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consensus.c
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/* The MIT License
Copyright (c) 2014-2024 Genome Research Ltd.
Author: Petr Danecek <[email protected]>
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.
*/
#include <stdio.h>
#include <stdlib.h>
#include <string.h>
#include <strings.h>
#include <assert.h>
#include <errno.h>
#include <getopt.h>
#include <unistd.h>
#include <ctype.h>
#include <htslib/vcf.h>
#include <htslib/kstring.h>
#include <htslib/synced_bcf_reader.h>
#include <htslib/kseq.h>
#include <htslib/bgzf.h>
#include "regidx.h"
#include "bcftools.h"
#include "rbuf.h"
#include "filter.h"
#include "smpl_ilist.h"
// Logic of the filters: include or exclude sites which match the filters?
#define FLT_INCLUDE 1
#define FLT_EXCLUDE 2
#define PICK_REF 1
#define PICK_ALT 2
#define PICK_LONG 4
#define PICK_SHORT 8
#define PICK_IUPAC 16
#define TO_UPPER 1
#define TO_LOWER 2
typedef struct
{
int num; // number of ungapped blocks in this chain
int *block_lengths; // length of the ungapped blocks in this chain
int *ref_gaps; // length of the gaps on the reference sequence between blocks
int *alt_gaps; // length of the gaps on the alternative sequence between blocks
int ori_pos;
int ref_last_block_ori; // start position on the reference sequence of the following ungapped block (0-based)
int alt_last_block_ori; // start position on the alternative sequence of the following ungapped block (0-based)
}
chain_t;
#define MASK_LC 1
#define MASK_UC 2
#define MASK_SKIP(x) (((x)->with!=MASK_LC && (x)->with!=MASK_UC) ? 1 : 0)
typedef struct
{
char *fname, with;
regidx_t *idx;
regitr_t *itr;
}
mask_t;
typedef struct
{
kstring_t fa_buf; // buffered reference sequence
int fa_ori_pos; // start position of the fa_buffer (wrt original sequence)
int fa_frz_pos; // protected position to avoid conflicting variants (last pos for SNPs/ins)
int fa_mod_off; // position difference of fa_frz_pos in the ori and modified sequence (ins positive)
int fa_frz_mod; // the fa_buf offset of the protected fa_frz_pos position, includes the modified sequence
int fa_end_pos; // region's end position in the original sequence
int fa_length; // region's length in the original sequence (in case end_pos not provided in the FASTA header)
int fa_case; // output upper case or lower case: TO_UPPER|TO_LOWER
int fa_src_pos; // last genomic coordinate read from the input fasta (0-based)
char prev_base; // this is only to validate the REF allele in the VCF - the modified fa_buf cannot be used for inserts following deletions, see 600#issuecomment-383186778
int prev_base_pos; // the position of prev_base
int prev_is_insert;
rbuf_t vcf_rbuf;
bcf1_t **vcf_buf;
int nvcf_buf, rid;
char *chr, *chr_prefix;
mask_t *mask;
int nmask;
int chain_id; // chain_id, to provide a unique ID to each chain in the chain output
chain_t *chain; // chain structure to store the sequence of ungapped blocks between the ref and alt sequences
// Note that the chain is re-initialised for each chromosome/seq_region
filter_t *filter;
char *filter_str;
int filter_logic; // include or exclude sites which match the filters? One of FLT_INCLUDE/FLT_EXCLUDE
bcf_srs_t *files;
bcf_hdr_t *hdr;
FILE *fp_out;
FILE *fp_chain;
char **argv;
int argc, output_iupac, iupac_GTs, haplotype, allele, isample, napplied;
uint8_t *iupac_bitmask, *iupac_als;
int miupac_bitmask, miupac_als, regions_overlap;
char *fname, *ref_fname, *sample, *sample_fname, *output_fname, *mask_fname, *chain_fname, missing_allele, absent_allele;
char mark_del, mark_ins, mark_snv;
smpl_ilist_t *smpl;
}
args_t;
static void destroy_chain(chain_t *chain)
{
if ( !chain ) return;
free(chain->ref_gaps);
free(chain->alt_gaps);
free(chain->block_lengths);
free(chain);
}
static chain_t* init_chain(chain_t *chain, int ref_ori_pos)
{
if ( chain ) destroy_chain(chain);
chain = (chain_t*) calloc(1,sizeof(chain_t));
