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1 #include <assert.h>
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2 #include <ctype.h>
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3 #include <string.h>
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4 #include "bam.h"
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5 #include "bam2bcf.h"
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6 #include "ksort.h"
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7 #include "kaln.h"
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8 #include "kprobaln.h"
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9 #include "khash.h"
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10 KHASH_SET_INIT_STR(rg)
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11
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12 #define MINUS_CONST 0x10000000
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13 #define INDEL_WINDOW_SIZE 50
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14
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15 void *bcf_call_add_rg(void *_hash, const char *hdtext, const char *list)
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16 {
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17 const char *s, *p, *q, *r, *t;
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18 khash_t(rg) *hash;
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19 if (list == 0 || hdtext == 0) return _hash;
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20 if (_hash == 0) _hash = kh_init(rg);
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21 hash = (khash_t(rg)*)_hash;
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22 if ((s = strstr(hdtext, "@RG\t")) == 0) return hash;
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23 do {
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24 t = strstr(s + 4, "@RG\t"); // the next @RG
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25 if ((p = strstr(s, "\tID:")) != 0) p += 4;
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26 if ((q = strstr(s, "\tPL:")) != 0) q += 4;
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27 if (p && q && (t == 0 || (p < t && q < t))) { // ID and PL are both present
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28 int lp, lq;
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29 char *x;
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30 for (r = p; *r && *r != '\t' && *r != '\n'; ++r); lp = r - p;
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31 for (r = q; *r && *r != '\t' && *r != '\n'; ++r); lq = r - q;
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32 x = calloc((lp > lq? lp : lq) + 1, 1);
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33 for (r = q; *r && *r != '\t' && *r != '\n'; ++r) x[r-q] = *r;
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34 if (strstr(list, x)) { // insert ID to the hash table
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35 khint_t k;
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36 int ret;
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37 for (r = p; *r && *r != '\t' && *r != '\n'; ++r) x[r-p] = *r;
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38 x[r-p] = 0;
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39 k = kh_get(rg, hash, x);
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40 if (k == kh_end(hash)) k = kh_put(rg, hash, x, &ret);
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41 else free(x);
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42 } else free(x);
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43 }
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44 s = t;
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45 } while (s);
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46 return hash;
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47 }
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48
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49 void bcf_call_del_rghash(void *_hash)
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50 {
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51 khint_t k;
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52 khash_t(rg) *hash = (khash_t(rg)*)_hash;
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53 if (hash == 0) return;
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54 for (k = kh_begin(hash); k < kh_end(hash); ++k)
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55 if (kh_exist(hash, k))
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56 free((char*)kh_key(hash, k));
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57 kh_destroy(rg, hash);
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58 }
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59
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60 static int tpos2qpos(const bam1_core_t *c, const uint32_t *cigar, int32_t tpos, int is_left, int32_t *_tpos)
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61 {
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62 int k, x = c->pos, y = 0, last_y = 0;
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63 *_tpos = c->pos;
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64 for (k = 0; k < c->n_cigar; ++k) {
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65 int op = cigar[k] & BAM_CIGAR_MASK;
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66 int l = cigar[k] >> BAM_CIGAR_SHIFT;
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67 if (op == BAM_CMATCH) {
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68 if (c->pos > tpos) return y;
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69 if (x + l > tpos) {
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70 *_tpos = tpos;
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71 return y + (tpos - x);
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72 }
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73 x += l; y += l;
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74 last_y = y;
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75 } else if (op == BAM_CINS || op == BAM_CSOFT_CLIP) y += l;
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76 else if (op == BAM_CDEL || op == BAM_CREF_SKIP) {
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77 if (x + l > tpos) {
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78 *_tpos = is_left? x : x + l;
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79 return y;
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80 }
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81 x += l;
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82 }
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83 }
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84 *_tpos = x;
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85 return last_y;
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86 }
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87 // FIXME: check if the inserted sequence is consistent with the homopolymer run
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88 // l is the relative gap length and l_run is the length of the homopolymer on the reference
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89 static inline int est_seqQ(const bcf_callaux_t *bca, int l, int l_run)
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90 {
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91 int q, qh;
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92 q = bca->openQ + bca->extQ * (abs(l) - 1);
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93 qh = l_run >= 3? (int)(bca->tandemQ * (double)abs(l) / l_run + .499) : 1000;
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94 return q < qh? q : qh;
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95 }
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96
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97 static inline int est_indelreg(int pos, const char *ref, int l, char *ins4)
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98 {
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99 int i, j, max = 0, max_i = pos, score = 0;
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100 l = abs(l);
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101 for (i = pos + 1, j = 0; ref[i]; ++i, ++j) {
