/* The MIT License Copyright (c) 2008, 2018 Genome Research Ltd (GRL). 2011 Heng Li Permission is hereby granted, free of charge, to any person obtaining a copy of this software and associated documentation files (the "Software"), to deal in the Software without restriction, including without limitation the rights to use, copy, modify, merge, publish, distribute, sublicense, and/or sell copies of the Software, and to permit persons to whom the Software is furnished to do so, subject to the following conditions: The above copyright notice and this permission notice shall be included in all copies or substantial portions of the Software. THE SOFTWARE IS PROVIDED "AS IS", WITHOUT WARRANTY OF ANY KIND, EXPRESS OR IMPLIED, INCLUDING BUT NOT LIMITED TO THE WARRANTIES OF MERCHANTABILITY, FITNESS FOR A PARTICULAR PURPOSE AND NONINFRINGEMENT. IN NO EVENT SHALL THE AUTHORS OR COPYRIGHT HOLDERS BE LIABLE FOR ANY CLAIM, DAMAGES OR OTHER LIABILITY, WHETHER IN AN ACTION OF CONTRACT, TORT OR OTHERWISE, ARISING FROM, OUT OF OR IN CONNECTION WITH THE SOFTWARE OR THE USE OR OTHER DEALINGS IN THE SOFTWARE. */ /* This program is separated from maq's read simulator with Colin * Hercus' modification to allow longer indels. */ #include #include #include #include #include #include #include #include #include #include #include #include #include "../version.h" #include "htslib/kseq.h" #include "htslib/hts_os.h" KSEQ_INIT(gzFile, gzread) const uint8_t nst_nt4_table[256] = { 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 5 /*'-'*/, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 0, 4, 1, 4, 4, 4, 2, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 3, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 0, 4, 1, 4, 4, 4, 2, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 3, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4 }; /* Simple normal random number generator, copied from genran.c */ double ran_normal(void) { static int iset = 0; static double gset; double fac, rsq, v1, v2; if (iset == 0) { do { v1 = 2.0 * drand48() - 1.0; v2 = 2.0 * drand48() - 1.0; rsq = v1 * v1 + v2 * v2; } while (rsq >= 1.0 || rsq == 0.0); fac = sqrt(-2.0 * log(rsq) / rsq); gset = v1 * fac; iset = 1; return v2 * fac; } else { iset = 0; return gset; } } /* wgsim */ enum muttype_t {NOCHANGE = 0, INSERT = 0x1000, SUBSTITUTE = 0xe000, DELETE = 0xf000}; typedef unsigned short mut_t; static mut_t mutmsk = (mut_t)0xf000; typedef struct { int l, m; /* length and maximum buffer size */ mut_t *s; /* sequence */ } mutseq_t; static double ERR_RATE = 0.02; static double MUT_RATE = 0.001; static double INDEL_FRAC = 0.15; static double INDEL_EXTEND = 0.3; static double MAX_N_RATIO = 0.05; void wgsim_mut_diref(const kseq_t *ks, int is_hap, mutseq_t *hap1, mutseq_t *hap2) { int i, deleting = 0; mutseq_t *ret[2]; ret[0] = hap1; ret[1] = hap2; ret[0]->l = ks->seq.l; ret[1]->l = ks->seq.l; ret[0]->m = ks->seq.m; ret[1]->m = ks->seq.m; ret[0]->s = (mut_t *)calloc(ks->seq.m, sizeof(mut_t)); ret[1]->s = (mut_t *)calloc(ks->seq.m, sizeof(mut_t)); for (i = 0; i != ks->seq.l; ++i) { int c; c = ret[0]->s[i] = ret[1]->s[i] = (mut_t)nst_nt4_table[(int)ks->seq.s[i]]; if (deleting) { if (drand48() < INDEL_EXTEND) { if (deleting & 1) ret[0]->s[i] |= DELETE; if (deleting & 2) ret[1]->s[i] |= DELETE; continue; } else deleting = 0; } if (c < 4 && drand48() < MUT_RATE) { // mutation if (drand48() >= INDEL_FRAC) { // substitution double r = drand48(); c = (c + (int)(r * 3.0 + 1)) & 3; if (is_hap || drand48() < 0.333333) { // hom ret[0]->s[i] = ret[1]->s[i] = SUBSTITUTE|c; } else { // het ret[drand48()<0.5?0:1]->s[i] = SUBSTITUTE|c; } } else { // indel if (drand48() < 0.5) { // deletion if (is_hap || drand48() < 0.333333) { // hom-del ret[0]->s[i] = ret[1]->s[i] = DELETE; deleting = 3; } else { // het-del deleting = drand48()<0.5?1:2; ret[deleting-1]->s[i] = DELETE; } } else { // insertion int num_ins = 0, ins = 0; do { num_ins++; ins = (ins << 2) | (int)(drand48() * 4.0); } while (num_ins < 4 && drand48() < INDEL_EXTEND); if (is_hap || drand48() < 0.333333) { // hom-ins ret[0]->s[i] = ret[1]->s[i] = (num_ins << 12) | (ins << 4) | c; } else { // het-ins ret[drand48()<0.5?0:1]->s[i] = (num_ins << 12) | (ins << 4) | c; } } } } } } void wgsim_print_mutref(const char *name, const kseq_t *ks, mutseq_t *hap1, mutseq_t *hap2) { int i, j = 0; // j keeps the end of the last deletion for (i = 0; i != ks->seq.l; ++i) { int c[3]; c[0] = nst_nt4_table[(int)ks->seq.s[i]]; c[1] = hap1->s[i]; c[2] = hap2->s[i]; if (c[0] >= 4) continue; if ((c[1] & mutmsk) != NOCHANGE || (c[2] & mutmsk) != NOCHANGE) { if (c[1] == c[2]) { // hom if ((c[1]&mutmsk) == SUBSTITUTE) { // substitution printf("%s\t%d\t%c\t%c\t-\n", name, i+1, "ACGTN"[c[0]], "ACGTN"[c[1]&0xf]); } else if ((c[1]&mutmsk) == DELETE) { // del if (i >= j) { printf("%s\t%d\t", name, i+1); for (j = i; j < ks->seq.l && hap1->s[j] == hap2->s[j] && (hap1->s[j]&mutmsk) == DELETE; ++j) putchar("ACGTN"[nst_nt4_table[(int)ks->seq.s[j]]]); printf("\t-\t-\n"); } } else if (((c[1] & mutmsk) >> 12) <= 4) { // ins printf("%s\t%d\t-\t", name, i+1); int n = (c[1]&mutmsk) >> 12, ins = c[1] >> 4; while (n > 0) { putchar("ACGTN"[ins & 0x3]); ins >>= 2; n--; } printf("\t-\n"); } // else: deleted base in a long deletion } else { // het if ((c[1]&mutmsk) == SUBSTITUTE || (c[2]&mutmsk) == SUBSTITUTE) { // substitution printf("%s\t%d\t%c\t%c\t+\n", name, i+1, "ACGTN"[c[0]], "XACMGRSVTWYHKDBN"[1<<(c[1]&0x3)|1<<(c[2]&0x3)]); } else if ((c[1]&mutmsk) == DELETE) { if (i >= j) { printf("%s\t%d\t", name, i+1); for (j = i; j < ks->seq.l && hap1->s[j] != hap2->s[j] && (hap1->s[j]&mutmsk) == DELETE; ++j) putchar("ACGTN"[nst_nt4_table[(int)ks->seq.s[j]]]); printf("\t-\t-\n"); } } else if ((c[2]&mutmsk) == DELETE) { if (i >= j) { printf("%s\t%d\t", name, i+1); for (j = i; j < ks->seq.l && hap1->s[j] != hap2->s[j] && (hap2->s[j]&mutmsk) == DELETE; ++j) putchar("ACGTN"[nst_nt4_table[(int)ks->seq.s[j]]]); printf("\t-\t-\n"); } } else if (((c[1] & mutmsk) >> 12) <= 4 && ((c[1] & mutmsk) >> 12) > 0) { // ins1 printf("%s\t%d\t-\t", name, i+1); int n = (c[1]&mutmsk) >> 12, ins = c[1] >> 4; while (n > 0) { putchar("ACGTN"[ins & 0x3]); ins >>= 2; n--; } printf("\t+\n"); } else if (((c[2] & mutmsk) >> 12) <= 4 || ((c[2] & mutmsk) >> 12) > 0) { // ins2 printf("%s\t%d\t-\t", name, i+1); int n = (c[2]&mutmsk) >> 12, ins = c[2] >> 4; while (n > 0) { putchar("ACGTN"[ins & 0x3]); ins >>= 2; n--; } printf("\t+\n"); } // else: deleted base in a long deletion } } } } void wgsim_core(FILE *fpout1, FILE *fpout2, const char *fn, int is_hap, uint64_t N, int dist, int std_dev, int size_l, int size_r) { kseq_t *ks; mutseq_t rseq[2]; gzFile fp_fa; uint64_t tot_len, ii; int i, l, n_ref; char *qstr; int size[2], Q, max_size; uint8_t *tmp_seq[2]; mut_t *target; int max_loop, max_loop_err = 0; fp_fa = gzopen(fn, "r"); if (fp_fa == NULL) { fprintf(stderr, "[wgsim] can't open '%s': %s\n", fn, strerror(errno)); return; } l = size_l > size_r? size_l : size_r; qstr = (char*)calloc(l+1, 1); tmp_seq[0] = (uint8_t*)calloc(l+2, 1); tmp_seq[1] = (uint8_t*)calloc(l+2, 1); size[0] = size_l; size[1] = size_r; max_size = size_l > size_r? size_l : size_r; Q = (ERR_RATE == 0.0)? 