chain->num = 0;
chain->block_lengths = NULL;
chain->ref_gaps = NULL;
chain->alt_gaps = NULL;
chain->ori_pos = ref_ori_pos;
chain->ref_last_block_ori = ref_ori_pos;
chain->alt_last_block_ori = ref_ori_pos;
return chain;
}
static void print_chain(args_t *args)
{
/*
Example chain format (see: https://genome.ucsc.edu/goldenPath/help/chain.html):
chain 1 500 + 480 500 1 501 + 480 501 1
12 3 1
1 0 3
484
chain line is:
- chain
- score (sum of the length of ungapped block in this case)
- ref_seqname (from the fasta header, parsed by htslib)
- ref_seqlength (from the fasta header)
- ref_strand (+ or -; always + for bcf-consensus)
- ref_start (as defined in the fasta header)
- ref_end (as defined in the fasta header)
- alt_seqname (same as ref_seqname as bcf-consensus only considers SNPs and indels)
- alt_seqlength (adjusted to match the length of the alt sequence)
- alt_strand (+ or -; always + for bcf-consensus)
- alt_start (same as ref_start, as no edits are recorded/applied before that position)
- alt_end (adjusted to match the length of the alt sequence)
- chain_num (just an auto-increment id)
the other (sorted) lines are:
- length of the ungapped alignment block
- gap on the ref sequence between this and the next block (all but the last line)
- gap on the alt sequence between this and the next block (all but the last line)
*/
chain_t *chain = args->chain;
int n = chain->num;
int ref_end_pos = args->fa_length + chain->ori_pos;
int last_block_size = ref_end_pos - chain->ref_last_block_ori;
int alt_end_pos = chain->alt_last_block_ori + last_block_size;
int score = 0;
for (n=0; n<chain->num; n++) {
score += chain->block_lengths[n];
}
score += last_block_size;
fprintf(args->fp_chain, "chain %d %s %d + %d %d %s %d + %d %d %d\n", score, args->chr, ref_end_pos, chain->ori_pos, ref_end_pos, args->chr, alt_end_pos, chain->ori_pos, alt_end_pos, ++args->chain_id);
for (n=0; n<chain->num; n++) {
fprintf(args->fp_chain, "%d %d %d\n", chain->block_lengths[n], chain->ref_gaps[n], chain->alt_gaps[n]);
}
fprintf(args->fp_chain, "%d\n\n", last_block_size);
}
static void push_chain_gap(chain_t *chain, int ref_start, int ref_len, int alt_start, int alt_len)
{
// fprintf(stderr, "push_chain_gap(chain=%p, ref_start=%d, ref_len=%d, alt_start=%d, alt_len=%d)\n", chain, ref_start, ref_len, alt_start, alt_len);
int num = chain->num;
if (num && ref_start <= chain->ref_last_block_ori) {
// In case this variant is back-to-back with the previous one
chain->ref_last_block_ori = ref_start + ref_len;
chain->alt_last_block_ori = alt_start + alt_len;
chain->ref_gaps[num-1] += ref_len;
chain->alt_gaps[num-1] += alt_len;
} else {
// Extend the ungapped blocks, store the gap length
chain->block_lengths = (int*) realloc(chain->block_lengths, (num + 1) * sizeof(int));
chain->ref_gaps = (int*) realloc(chain->ref_gaps, (num + 1) * sizeof(int));
chain->alt_gaps = (int*) realloc(chain->alt_gaps, (num + 1) * sizeof(int));
chain->block_lengths[num] = ref_start - chain->ref_last_block_ori;
chain->ref_gaps[num] = ref_len;
chain->alt_gaps[num] = alt_len;
// Update the start positions of the next block
chain->ref_last_block_ori = ref_start + ref_len;
chain->alt_last_block_ori = alt_start + alt_len;
// Increment the number of ungapped blocks
chain->num++;
}
}
static void init_data(args_t *args)
{
args->files = bcf_sr_init();
args->files->require_index = 1;
if ( !bcf_sr_add_reader(args->files,args->fname) ) error("Failed to read from %s: %s\n", !strcmp("-",args->fname)?"standard input":args->fname, bcf_sr_strerror(args->files->errnum));
args->hdr = args->files->readers[0].header;
args->isample = -1;
if ( !args->sample )
args->smpl = smpl_ilist_init(args->hdr,NULL,0,SMPL_NONE|SMPL_VERBOSE);
else if ( args->sample && strcmp("-",args->sample) )
{
args->smpl = smpl_ilist_init(args->hdr,args->sample,0,SMPL_NONE|SMPL_VERBOSE);
if ( args->smpl && !args->smpl->n ) error("No matching sample found\n");