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102 if (ins4) score += (toupper(ref[i]) != "ACGTN"[(int)ins4[j%l]])? -10 : 1;
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103 else score += (toupper(ref[i]) != toupper(ref[pos+1+j%l]))? -10 : 1;
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104 if (score < 0) break;
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105 if (max < score) max = score, max_i = i;
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106 }
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107 return max_i - pos;
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108 }
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109
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110 int bcf_call_gap_prep(int n, int *n_plp, bam_pileup1_t **plp, int pos, bcf_callaux_t *bca, const char *ref,
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111 const void *rghash)
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112 {
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113 extern void ks_introsort_uint32_t(int, uint32_t*);
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114 int i, s, j, k, t, n_types, *types, max_rd_len, left, right, max_ins, *score1, *score2, max_ref2;
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115 int N, K, l_run, ref_type, n_alt;
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116 char *inscns = 0, *ref2, *query, **ref_sample;
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117 khash_t(rg) *hash = (khash_t(rg)*)rghash;
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118 if (ref == 0 || bca == 0) return -1;
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119 // mark filtered reads
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120 if (rghash) {
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121 N = 0;
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122 for (s = N = 0; s < n; ++s) {
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123 for (i = 0; i < n_plp[s]; ++i) {
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124 bam_pileup1_t *p = plp[s] + i;
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125 const uint8_t *rg = bam_aux_get(p->b, "RG");
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126 p->aux = 1; // filtered by default
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127 if (rg) {
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128 khint_t k = kh_get(rg, hash, (const char*)(rg + 1));
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129 if (k != kh_end(hash)) p->aux = 0, ++N; // not filtered
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130 }
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131 }
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132 }
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133 if (N == 0) return -1; // no reads left
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134 }
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135 // determine if there is a gap
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136 for (s = N = 0; s < n; ++s) {
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137 for (i = 0; i < n_plp[s]; ++i)
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138 if (plp[s][i].indel != 0) break;
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139 if (i < n_plp[s]) break;
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140 }
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141 if (s == n) return -1; // there is no indel at this position.
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142 for (s = N = 0; s < n; ++s) N += n_plp[s]; // N is the total number of reads
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143 { // find out how many types of indels are present
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144 int m, n_alt = 0, n_tot = 0;
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145 uint32_t *aux;
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146 aux = calloc(N + 1, 4);
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147 m = max_rd_len = 0;
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148 aux[m++] = MINUS_CONST; // zero indel is always a type
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149 for (s = 0; s < n; ++s) {
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150 for (i = 0; i < n_plp[s]; ++i) {
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151 const bam_pileup1_t *p = plp[s] + i;
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152 if (rghash == 0 || p->aux == 0) {
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153 ++n_tot;
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154 if (p->indel != 0) {
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155 ++n_alt;
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156 aux[m++] = MINUS_CONST + p->indel;
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157 }
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158 }
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159 j = bam_cigar2qlen(&p->b->core, bam1_cigar(p->b));
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160 if (j > max_rd_len) max_rd_len = j;
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161 }
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162 }
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163 ks_introsort(uint32_t, m, aux);
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164 // squeeze out identical types
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165 for (i = 1, n_types = 1; i < m; ++i)
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166 if (aux[i] != aux[i-1]) ++n_types;
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167 if (n_types == 1 || (double)n_alt / n_tot < bca->min_frac || n_alt < bca->min_support) { // then skip
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168 free(aux); return -1;
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169 }
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170 types = (int*)calloc(n_types, sizeof(int));
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171 t = 0;
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172 types[t++] = aux[0] - MINUS_CONST;
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173 for (i = 1; i < m; ++i)
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174 if (aux[i] != aux[i-1])
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175 types[t++] = aux[i] - MINUS_CONST;
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176 free(aux);
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177 for (t = 0; t < n_types; ++t)
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178 if (types[t] == 0) break;
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179 ref_type = t; // the index of the reference type (0)
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180 assert(n_types < 64);
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181 }
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182 { // calculate left and right boundary
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183 left = pos > INDEL_WINDOW_SIZE? pos - INDEL_WINDOW_SIZE : 0;
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184 right = pos + INDEL_WINDOW_SIZE;
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185 if (types[0] < 0) right -= types[0];
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186 // in case the alignments stand out the reference
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187 for (i = pos; i < right; ++i)
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188 if (ref[i] == 0) break;
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189 right = i;
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190 }
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191 /* The following block fixes a long-existing flaw in the INDEL
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192 * calling model: the interference of nearby SNPs. However, it also
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193 * reduces the power because sometimes, substitutions caused by
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194 * indels are not distinguishable from true mutations. Multiple
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195 * sequence realignment helps to increase the power.
