#include #include #include "bwtaln.h" #include "utils.h" #include "bamlite.h" #include "kseq.h" KSEQ_INIT(gzFile, gzread) extern unsigned char nst_nt4_table[256]; static char bam_nt16_nt4_table[] = { 4, 0, 1, 4, 2, 4, 4, 4, 3, 4, 4, 4, 4, 4, 4, 4 }; struct __bwa_seqio_t { // for BAM input int is_bam, which; // 1st bit: read1, 2nd bit: read2, 3rd: SE bamFile fp; // for fastq input kseq_t *ks; }; bwa_seqio_t *bwa_bam_open(const char *fn, int which) { bwa_seqio_t *bs; bam_header_t *h; bs = (bwa_seqio_t*)calloc(1, sizeof(bwa_seqio_t)); bs->is_bam = 1; bs->which = which; bs->fp = bam_open(fn, "r"); h = bam_header_read(bs->fp); bam_header_destroy(h); return bs; } bwa_seqio_t *bwa_seq_open(const char *fn) { gzFile fp; bwa_seqio_t *bs; bs = (bwa_seqio_t*)calloc(1, sizeof(bwa_seqio_t)); fp = xzopen(fn, "r"); bs->ks = kseq_init(fp); return bs; } void bwa_seq_close(bwa_seqio_t *bs) { if (bs == 0) return; if (bs->is_bam) bam_close(bs->fp); else { gzclose(bs->ks->f->f); kseq_destroy(bs->ks); } free(bs); } void seq_reverse(int len, ubyte_t *seq, int is_comp) { int i; if (is_comp) { for (i = 0; i < len>>1; ++i) { char tmp = seq[len-1-i]; if (tmp < 4) tmp = 3 - tmp; seq[len-1-i] = (seq[i] >= 4)? seq[i] : 3 - seq[i]; seq[i] = tmp; } if (len&1) seq[i] = (seq[i] >= 4)? seq[i] : 3 - seq[i]; } else { for (i = 0; i < len>>1; ++i) { char tmp = seq[len-1-i]; seq[len-1-i] = seq[i]; seq[i] = tmp; } } } int bwa_trim_read(int trim_qual, bwa_seq_t *p) { int s = 0, l, max = 0, max_l = p->len - 1; if (trim_qual < 1 || p->qual == 0) return 0; for (l = p->len - 1; l >= BWA_MIN_RDLEN - 1; --l) { s += trim_qual - (p->qual[l] - 33); if (s < 0) break; if (s > max) { max = s; max_l = l; } } p->clip_len = p->len = max_l + 1; return p->full_len - p->len; } static bwa_seq_t *bwa_read_bam(bwa_seqio_t *bs, int n_needed, int *n, int is_comp, int trim_qual) { bwa_seq_t *seqs, *p; int n_seqs, l, i; long n_trimmed = 0, n_tot = 0; bam1_t *b; b = bam_init1(); n_seqs = 0; seqs = (bwa_seq_t*)calloc(n_needed, sizeof(bwa_seq_t)); while (bam_read1(bs->fp, b) >= 0) { uint8_t *s, *q; int go = 0; if ((bs->which & 1) && (b->core.flag & BAM_FREAD1)) go = 1; if ((bs->which & 2) && (b->core.flag & BAM_FREAD2)) go = 1; if ((bs->which & 4) && !(b->core.flag& BAM_FREAD1) && !(b->core.flag& BAM_FREAD2))go = 1; if (go == 0) continue; l = b->core.l_qseq; p = &seqs[n_seqs++]; p->tid = -1; // no assigned to a thread p->qual = 0; p->full_len = p->clip_len = p->len = l; n_tot += p->full_len; s = bam1_seq(b); q = bam1_qual(b); p->seq = (ubyte_t*)calloc(p->len + 1, 1); p->qual = (ubyte_t*)calloc(p->len + 1, 1); for (i = 0; i != p->full_len; ++i) { p->seq[i] = bam_nt16_nt4_table[(int)bam1_seqi(s, i)]; p->qual[i] = q[i] + 33 < 126? q[i] + 33 : 126; } if (bam1_strand(b)) { // then reverse seq_reverse(p->len, p->seq, 1); seq_reverse(p->len, p->qual, 0); } if (trim_qual >= 1) n_trimmed += bwa_trim_read(trim_qual, p); p->rseq = (ubyte_t*)calloc(p->full_len, 1); memcpy(p->rseq, p->seq, p->len); seq_reverse(p->len, p->seq, 0); // *IMPORTANT*: will be reversed back in bwa_refine_gapped() seq_reverse(p->len, p->rseq, is_comp); p->name = strdup((const char*)bam1_qname(b)); if (n_seqs == n_needed) break; } *n = n_seqs; if (n_seqs && trim_qual >= 1) fprintf(stderr, "[bwa_read_seq] %.1f%% bases are trimmed.