}
else if ( args->sample_fname )
{
args->smpl = smpl_ilist_init(args->hdr,args->sample_fname,1,SMPL_NONE|SMPL_VERBOSE);
if ( args->smpl && !args->smpl->n ) error("No matching sample found\n");
}
if ( args->smpl )
{
if ( args->haplotype || args->allele )
{
if ( args->smpl->n > 1 ) error("Too many samples, only one can be used with -H\n");
args->isample = args->smpl->idx[0];
}
else
args->iupac_GTs = 1;
}
int i;
for (i=0; i<args->nmask; i++)
{
mask_t *mask = &args->mask[i];
mask->idx = regidx_init(mask->fname,NULL,NULL,0,NULL);
if ( !mask->idx ) error("Failed to initialize mask regions\n");
mask->itr = regitr_init(mask->idx);
}
// In case we want to store the chains
if ( args->chain_fname )
{
args->fp_chain = fopen(args->chain_fname,"w");
if ( ! args->fp_chain ) error("Failed to create %s: %s\n", args->chain_fname, strerror(errno));
args->chain_id = 0;
}
rbuf_init(&args->vcf_rbuf, 100);
args->vcf_buf = (bcf1_t**) calloc(args->vcf_rbuf.m, sizeof(bcf1_t*));
if ( args->output_fname ) {
args->fp_out = fopen(args->output_fname,"w");
if ( ! args->fp_out ) error("Failed to create %s: %s\n", args->output_fname, strerror(errno));
}
else args->fp_out = stdout;
if ( args->isample<0 && !args->iupac_GTs ) fprintf(stderr,"Note: the --samples option not given, applying all records regardless of the genotype\n");
if ( args->filter_str )
args->filter = filter_init(args->hdr, args->filter_str);
args->rid = -1;
}
static void add_mask(args_t *args, char *fname)
{
args->nmask++;
args->mask = (mask_t*)realloc(args->mask,args->nmask*sizeof(*args->mask));
mask_t *mask = &args->mask[args->nmask-1];
mask->fname = fname;
mask->with = 'N';
}
static void add_mask_with(args_t *args, char *with)
{
if ( !args->nmask ) error("The --mask-with option must follow --mask\n");
mask_t *mask = &args->mask[args->nmask-1];
if ( !strcasecmp(with,"uc") ) mask->with = MASK_UC;
else if ( !strcasecmp(with,"lc") ) mask->with = MASK_LC;
else if ( strlen(with)!=1 ) error("Expected \"lc\", \"uc\", or a single character with the --mask-with option\n");
else mask->with = *with;
}
static void destroy_data(args_t *args)
{
free(args->iupac_als);
free(args->iupac_bitmask);
if (args->filter) filter_destroy(args->filter);
if ( args->smpl ) smpl_ilist_destroy(args->smpl);
bcf_sr_destroy(args->files);
int i;
for (i=0; i<args->vcf_rbuf.m; i++)
if ( args->vcf_buf[i] ) bcf_destroy1(args->vcf_buf[i]);
free(args->vcf_buf);
free(args->fa_buf.s);
free(args->chr);
for (i=0; i<args->nmask; i++)
{
mask_t *mask = &args->mask[i];
regidx_destroy(mask->idx);
regitr_destroy(mask->itr);
}
free(args->mask);
if ( args->chain_fname )
if ( fclose(args->fp_chain) ) error("Close failed: %s\n", args->chain_fname);
if ( fclose(args->fp_out) ) error("Close failed: %s\n", args->output_fname);
destroy_chain(args->chain);
}
static void init_region(args_t *args, char *line)
{
char *ss, *se = line;
while ( *se && !isspace(*se) && *se!=':' ) se++;
hts_pos_t from = 0, to = 0;
char tmp = 0, *tmp_ptr = NULL;
if ( *se )
{
tmp = *se; *se = 0; tmp_ptr = se;
ss = ++se;
from = strtol(ss,&se,10);
if ( ss==se || !*se || *se!='-' ) from = 0;
else
{
from--;
ss = ++se;
to = strtol(ss,&se,10);
if ( ss==se || (*se && !isspace(*se)) ) { from = 0; to = 0; }
else to--;
}
}
free(args->chr);
args->chr = strdup(line);
args->rid = bcf_hdr_name2id(args->hdr,line);
if ( args->rid<0 ) fprintf(stderr,"Warning: Sequence \"%s\" not in %s\n", line,args->fname);
args->prev_base_pos = -1;
args->fa_buf.l = 0;
args->fa_length = 0;
args->fa_end_pos = to; // 0-based
args->fa_ori_pos = from; // 0-based
args->fa_src_pos = from; // 0-based
args->fa_mod_off = 0;
args->fa_frz_pos = -1;
args->fa_frz_mod = -1;
args->fa_case = -1;
args->vcf_rbuf.n = 0;
// bcf_sr_set_regions accepts 1-based coordinates
kstring_t str = {0,0,0};
if ( !from ) from = 1;
else from++;
#ifndef MAX_CSI_COOR
#define MAX_CSI_COOR ((1LL << (14 + 30)) - 1)
#endif