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196 */
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197 { // construct per-sample consensus
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198 int L = right - left + 1, max_i, max2_i;
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199 uint32_t *cns, max, max2;
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200 char *ref0, *r;
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201 ref_sample = calloc(n, sizeof(void*));
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202 cns = calloc(L, 4);
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203 ref0 = calloc(L, 1);
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204 for (i = 0; i < right - left; ++i)
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205 ref0[i] = bam_nt16_table[(int)ref[i+left]];
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206 for (s = 0; s < n; ++s) {
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207 r = ref_sample[s] = calloc(L, 1);
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208 memset(cns, 0, sizeof(int) * L);
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209 // collect ref and non-ref counts
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210 for (i = 0; i < n_plp[s]; ++i) {
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211 bam_pileup1_t *p = plp[s] + i;
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212 bam1_t *b = p->b;
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213 uint32_t *cigar = bam1_cigar(b);
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214 uint8_t *seq = bam1_seq(b);
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215 int x = b->core.pos, y = 0;
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216 for (k = 0; k < b->core.n_cigar; ++k) {
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217 int op = cigar[k]&0xf;
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218 int j, l = cigar[k]>>4;
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219 if (op == BAM_CMATCH) {
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220 for (j = 0; j < l; ++j)
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221 if (x + j >= left && x + j < right)
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222 cns[x+j-left] += (bam1_seqi(seq, y+j) == ref0[x+j-left])? 1 : 0x10000;
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223 x += l; y += l;
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224 } else if (op == BAM_CDEL || op == BAM_CREF_SKIP) x += l;
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225 else if (op == BAM_CINS || op == BAM_CSOFT_CLIP) y += l;
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226 }
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227 }
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228 // determine the consensus
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229 for (i = 0; i < right - left; ++i) r[i] = ref0[i];
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230 max = max2 = 0; max_i = max2_i = -1;
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231 for (i = 0; i < right - left; ++i) {
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232 if (cns[i]>>16 >= max>>16) max2 = max, max2_i = max_i, max = cns[i], max_i = i;
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233 else if (cns[i]>>16 >= max2>>16) max2 = cns[i], max2_i = i;
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234 }
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235 if ((double)(max&0xffff) / ((max&0xffff) + (max>>16)) >= 0.7) max_i = -1;
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236 if ((double)(max2&0xffff) / ((max2&0xffff) + (max2>>16)) >= 0.7) max2_i = -1;
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237 if (max_i >= 0) r[max_i] = 15;
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238 if (max2_i >= 0) r[max2_i] = 15;
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239 // for (i = 0; i < right - left; ++i) fputc("=ACMGRSVTWYHKDBN"[(int)r[i]], stderr); fputc('\n', stderr);
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240 }
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241 free(ref0); free(cns);
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242 }
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243 { // the length of the homopolymer run around the current position
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244 int c = bam_nt16_table[(int)ref[pos + 1]];
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245 if (c == 15) l_run = 1;
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246 else {
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247 for (i = pos + 2; ref[i]; ++i)
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248 if (bam_nt16_table[(int)ref[i]] != c) break;
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249 l_run = i;
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250 for (i = pos; i >= 0; --i)
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251 if (bam_nt16_table[(int)ref[i]] != c) break;
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252 l_run -= i + 1;
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253 }
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254 }
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255 // construct the consensus sequence
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256 max_ins = types[n_types - 1]; // max_ins is at least 0
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257 if (max_ins > 0) {
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258 int *inscns_aux = calloc(4 * n_types * max_ins, sizeof(int));
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259 // count the number of occurrences of each base at each position for each type of insertion
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260 for (t = 0; t < n_types; ++t) {
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261 if (types[t] > 0) {
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262 for (s = 0; s < n; ++s) {
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263 for (i = 0; i < n_plp[s]; ++i) {