\n", 100.0f * n_trimmed/n_tot); if (n_seqs == 0) { free(seqs); bam_destroy1(b); return 0; } bam_destroy1(b); return seqs; } #define BARCODE_LOW_QUAL 13 bwa_seq_t *bwa_read_seq(bwa_seqio_t *bs, int n_needed, int *n, int mode, int trim_qual) { bwa_seq_t *seqs, *p; kseq_t *seq = bs->ks; int n_seqs, l, i, is_comp = mode&BWA_MODE_COMPREAD, is_64 = mode&BWA_MODE_IL13, l_bc = mode>>24; long n_trimmed = 0, n_tot = 0; if (l_bc > 15) { fprintf(stderr, "[%s] the maximum barcode length is 15.\n", __func__); return 0; } if (bs->is_bam) return bwa_read_bam(bs, n_needed, n, is_comp, trim_qual); // l_bc has no effect for BAM input n_seqs = 0; seqs = (bwa_seq_t*)calloc(n_needed, sizeof(bwa_seq_t)); while ((l = kseq_read(seq)) >= 0) { if (is_64 && seq->qual.l) for (i = 0; i < seq->qual.l; ++i) seq->qual.s[i] -= 31; if (seq->seq.l <= l_bc) continue; // sequence length equals or smaller than the barcode length p = &seqs[n_seqs++]; if (l_bc) { // then trim barcode for (i = 0; i < l_bc; ++i) p->bc[i] = (seq->qual.l && seq->qual.s[i]-33 < BARCODE_LOW_QUAL)? tolower(seq->seq.s[i]) : toupper(seq->seq.s[i]); p->bc[i] = 0; for (; i < seq->seq.l; ++i) seq->seq.s[i - l_bc] = seq->seq.s[i]; seq->seq.l -= l_bc; seq->seq.s[seq->seq.l] = 0; if (seq->qual.l) { for (i = l_bc; i < seq->qual.l; ++i) seq->qual.s[i - l_bc] = seq->qual.s[i]; seq->qual.l -= l_bc; seq->qual.s[seq->qual.l] = 0; } l = seq->seq.l; } else p->bc[0] = 0; p->tid = -1; // no assigned to a thread p->qual = 0; p->full_len = p->clip_len = p->len = l; n_tot += p->full_len; p->seq = (ubyte_t*)calloc(p->len, 1); for (i = 0; i != p->full_len; ++i) p->seq[i] = nst_nt4_table[(int)seq->seq.s[i]]; if (seq->qual.l) { // copy quality p->qual = (ubyte_t*)strdup((char*)seq->qual.s); if (trim_qual >= 1) n_trimmed += bwa_trim_read(trim_qual, p); } p->rseq = (ubyte_t*)calloc(p->full_len, 1); memcpy(p->rseq, p->seq, p->len); seq_reverse(p->len, p->seq, 0); // *IMPORTANT*: will be reversed back in bwa_refine_gapped() seq_reverse(p->len, p->rseq, is_comp); p->name = strdup((const char*)seq->name.s); { // trim /[12]$ int t = strlen(p->name); if (t > 2 && p->name[t-2] == '/' && (p->name[t-1] == '1' || p->name[t-1] == '2')) p->name[t-2] = '\0'; } if (n_seqs == n_needed) break; } *n = n_seqs; if (n_seqs && trim_qual >= 1) fprintf(stderr, "[bwa_read_seq] %.1f%% bases are trimmed.\n", 100.0f * n_trimmed/n_tot); if (n_seqs == 0) { free(seqs); return 0; } return seqs; } void bwa_free_read_seq(int n_seqs, bwa_seq_t *seqs) { int i, j; for (i = 0; i != n_seqs; ++i) { bwa_seq_t *p = seqs + i; for (j = 0; j < p->n_multi; ++j) if (p->multi[j].cigar) free(p->multi[j].cigar); free(p->name); free(p->seq); free(p->rseq); free(p->qual); free(p->aln); free(p->md); free(p->multi); free(p->cigar); } free(seqs); }