if ( to==0 ) to = MAX_CSI_COOR - 1;
else to++;
ksprintf(&str,"%s:%"PRIhts_pos"-%"PRIhts_pos,line,from,to);
bcf_sr_set_opt(args->files,BCF_SR_REGIONS_OVERLAP,args->regions_overlap);
bcf_sr_set_regions(args->files,str.s,0);
free(str.s);
if ( tmp_ptr ) *tmp_ptr = tmp;
fprintf(args->fp_out,">%s%s\n",args->chr_prefix?args->chr_prefix:"",line);
if ( args->chain_fname )
args->chain = init_chain(args->chain, args->fa_ori_pos);
}
static bcf1_t **next_vcf_line(args_t *args)
{
if ( args->vcf_rbuf.n )
{
int i = rbuf_shift(&args->vcf_rbuf);
return &args->vcf_buf[i];
}
while ( bcf_sr_next_line(args->files) )
{
if ( args->filter )
{
int is_ok = filter_test(args->filter, bcf_sr_get_line(args->files,0), NULL);
if ( args->filter_logic & FLT_EXCLUDE ) is_ok = is_ok ? 0 : 1;
if ( !is_ok ) continue;
}
return &args->files->readers[0].buffer[0];
}
return NULL;
}
static void unread_vcf_line(args_t *args, bcf1_t **rec_ptr)
{
bcf1_t *rec = *rec_ptr;
if ( args->vcf_rbuf.n >= args->vcf_rbuf.m )
error("FIXME: too many overlapping records near %s:%"PRId64"\n", bcf_seqname(args->hdr,rec),(int64_t) rec->pos+1);
// Insert the new record in the buffer. The line would be overwritten in
// the next bcf_sr_next_line call, therefore we need to swap it with an
// unused one
int i = rbuf_append(&args->vcf_rbuf);
if ( !args->vcf_buf[i] ) args->vcf_buf[i] = bcf_init1();
bcf1_t *tmp = rec; *rec_ptr = args->vcf_buf[i]; args->vcf_buf[i] = tmp;
}
static void flush_fa_buffer(args_t *args, int len)
{
if ( !args->fa_buf.l ) return;
int nwr = 0;
while ( nwr + 60 <= args->fa_buf.l )
{
if ( fwrite(args->fa_buf.s+nwr,1,60,args->fp_out) != 60 ) error("Could not write: %s\n", args->output_fname);
if ( fwrite("\n",1,1,args->fp_out) != 1 ) error("Could not write: %s\n", args->output_fname);
nwr += 60;
}
if ( nwr )
args->fa_ori_pos += nwr;
args->fa_frz_mod -= nwr;
if ( len )
{
// not finished on this chr yet and the buffer cannot be emptied completely
if ( nwr && nwr < args->fa_buf.l )
memmove(args->fa_buf.s,args->fa_buf.s+nwr,args->fa_buf.l-nwr);
args->fa_buf.l -= nwr;
return;
}
// empty the whole buffer
if ( nwr == args->fa_buf.l ) { args->fa_buf.l = 0; return; }
if ( fwrite(args->fa_buf.s+nwr,1,args->fa_buf.l - nwr,args->fp_out) != args->fa_buf.l - nwr ) error("Could not write: %s\n", args->output_fname);
if ( fwrite("\n",1,1,args->fp_out) != 1 ) error("Could not write: %s\n", args->output_fname);
args->fa_ori_pos += args->fa_buf.l - nwr - args->fa_mod_off;
args->fa_mod_off = 0;
args->fa_buf.l = 0;
}
static void apply_absent(args_t *args, hts_pos_t pos)
{
if ( !args->fa_buf.l || pos <= args->fa_frz_pos + 1 || pos <= args->fa_ori_pos ) return;
int ie = pos && pos - args->fa_ori_pos + args->fa_mod_off < args->fa_buf.l ? pos - args->fa_ori_pos + args->fa_mod_off : args->fa_buf.l;
int ib = args->fa_frz_mod < 0 ? 0 : args->fa_frz_mod;
int i;
for (i=ib; i<ie; i++)
args->fa_buf.s[i] = args->absent_allele;
}
static void freeze_ref(args_t *args, bcf1_t *rec)
{
if ( args->fa_frz_pos >= rec->pos + rec->rlen - 1 ) return;
args->fa_frz_pos = rec->pos + rec->rlen - 1;
args->fa_frz_mod = rec->pos - args->fa_ori_pos + args->fa_mod_off + rec->rlen;
}
static char *mark_del(char *ref, int rlen, char *alt, int mark)
{
char *out = malloc(rlen+1);
int i;
if ( alt )
{
int nalt = strlen(alt);
for (i=0; i<nalt; i++) out[i] = alt[i];
}
else // symbolic <DEL>
{
int nref = strlen(ref);
for (i=0; i<nref; i++) out[i] = ref[i];
}
for (; i<rlen; i++) out[i] = mark;
out[rlen] = 0;
return out;
}
static void mark_ins(char *ref, char *alt, char mark)
{
int i, nref = strlen(ref), nalt = strlen(alt);
if ( mark==TO_LOWER )
for (i=nref; i<nalt; i++) alt[i] = tolower(alt[i]);
else if ( mark==TO_UPPER )
for (i=nref; i<nalt; i++) alt[i] = toupper(alt[i]);
else if ( mark )
for (i=nref; i<nalt; i++) alt[i] = mark;
}
static void mark_snv(char *ref, char *alt, char mark)
{
int i, nref = strlen(ref), nalt = strlen(alt);
int n = nref < nalt ? nref : nalt;