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264 bam_pileup1_t *p = plp[s] + i;
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265 if (p->indel == types[t]) {
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266 uint8_t *seq = bam1_seq(p->b);
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267 for (k = 1; k <= p->indel; ++k) {
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268 int c = bam_nt16_nt4_table[bam1_seqi(seq, p->qpos + k)];
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269 if (c < 4) ++inscns_aux[(t*max_ins+(k-1))*4 + c];
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270 }
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271 }
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272 }
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273 }
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274 }
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275 }
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276 // use the majority rule to construct the consensus
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277 inscns = calloc(n_types * max_ins, 1);
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278 for (t = 0; t < n_types; ++t) {
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279 for (j = 0; j < types[t]; ++j) {
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280 int max = 0, max_k = -1, *ia = &inscns_aux[(t*max_ins+j)*4];
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281 for (k = 0; k < 4; ++k)
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282 if (ia[k] > max)
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283 max = ia[k], max_k = k;
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284 inscns[t*max_ins + j] = max? max_k : 4;
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285 }
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286 }
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287 free(inscns_aux);
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288 }
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289 // compute the likelihood given each type of indel for each read
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290 max_ref2 = right - left + 2 + 2 * (max_ins > -types[0]? max_ins : -types[0]);
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291 ref2 = calloc(max_ref2, 1);
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292 query = calloc(right - left + max_rd_len + max_ins + 2, 1);
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293 score1 = calloc(N * n_types, sizeof(int));
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294 score2 = calloc(N * n_types, sizeof(int));
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295 bca->indelreg = 0;
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296 for (t = 0; t < n_types; ++t) {
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297 int l, ir;
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298 kpa_par_t apf1 = { 1e-4, 1e-2, 10 }, apf2 = { 1e-6, 1e-3, 10 };
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299 apf1.bw = apf2.bw = abs(types[t]) + 3;
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300 // compute indelreg
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301 if (types[t] == 0) ir = 0;
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302 else if (types[t] > 0) ir = est_indelreg(pos, ref, types[t], &inscns[t*max_ins]);
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303 else ir = est_indelreg(pos, ref, -types[t], 0);
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304 if (ir > bca->indelreg) bca->indelreg = ir;
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305 // fprintf(stderr, "%d, %d, %d\n", pos, types[t], ir);
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306 // realignment
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307 for (s = K = 0; s < n; ++s) {
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308 // write ref2
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309 for (k = 0, j = left; j <= pos; ++j)
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310 ref2[k++] = bam_nt16_nt4_table[(int)ref_sample[s][j-left]];
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311 if (types[t] <= 0) j += -types[t];
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312 else for (l = 0; l < types[t]; ++l)
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313 ref2[k++] = inscns[t*max_ins + l];
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314 for (; j < right && ref[j]; ++j)
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315 ref2[k++] = bam_nt16_nt4_table[(int)ref_sample[s][j-left]];
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316 for (; k < max_ref2; ++k) ref2[k] = 4;
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317 if (j < right) right = j;
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318 // align each read to ref2
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319 for (i = 0; i < n_plp[s]; ++i, ++K) {
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320 bam_pileup1_t *p = plp[s] + i;
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321 int qbeg, qend, tbeg, tend, sc, kk;
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322 uint8_t *seq = bam1_seq(p->b);
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323 uint32_t *cigar = bam1_cigar(p->b);
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324 if (p->b->core.flag&4) continue; // unmapped reads
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325 // FIXME: the following loop should be better moved outside; nonetheless, realignment should be much slower anyway.
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326 for (kk = 0; kk < p->b->core.n_cigar; ++kk)
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327 if ((cigar[kk]&BAM_CIGAR_MASK) == BAM_CREF_SKIP) break;
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328 if (kk < p->b->core.n_cigar) continue;
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329 // FIXME: the following skips soft clips, but using them may be more sensitive.