'I' : (int)(-10.0 * log(ERR_RATE) / log(10.0) + 0.499) + 33; ks = kseq_init(fp_fa); tot_len = n_ref = 0; fprintf(stderr, "[%s] calculating the total length of the reference sequence...\n", __func__); while ((l = kseq_read(ks)) >= 0) { tot_len += l; ++n_ref; } fprintf(stderr, "[%s] %d sequences, total length: %llu\n", __func__, n_ref, (long long)tot_len); kseq_destroy(ks); gzclose(fp_fa); fp_fa = gzopen(fn, "r"); ks = kseq_init(fp_fa); while ((l = kseq_read(ks)) >= 0) { uint64_t n_pairs = (uint64_t)((long double)l / tot_len * N + 0.5); if (l < dist + 3 * std_dev) { fprintf(stderr, "[%s] skip sequence '%s' as it is shorter than %d!\n", __func__, ks->name.s, dist + 3 * std_dev); continue; } // generate mutations and print them out wgsim_mut_diref(ks, is_hap, rseq, rseq+1); wgsim_print_mutref(ks->name.s, ks, rseq, rseq+1); for (ii = 0; max_loop = 1000, ii != n_pairs; ++ii) { // the core loop double ran; int d, pos, s[2], is_flip; int n_sub[2], n_indel[2], n_err[2], ext_coor[2], j, k; FILE *fpo[2]; try_again: is_flip = 0; do { // avoid boundary failure ran = ran_normal(); ran = ran * std_dev + dist; d = (int)(ran + 0.5); d = d > max_size? d : max_size; pos = (int)((l - d + 1) * drand48()); } while (pos < 0 || pos >= ks->seq.l || pos + d - 1 >= ks->seq.l); // flip or not if (drand48() < 0.5) { fpo[0] = fpout1; fpo[1] = fpout2; s[0] = size[0]; s[1] = size[1]; } else { fpo[1] = fpout1; fpo[0] = fpout2; s[1] = size[0]; s[0] = size[1]; is_flip = 1; } // generate the read sequences target = rseq[drand48()<0.5?0:1].s; // haplotype from which the reads are generated n_sub[0] = n_sub[1] = n_indel[0] = n_indel[1] = n_err[0] = n_err[1] = 0; // forward read for (i = pos, k = 0, ext_coor[0] = -10; i >= 0 && i < ks->seq.l && k < s[0]; ++i) { int c = target[i], mut_type = c & mutmsk; if (ext_coor[0] < 0) { if (mut_type != NOCHANGE && mut_type != SUBSTITUTE) continue; ext_coor[0] = i; } if (mut_type == DELETE) ++n_indel[0]; else if (mut_type == NOCHANGE || mut_type == SUBSTITUTE) { tmp_seq[0][k++] = c & 0xf; if (mut_type == SUBSTITUTE) ++n_sub[0]; } else { int n, ins; ++n_indel[0]; tmp_seq[0][k++] = c & 0xf; for (n = mut_type>>12, ins = c>>4; n > 0 && k < s[0]; --n, ins >>= 2) tmp_seq[0][k++] = ins & 0x3; } } if (k != s[0]) ext_coor[0] = -10; // reverse read for (i = pos + d - 1, k = 0, ext_coor[1] = -10; i >= 0 && i < ks->seq.l && k < s[1]; --i) { int c = target[i], mut_type = c & mutmsk; if (ext_coor[1] < 0) { if (mut_type != NOCHANGE && mut_type != SUBSTITUTE) continue; ext_coor[1] = i; } if (mut_type == DELETE) ++n_indel[1]; else if (mut_type == NOCHANGE || mut_type == SUBSTITUTE) { tmp_seq[1][k++] = c & 0xf; if (mut_type == SUBSTITUTE) ++n_sub[1]; } else { int n, ins; ++n_indel[1]; for (n = mut_type>>12, ins = c>>4; n > 0 && k < s[1];) tmp_seq[1][k++] = (ins>>(2*--n)) & 0x3; tmp_seq[1][k++] = c & 0xf; } } if (k != s[1]) ext_coor[1] = -10; for (k = 0; k < s[1]; ++k) tmp_seq[1][k] = tmp_seq[1][k] < 4? 