if ( mark==TO_LOWER )
{
for (i=0; i<n; i++)
if ( tolower(ref[i])!=tolower(alt[i]) ) alt[i] = tolower(alt[i]);
}
else if ( mark==TO_UPPER)
{
for (i=0; i<n; i++)
if ( tolower(ref[i])!=tolower(alt[i]) ) alt[i] = toupper(alt[i]);
}
else if ( mark==TO_UPPER)
{
for (i=0; i<n; i++)
if ( tolower(ref[i])!=tolower(alt[i]) ) alt[i] = toupper(alt[i]);
}
else if ( mark )
{
for (i=0; i<n; i++)
if ( tolower(ref[i])!=tolower(alt[i]) ) alt[i] = mark;
}
}
static void iupac_init(args_t *args, bcf1_t *rec)
{
int i;
hts_resize(uint8_t, rec->n_allele, &args->miupac_als, &args->iupac_als, 0);
for (i=0; i<args->miupac_als; i++) args->iupac_als[i] = 0;
}
static int iupac_add_gt(args_t *args, bcf1_t *rec, uint8_t *gt, int ngt)
{
int i, is_set = 0;
for (i=0; i<ngt; i++)
{
if ( bcf_gt_is_missing(gt[i]) ) continue;
if ( gt[i]==(uint8_t)bcf_int8_vector_end ) break;
int ial = bcf_gt_allele(gt[i]);
if ( ial >= rec->n_allele ) error("Invalid VCF, too few ALT alleles at %s:%"PRId64"\n", bcf_seqname(args->hdr,rec),(int64_t) rec->pos+1);
args->iupac_als[ial] = 1;
is_set = 1;
}
return is_set;
}
static int iupac_set_allele(args_t *args, bcf1_t *rec)
{
int i,j, max_len = 0, alt_len = 0, ialt = -1, fallback_alt = -1;
for (i=0; i<rec->n_allele; i++)
{
if ( !args->iupac_als[i] ) continue;
if ( fallback_alt <=0 ) fallback_alt = i;
int l = strlen(rec->d.allele[i]);
for (j=0; j<l; j++)
if ( iupac2bitmask(rec->d.allele[i][j]) < 0 ) break;
if ( j<l ) continue; // symbolic allele, breakpoint or invalid character in the allele
if ( l>max_len )
{
hts_resize(uint8_t, l, &args->miupac_bitmask, &args->iupac_bitmask, HTS_RESIZE_CLEAR);
for (j=max_len; j<l; j++) args->iupac_bitmask[j] = 0;
max_len = l;
}
if ( i>0 && l>alt_len )
{
alt_len = l;
ialt = i;
}
for (j=0; j<l; j++)
args->iupac_bitmask[j] |= iupac2bitmask(rec->d.allele[i][j]);
}
if ( alt_len > 0 )
{
for (j=0; j<alt_len; j++) rec->d.allele[ialt][j] = bitmask2iupac(args->iupac_bitmask[j]);
return ialt;
}
if ( fallback_alt >= 0 ) return fallback_alt;
return ialt;
}
static void apply_variant(args_t *args, bcf1_t *rec)
{
static int warned_haplotype = 0;
if ( args->absent_allele ) apply_absent(args, rec->pos);
if ( rec->n_allele==1 && !args->missing_allele && !args->absent_allele ) { return; }
int i,j;
if ( args->mask )
{
char *chr = (char*)bcf_hdr_id2name(args->hdr,args->rid);
int start = rec->pos;
int end = rec->pos + rec->rlen - 1;
for (i=0; i<args->nmask; i++)
{
mask_t *mask = &args->mask[i];
if ( MASK_SKIP(mask) && regidx_overlap(mask->idx, chr,start,end,NULL) ) return;
}
}
int ialt = 1; // the alternate allele
if ( args->iupac_GTs )
{
bcf_unpack(rec, BCF_UN_FMT);
bcf_fmt_t *fmt = bcf_get_fmt(args->hdr, rec, "GT");
if ( !fmt ) return;
if ( fmt->type!=BCF_BT_INT8 )
error("Todo: GT field represented with BCF_BT_INT8, too many alleles at %s:%"PRId64"?\n",bcf_seqname(args->hdr,rec),(int64_t) rec->pos+1);
ialt = -1;
int is_set = 0;
iupac_init(args,rec);
for (i=0; i<args->smpl->n; i++)
{
uint8_t *ptr = fmt->p + fmt->size*args->smpl->idx[i];
is_set += iupac_add_gt(args, rec, ptr, fmt->n);
}
if ( !is_set && !args->missing_allele ) return;
if ( is_set ) ialt = iupac_set_allele(args, rec);
}
else if ( args->isample >= 0 )
{
bcf_unpack(rec, BCF_UN_FMT);
bcf_fmt_t *fmt = bcf_get_fmt(args->hdr, rec, "GT");
if ( !fmt ) return;
if ( fmt->type!=BCF_BT_INT8 )
error("Todo: GT field represented with BCF_BT_INT8, too many alleles at %s:%"PRId64"?\n",bcf_seqname(args->hdr,rec),(int64_t) rec->pos+1);
uint8_t *ptr = fmt->p + fmt->size*args->isample;
enum { use_hap, use_iupac, pick_one } action = use_hap;
if ( args->allele==PICK_IUPAC )
{
if ( !args->haplotype ) action = use_iupac;
if ( !bcf_gt_is_phased(ptr[0]) && !bcf_gt_is_phased(ptr[fmt->n-1]) ) action = use_iupac;
}
else if ( args->output_iupac ) action = use_iupac;
else if ( !args->haplotype ) action = pick_one;
if ( action==use_hap )
{
if ( args->haplotype > fmt->n )
{