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330 // determine the start and end of sequences for alignment
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331 qbeg = tpos2qpos(&p->b->core, bam1_cigar(p->b), left, 0, &tbeg);
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332 qend = tpos2qpos(&p->b->core, bam1_cigar(p->b), right, 1, &tend);
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333 if (types[t] < 0) {
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334 int l = -types[t];
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335 tbeg = tbeg - l > left? tbeg - l : left;
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336 }
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337 // write the query sequence
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338 for (l = qbeg; l < qend; ++l)
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339 query[l - qbeg] = bam_nt16_nt4_table[bam1_seqi(seq, l)];
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340 { // do realignment; this is the bottleneck
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341 const uint8_t *qual = bam1_qual(p->b), *bq;
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342 uint8_t *qq;
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343 qq = calloc(qend - qbeg, 1);
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344 bq = (uint8_t*)bam_aux_get(p->b, "ZQ");
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345 if (bq) ++bq; // skip type
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346 for (l = qbeg; l < qend; ++l) {
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347 qq[l - qbeg] = bq? qual[l] + (bq[l] - 64) : qual[l];
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348 if (qq[l - qbeg] > 30) qq[l - qbeg] = 30;
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349 if (qq[l - qbeg] < 7) qq[l - qbeg] = 7;
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350 }
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351 sc = kpa_glocal((uint8_t*)ref2 + tbeg - left, tend - tbeg + abs(types[t]),
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352 (uint8_t*)query, qend - qbeg, qq, &apf1, 0, 0);
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353 l = (int)(100. * sc / (qend - qbeg) + .499); // used for adjusting indelQ below
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354 if (l > 255) l = 255;
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355 score1[K*n_types + t] = score2[K*n_types + t] = sc<<8 | l;
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356 if (sc > 5) {
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357 sc = kpa_glocal((uint8_t*)ref2 + tbeg - left, tend - tbeg + abs(types[t]),
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358 (uint8_t*)query, qend - qbeg, qq, &apf2, 0, 0);
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359 l = (int)(100. * sc / (qend - qbeg) + .499);
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360 if (l > 255) l = 255;
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361 score2[K*n_types + t] = sc<<8 | l;
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362 }
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363 free(qq);
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364 }
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365 /*
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366 for (l = 0; l < tend - tbeg + abs(types[t]); ++l)
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367 fputc("ACGTN"[(int)ref2[tbeg-left+l]], stderr);
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368 fputc('\n', stderr);
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369 for (l = 0; l < qend - qbeg; ++l) fputc("ACGTN"[(int)query[l]], stderr);
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370 fputc('\n', stderr);
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371 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);
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372 */
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373 }
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374 }
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375 }
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376 free(ref2); free(query);
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377 { // compute indelQ
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378 int *sc, tmp, *sumq;
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379 sc = alloca(n_types * sizeof(int));
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380 sumq = alloca(n_types * sizeof(int));
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381 memset(sumq, 0, sizeof(int) * n_types);
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382 for (s = K = 0; s < n; ++s) {
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383 for (i = 0; i < n_plp[s]; ++i, ++K) {
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384 bam_pileup1_t *p = plp[s] + i;
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385 int *sct = &score1[K*n_types], indelQ1, indelQ2, seqQ, indelQ;
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386 for (t = 0; t < n_types; ++t) sc[t] = sct[t]<<6 | t;
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387 for (t = 1; t < n_types; ++t) // insertion sort
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388 for (j = t; j > 0 && sc[j] < sc[j-1]; --j)
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389 tmp = sc[j], sc[j] = sc[j-1], sc[j-1] = tmp;
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390 /* errmod_cal() assumes that if the call is wrong, the
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391 * likelihoods of other events are equal. This is about
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392 * right for substitutions, but is not desired for
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393 * indels. To reuse errmod_cal(), I have to make
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394 * compromise for multi-allelic indels.
