&0x3f] += indelQ;
+ 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;
+ p->aux = (sc[0]&0x3f)<<16 | seqQ<<8 | indelQ;
+ sumq[sc[0]&0x3f] += indelQ < seqQ? indelQ : seqQ;
+// fprintf(stderr, "pos=%d read=%d:%d name=%s call=%d q=%d\n", pos, s, i, bam1_qname(p->b), types[sc[0]&0x3f], indelQ);
}
}
- // determine bca->indel_types[]
+ // 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)
+ 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;
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)
+ 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);
+ }
+ }
}
- // FIXME: to set the inserted sequence
- free(score);
+ free(score1); free(score2);
// free
free(types); free(inscns);
- return 0;
+ return n_alt > 0? 0 : -1;
}