if ( bcf_gt_is_missing(ptr[fmt->n-1]) || bcf_gt_is_missing(ptr[0]) )
{
if ( !args->missing_allele ) return;
ialt = -1;
}
else
{
if ( !warned_haplotype )
{
fprintf(stderr, "Can't apply %d-th haplotype at %s:%"PRId64". (This warning is printed only once.)\n", args->haplotype,bcf_seqname(args->hdr,rec),(int64_t) rec->pos+1);
warned_haplotype = 1;
}
return;
}
}
else
{
ialt = (int8_t)ptr[args->haplotype-1];
if ( bcf_gt_is_missing(ialt) || ialt==bcf_int8_vector_end )
{
if ( !args->missing_allele ) return;
ialt = -1;
}
else
ialt = bcf_gt_allele(ialt);
}
}
else if ( action==use_iupac )
{
ialt = -1;
iupac_init(args,rec);
int is_set = iupac_add_gt(args, rec, ptr, fmt->n);
if ( !is_set && !args->missing_allele ) return;
if ( is_set ) ialt = iupac_set_allele(args, rec);
}
else
{
int is_hom = 1;
for (i=0; i<fmt->n; i++)
{
if ( bcf_gt_is_missing(ptr[i]) )
{
if ( !args->missing_allele ) return; // ignore missing or half-missing genotypes
ialt = -1;
break;
}
if ( ptr[i]==(uint8_t)bcf_int8_vector_end ) break;
ialt = bcf_gt_allele(ptr[i]);
if ( i>0 && ialt!=bcf_gt_allele(ptr[i-1]) ) { is_hom = 0; break; }
}
if ( !is_hom )
{
int prev_len = 0, jalt;
for (i=0; i<fmt->n; i++)
{
if ( ptr[i]==(uint8_t)bcf_int8_vector_end ) break;
jalt = bcf_gt_allele(ptr[i]);
if ( rec->n_allele <= jalt ) error("Broken VCF, too few alts at %s:%"PRId64"\n", bcf_seqname(args->hdr,rec),(int64_t) rec->pos+1);
if ( args->allele & (PICK_LONG|PICK_SHORT) )
{
int len = jalt==0 ? rec->rlen : strlen(rec->d.allele[jalt]);
if ( i==0 ) ialt = jalt, prev_len = len;
else if ( len == prev_len )
{
if ( args->allele & PICK_REF && jalt==0 ) ialt = jalt, prev_len = len;
else if ( args->allele & PICK_ALT && ialt==0 ) ialt = jalt, prev_len = len;
}
else if ( args->allele & PICK_LONG && len > prev_len ) ialt = jalt, prev_len = len;
else if ( args->allele & PICK_SHORT && len < prev_len ) ialt = jalt, prev_len = len;
}
else
{
if ( args->allele & PICK_REF && jalt==0 ) ialt = jalt;
else if ( args->allele & PICK_ALT && ialt==0 ) ialt = jalt;
}
}
}
}
if ( !ialt )
{
// ref allele
if ( args->absent_allele ) freeze_ref(args,rec);
return;
}
if ( rec->n_allele <= ialt ) error("Broken VCF, too few alts at %s:%"PRId64"\n", bcf_seqname(args->hdr,rec),(int64_t) rec->pos+1);
}
else if ( args->output_iupac && rec->n_allele>1 )
{
int ialt, alen = 0, mlen = 0;
for (i=0; i<rec->n_allele; i++)
{
int l = strlen(rec->d.allele[i]);
for (j=0; j<l; j++)
if ( iupac2bitmask(rec->d.allele[i][j]) < 0 ) break;
if ( j<l ) continue; // symbolic allele, breakpoint or invalid character in the allele
if ( l > mlen )
{
hts_expand(uint8_t,l,args->miupac_bitmask,args->iupac_bitmask);
for (j=mlen; j<l; j++) args->iupac_bitmask[j] = 0;
mlen = l;
}
if ( i>0 && l>alen )
{
alen = l;
ialt = i;
}
for (j=0; j<l; j++)
args->iupac_bitmask[j] |= iupac2bitmask(rec->d.allele[i][j]);
}
if ( alen > 0 )
for (j=0; j<alen; j++) rec->d.allele[ialt][j] = bitmask2iupac(args->iupac_bitmask[j]);
else
ialt = 1;
}
if ( rec->n_allele==1 && ialt!=-1 )
{
// non-missing reference
if ( args->absent_allele ) freeze_ref(args,rec);
return;
}
if ( ialt==-1 )
{
char alleles[4];
alleles[0] = rec->d.allele[0][0];
alleles[1] = ',';
alleles[2] = args->missing_allele;
alleles[3] = 0;
bcf_update_alleles_str(args->hdr, rec, alleles);
ialt = 1;
}
// For some variant types POS+REF refer to the base *before* the event; in such case set trim_beg
int trim_beg = 0;
int var_type = bcf_get_variant_type(rec,ialt);
int var_len = rec->d.var[ialt].n;
if ( var_type & VCF_INDEL )
{
// normally indel starts one base after, but not if the first base of the fa reference is deleted
if ( rec->d.allele[0][0] == rec->d.allele[ialt][0] )
trim_beg = 1;
else
trim_beg = 0;
}
else if ( (var_type & VCF_OTHER) && !strcasecmp(rec->d.allele[ialt],"<DEL>") )
{
trim_beg = 1;
var_len = 1 - rec->rlen;
}
else if ( (var_type & VCF_OTHER) && !strncasecmp(rec->d.allele[ialt],"<INS",4) ) trim_beg = 1;
// Overlapping variant?