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395 */
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396 if ((sc[0]&0x3f) == ref_type) {
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397 indelQ1 = (sc[1]>>14) - (sc[0]>>14);
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398 seqQ = est_seqQ(bca, types[sc[1]&0x3f], l_run);
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399 } else {
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400 for (t = 0; t < n_types; ++t) // look for the reference type
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401 if ((sc[t]&0x3f) == ref_type) break;
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402 indelQ1 = (sc[t]>>14) - (sc[0]>>14);
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403 seqQ = est_seqQ(bca, types[sc[0]&0x3f], l_run);
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404 }
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405 tmp = sc[0]>>6 & 0xff;
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406 indelQ1 = tmp > 111? 0 : (int)((1. - tmp/111.) * indelQ1 + .499); // reduce indelQ
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407 sct = &score2[K*n_types];
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408 for (t = 0; t < n_types; ++t) sc[t] = sct[t]<<6 | t;
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409 for (t = 1; t < n_types; ++t) // insertion sort
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410 for (j = t; j > 0 && sc[j] < sc[j-1]; --j)
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411 tmp = sc[j], sc[j] = sc[j-1], sc[j-1] = tmp;
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412 if ((sc[0]&0x3f) == ref_type) {
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413 indelQ2 = (sc[1]>>14) - (sc[0]>>14);
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414 } else {
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415 for (t = 0; t < n_types; ++t) // look for the reference type
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416 if ((sc[t]&0x3f) == ref_type) break;
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417 indelQ2 = (sc[t]>>14) - (sc[0]>>14);
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418 }
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419 tmp = sc[0]>>6 & 0xff;
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420 indelQ2 = tmp > 111? 0 : (int)((1. - tmp/111.) * indelQ2 + .499);
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421 // pick the smaller between indelQ1 and indelQ2
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422 indelQ = indelQ1 < indelQ2? indelQ1 : indelQ2;
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423 if (indelQ > 255) indelQ = 255;
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424 if (seqQ > 255) seqQ = 255;
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425 p->aux = (sc[0]&0x3f)<<16 | seqQ<<8 | indelQ; // use 22 bits in total
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426 sumq[sc[0]&0x3f] += indelQ < seqQ? indelQ : seqQ;
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427 // 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);
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428 }
|
|
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429 }
|
|
|
430 // determine bca->indel_types[] and bca->inscns
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|
|
431 bca->maxins = max_ins;
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|
432 bca->inscns = realloc(bca->inscns, bca->maxins * 4);
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433 for (t = 0; t < n_types; ++t)
|
|
|
434 sumq[t] = sumq[t]<<6 | t;
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|
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435 for (t = 1; t < n_types; ++t) // insertion sort
|
|
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436 for (j = t; j > 0 && sumq[j] > sumq[j-1]; --j)
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|
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437 tmp = sumq[j], sumq[j] = sumq[j-1], sumq[j-1] = tmp;
|
|
|
438 for (t = 0; t < n_types; ++t) // look for the reference type
|
|
|
439 if ((sumq[t]&0x3f) == ref_type) break;
|
|
|
440 if (t) { // then move the reference type to the first
|
|
|
441 tmp = sumq[t];
|
|
|
442 for (; t > 0; --t) sumq[t] = sumq[t-1];
|
|
|
443 sumq[0] = tmp;
|
|
|
444 }
|
|
|
445 for (t = 0; t < 4; ++t) bca->indel_types[t] = B2B_INDEL_NULL;
|
|
|
446 for (t = 0; t < 4 && t < n_types; ++t) {
|
|
|
447 bca->indel_types[t] = types[sumq[t]&0x3f];
|
|
|
448 memcpy(&bca->inscns[t * bca->maxins], &inscns[(sumq[t]&0x3f) * max_ins], bca->maxins);
|
|
|
449 }
|
|
|
450 // update p->aux
|
|
|
451 for (s = n_alt = 0; s < n; ++s) {
|
|
|
452 for (i = 0; i < n_plp[s]; ++i) {
|
|
|
453 bam_pileup1_t *p = plp[s] + i;
|
|
|
454 int x = types[p->aux>>16&0x3f];
|
|
|
455 for (j = 0; j < 4; ++j)
|
|
|
456 if (x == bca->indel_types[j]) break;
|
|
|
457 p->aux = j<<16 | (j == 4? 0 : (p->aux&0xffff));
|
|
|
458 if ((p->aux>>16&0x3f) > 0) ++n_alt;
|
|
|
459 // 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);
|
|
|
460 }
|
|
|
461 }
|
|
|
462 }
|
|
|
463 free(score1); free(score2);
|
|
|
464 // free
|
|
|
465 for (i = 0; i < n; ++i) free(ref_sample[i]);
|
|
|
466 free(ref_sample);
|
|
|
467 free(types); free(inscns);
|
|
|
468 return n_alt > 0? 0 : -1;
|
|
|
469 }
|