3 - tmp_seq[1][k] : 4; // complement if (ext_coor[0] < 0 || ext_coor[1] < 0) { // fail to generate the read(s) --ii; continue; } // generate sequencing errors for (j = 0; j < 2; ++j) { int n_n = 0; for (i = 0; i < s[j]; ++i) { int c = tmp_seq[j][i]; if (c >= 4) { // actually c should be never larger than 4 if everything is correct c = 4; ++n_n; } else if (drand48() < ERR_RATE) { // c = (c + (int)(drand48() * 3.0 + 1)) & 3; // random sequencing errors c = (c + 1) & 3; // recurrent sequencing errors ++n_err[j]; } tmp_seq[j][i] = c; } if ((double)n_n / s[j] > MAX_N_RATIO) break; } if (j < 2) { // too many ambiguous bases on one of the reads if (max_loop--) goto try_again; if (!max_loop_err) { fprintf(stderr, "Failed to produce a sequence with insufficient Ns. " "Omitting some sequence-pairs\n"); max_loop_err = 1; } continue; } // print for (j = 0; j < 2; ++j) { for (i = 0; i < s[j]; ++i) qstr[i] = Q; qstr[i] = 0; fprintf(fpo[j], "@%s_%u_%u_%d:%d:%d_%d:%d:%d_%llx/%d\n", ks->name.s, ext_coor[0]+1, ext_coor[1]+1, n_err[0], n_sub[0], n_indel[0], n_err[1], n_sub[1], n_indel[1], (long long)ii, j==0? is_flip+1 : 2-is_flip); for (i = 0; i < s[j]; ++i) fputc("ACGTN"[(int)tmp_seq[j][i]], fpo[j]); fprintf(fpo[j], "\n+\n%s\n", qstr); } } free(rseq[0].s); free(rseq[1].s); } kseq_destroy(ks); gzclose(fp_fa); free(qstr); free(tmp_seq[0]); free(tmp_seq[1]); } static int simu_usage(void) { fprintf(stderr, "\n"); fprintf(stderr, "Program: wgsim (short read simulator)\n"); fprintf(stderr, "Version: %s\n", SAMTOOLS_VERSION); fprintf(stderr, "Contact: Heng Li \n\n"); fprintf(stderr, "Usage: wgsim [options] \n\n"); fprintf(stderr, "Options: -e FLOAT base error rate [%.3f]\n", ERR_RATE); fprintf(stderr, " -d INT outer distance between the two ends [500]\n"); fprintf(stderr, " -s INT standard deviation [50]\n"); fprintf(stderr, " -N INT number of read pairs [1000000]\n"); fprintf(stderr, " -1 INT length of the first read [70]\n"); fprintf(stderr, " -2 INT length of the second read [70]\n"); fprintf(stderr, " -r FLOAT rate of mutations [%.4f]\n", MUT_RATE); fprintf(stderr, " -R FLOAT fraction of indels [%.2f]\n", INDEL_FRAC); fprintf(stderr, " -X FLOAT probability an indel is extended [%.2f]\n", INDEL_EXTEND); fprintf(stderr, " -S INT seed for random generator [0, use the current time]\n"); fprintf(stderr, " -A FLOAT discard if the fraction of ambiguous bases higher than FLOAT [%.2f]\n", MAX_N_RATIO); fprintf(stderr, " -h haplotype mode\n"); fprintf(stderr, "\n"); return 1; } int main(int argc, char *argv[]) { int64_t N; int dist, std_dev, c, size_l, size_r, is_hap = 0; FILE *fpout1, *fpout2; int seed = 0; N = 1000000; dist = 500; std_dev = 50; size_l = size_r = 70; while ((c = getopt(argc, argv, "e:d:s:N:1:2:r:R:hX:S:A:")) >= 0) { switch (c) { case 'd': dist = atoi(optarg); break; case 's': std_dev = atoi(optarg); break; case 'N': N = atoi(optarg); break; case '1': size_l = atoi(optarg); break; case '2': size_r = atoi(optarg); break; case 'e': ERR_RATE = atof(optarg); break; case 'r': MUT_RATE = atof(optarg); break; case 'R': INDEL_FRAC = atof(optarg); break; case 'X': INDEL_EXTEND = atof(optarg); break; case 'A': MAX_N_RATIO = atof(optarg); break; case 'S': seed = atoi(optarg); break; case 'h': is_hap = 1; break; } } if (argc - optind < 3) return simu_usage(); fpout1 = fopen(argv[optind+1], "w"); fpout2 = fopen(argv[optind+2], "w"); if (!fpout1 || !fpout2) { fprintf(stderr, "[wgsim] file open error\n"); return 1; } if (seed <= 0) seed = time(0)&0x7fffffff; fprintf(stderr, "[wgsim] seed = %d\n", seed); hts_srand48(seed); wgsim_core(fpout1, fpout2, argv[optind], is_hap, N, dist, std_dev, size_l, size_r); fclose(fpout1); fclose(fpout2); return 0; }