if ( rec->pos <= args->fa_frz_pos )
{
// Can be still OK iff this is an insertion (and which does not follow another insertion, see #888).
// This still may not be enough for more complicated cases with multiple duplicate positions
// and other types in between. In such case let the user normalize the VCF and remove duplicates.
int overlap = 0;
if ( rec->pos < args->fa_frz_pos || !trim_beg || var_len==0 || args->prev_is_insert ) overlap = 1;
if ( overlap )
{
fprintf(stderr,"The site %s:%"PRId64" overlaps with another variant, skipping...\n", bcf_seqname(args->hdr,rec),(int64_t) rec->pos+1);
return;
}
}
char *ref_allele = rec->d.allele[0];
char *alt_allele = rec->d.allele[ialt];
int rmme_alt = 0;
int len_diff = 0, alen = 0;
int idx = rec->pos - args->fa_ori_pos + args->fa_mod_off; // position of the variant within the modified fasta sequence
if ( idx<0 )
{
if ( alt_allele[0]=='<' ) // symbolic allele
{
rec->pos -= idx + 1;
rec->rlen += idx + 1;
idx = -1;
}
else if ( strlen(ref_allele) < -idx ) // the ref allele is shorter but overlaps the fa sequence? This should never happen
{
assert(0);
fprintf(stderr,"Warning: ignoring overlapping variant starting at %s:%"PRId64"\n", bcf_seqname(args->hdr,rec),(int64_t) rec->pos+1);
return;
}
else if ( strlen(alt_allele) > -idx ) // the ref allele overlaps the fa and so does the alt allele
{
rec->pos -= idx;
rec->rlen += idx;
ref_allele -= idx;
alt_allele -= idx;
idx = 0;
}
else // the ref allele overlaps the fa but alt allele does not: trim to leave one base before
{
rec->pos -= idx + 1;
rec->rlen += idx + 1;
ref_allele -= idx + 1;
alt_allele += strlen(alt_allele) - 1;
idx = -1;
}
}
if ( rec->rlen > args->fa_buf.l - idx )
{
rec->rlen = args->fa_buf.l - idx;
if ( alt_allele[0]!='<' )
{
alen = strlen(alt_allele);
if ( alen > rec->rlen )
{
fprintf(stderr,"Warning: trimming variant \"%s\" starting at %s:%"PRId64"\n", alt_allele,bcf_seqname(args->hdr,rec),(int64_t) rec->pos+1);
alt_allele[rec->rlen] = 0;
}
}
}
if ( idx>0 && idx>=args->fa_buf.l )
error("FIXME: %s:%"PRId64" .. idx=%d, ori_pos=%d, len=%"PRIu64", off=%d\n",bcf_seqname(args->hdr,rec),(int64_t) rec->pos+1,idx,args->fa_ori_pos,(uint64_t)args->fa_buf.l,args->fa_mod_off);
// sanity check the reference base
if ( alt_allele[0]=='<' )
{
// TODO: symbolic deletions probably need more work above with PICK_SHORT|PICK_LONG
if ( strcasecmp(alt_allele,"<DEL>") && strcasecmp(alt_allele,"<*>") && strcasecmp(alt_allele,"<NON_REF>") )
error("Symbolic alleles other than <DEL>, <*> or <NON_REF> are currently not supported, e.g. \"%s\" at %s:%"PRId64".\n"
"Please use filtering expressions to exclude such sites, for example by running with: -e 'ALT~\"<.*>\"'\n",
alt_allele,bcf_seqname(args->hdr,rec),(int64_t) rec->pos+1);
if ( !strcasecmp(alt_allele,"<DEL>") )
{
static int multibase_ref_del_warned = 0;
if ( ref_allele[1]!=0 && !multibase_ref_del_warned )
{
fprintf(stderr,
"Warning: one REF base is expected with <DEL>, assuming the actual deletion starts at POS+1 at %s:%"PRId64".\n"
" (This warning is printed only once.)\n", bcf_seqname(args->hdr,rec),(int64_t) rec->pos+1);
multibase_ref_del_warned = 1;
}
if ( args->mark_del ) // insert dashes instead of delete sequence
{
alt_allele = mark_del(ref_allele, rec->rlen, NULL, args->mark_del);
alen = rec->rlen;
len_diff = 0;
rmme_alt = 1;
}
else
{
len_diff = 1-rec->rlen;
alt_allele = ref_allele; // according to VCF spec, the first REF base must precede the event
alen = 1;
}
}
else
{
// <*> or <NON_REF> .. gVCF, evidence for the reference allele throughout the whole block
freeze_ref(args,rec);
return;
}
}
else if ( idx>=0 && strncasecmp(ref_allele,args->fa_buf.s+idx,rec->rlen) )
{
// This is hacky, handle a special case: if SNP or an insert follows a deletion (AAC>A, C>CAA),
// the reference base in fa_buf is lost and the check fails. We do not keep a buffer
// with the original sequence as it should not be necessary, we should encounter max
// one base overlap
int fail = 1;
if ( args->prev_base_pos==rec->pos && toupper(ref_allele[0])==toupper(args->prev_base) )
{
if ( rec->rlen==1 ) fail = 0;
else if ( !strncasecmp(ref_allele+1,args->fa_buf.s+idx+1,rec->rlen-1) ) fail = 0;
}
if ( fail )
{
char tmp = 0;
if ( args->fa_buf.l - idx > rec->rlen )
{
tmp = args->fa_buf.s[idx+rec->rlen];
args->fa_buf.s[idx+rec->rlen] = 0;
}
error(
"The fasta sequence does not match the REF allele at %s:%"PRId64":\n"
" REF .vcf: [%s]\n"
" ALT .vcf: [%s]\n"
" REF .fa : [%s]%c%s\n",
bcf_seqname(args->hdr,rec),(int64_t) rec->pos+1, ref_allele, alt_allele, args->fa_buf.s+idx,
tmp?tmp:' ',tmp?args->fa_buf.s+idx+rec->rlen+1:""
);
}
alen = strlen(alt_allele);
len_diff = alen - rec->rlen;
if ( args->mark_del && len_diff<0 )
{
alt_allele = mark_del(ref_allele, rec->rlen, alt_allele, args->mark_del);
alen = rec->rlen;
len_diff = 0;
rmme_alt = 1;
}
}
else
{
alen = strlen(alt_allele);
len_diff = alen - rec->rlen;
if ( args->mark_del && len_diff<0 )
{
alt_allele = mark_del(ref_allele, rec->rlen, alt_allele, args->mark_del);
alen = rec->rlen;
len_diff = 0;
rmme_alt = 1;
}
}
int safe_idx = idx<0 ? 0 : idx; // idx can be negative in case of overlapping deletion
args->fa_case = toupper(args->fa_buf.s[safe_idx])==args->fa_buf.s[safe_idx] ? TO_UPPER : TO_LOWER;
if ( args->fa_case==TO_UPPER )
for (i=0; i<alen; i++) alt_allele[i] = toupper(alt_allele[i]);
else
for (i=0; i<alen; i++) alt_allele[i] = tolower(alt_allele[i]);
if ( args->mark_ins && len_diff>0 )
mark_ins(ref_allele, alt_allele, args->mark_ins);
if ( args->mark_snv )
mark_snv(ref_allele, alt_allele, args->mark_snv);
if ( len_diff <= 0 )
{
// deletion or same size event
assert( args->fa_buf.l >= idx+rec->rlen );
args->prev_base = args->fa_buf.s[idx+rec->rlen-1];
args->prev_base_pos = rec->pos + rec->rlen - 1;
args->prev_is_insert = 0;
args->fa_frz_mod = idx + alen;
for (i=trim_beg; i<alen; i++)
args->fa_buf.s[idx+i] = alt_allele[i];
if ( len_diff )
memmove(args->fa_buf.s+idx+alen,args->fa_buf.s+idx+rec->rlen,args->fa_buf.l-idx-rec->rlen);
}
else
{
args->prev_is_insert = 1;
args->prev_base_pos = rec->pos;
// insertion
ks_resize(&args->fa_buf, args->fa_buf.l + len_diff);
memmove(args->fa_buf.s + idx + rec->rlen + len_diff, args->fa_buf.s + idx + rec->rlen, args->fa_buf.l - idx - rec->rlen);
// This can get tricky, make sure the bases unchanged by the insertion do not overwrite preceeding variants.
// For example, here we want to get TAA:
// POS REF ALT
// 1 C T
// 1 C CAA
int ibeg = 0;
while ( ibeg<alen && ref_allele[ibeg]==alt_allele[ibeg] && rec->pos + ibeg <= args->prev_base_pos ) ibeg++;
for (i=ibeg; i<alen; i++)
args->fa_buf.s[idx+i] = alt_allele[i];
args->fa_frz_mod = idx + alen - ibeg + 1;
}
if (args->chain && len_diff != 0)
{
// If first nucleotide of both REF and ALT are the same... (indels typically include the nucleotide before the variant)