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csq.c
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csq.c
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/* The MIT License
Copyright (c) 2016-2024 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.
*/
/*
Things that would be nice to have
- dynamic N_REF_PAD
- for stop-lost events (also in frameshifts) report the number of truncated aa's
- memory could be greatly reduced by indexing gff (but it is quite compact already)
- deletions that go beyond transcript boundaries are not checked at sequence level
- alloc tscript->ref in hap_finalize, introduce fa_off_beg:16,fa_off_end:16
- see test/csq/ENST00000573314/insertion-overlap.vcf #1476288882
Read about transcript types here
http://vega.sanger.ac.uk/info/about/gene_and_transcript_types.html
https://www.ensembl.org/info/genome/variation/prediction/predicted_data.html
https://www.gencodegenes.org/pages/biotypes.html
List of supported biotypes
antisense
IG_C_gene
IG_D_gene
IG_J_gene
IG_LV_gene
IG_V_gene
lincRNA
lncRNA .. generic term for 3prime_overlapping_ncRNA, antisense, bidirectional_promoter_lncRNA, lincRNA, macro_lncRNA, non_coding, processed_transcript, sense_intronic, sense_overlapping
macro_lncRNA
miRNA
misc_RNA
Mt_rRNA
Mt_tRNA
polymorphic_pseudogene
processed_transcript
protein_coding, mRNA
ribozyme
rRNA
sRNA
scRNA
scaRNA
sense_intronic
sense_overlapping
snRNA
snoRNA
TR_C_gene
TR_D_gene
TR_J_gene
TR_V_gene
The gff parsing logic
We collect features such by combining gff lines A,B,C as follows:
A .. gene line with a supported biotype
A.ID=~/^gene:/
B .. transcript line referencing A with supported biotype
B.ID=~/^transcript:/ && B.Parent=~/^gene:A.ID/
C .. corresponding CDS, exon, and UTR lines:
C[3] in {"CDS","exon","three_prime_UTR","five_prime_UTR"} && C.Parent=~/^transcript:B.ID/
For coding biotypes ("protein_coding" or "polymorphic_pseudogene") the
complete chain link C -> B -> A is required. For the rest, link B -> A suffices.
The supported consequence types, sorted by impact:
splice_acceptor_variant .. end region of an intron changed (2bp at the 3' end of an intron)
splice_donor_variant .. start region of an intron changed (2bp at the 5' end of an intron)
stop_gained .. DNA sequence variant resulting in a stop codon
frameshift_variant .. number of inserted/deleted bases not a multiple of three, disrupted translational frame
stop_lost .. elongated transcript, stop codon changed
start_lost .. the first codon changed
inframe_altering .. combination of indels leading to unchanged reading frame and length
inframe_insertion .. inserted coding sequence, unchanged reading frame
inframe_deletion .. deleted coding sequence, unchanged reading frame
missense_variant .. amino acid (aa) change, unchanged length
splice_region_variant .. change within 1-3 bases of the exon or 3-8 bases of the intron
synonymous_variant .. DNA sequence variant resulting in no amino acid change
stop_retained_variant .. different stop codon
start_retained_variant .. start codon retained by indel realignment
non_coding_variant .. variant in non-coding sequence, such as RNA gene
5_prime_UTR_variant
3_prime_UTR_variant
intron_variant .. reported only if none of the above
intergenic_variant .. reported only if none of the above
The annotation algorithm.
The algorithm checks if the variant falls in a region of a supported type. The
search is performed in the following order, until a match is found:
1. idx_cds(gf_cds_t) - lookup CDS by position, create haplotypes, call consequences
2. idx_utr(gf_utr_t) - check UTR hits
3. idx_exon(gf_exon_t) - check for splice variants
4. idx_tscript(tscript_t) - check for intronic variants, RNAs, etc.
These regidx indexes are created by parsing a gff3 file as follows:
1. create the array "ftr" of all UTR, CDS, exons. This will be
processed later and pruned based on transcript types we want to keep.
In the same go, create the hash "id2tr" of transcripts to keep
(based on biotype) which maps from transcript_id to a transcript. At
the same time also build the hash "gid2gene" which maps from gene_id to
gf_gene_t pointer.
2. build "idx_cds", "idx_tscript", "idx_utr" and "idx_exon" indexes.
Use only features from "ftr" which are present in "id2tr".
3. clean data that won't be needed anymore: ftr, id2tr, gid2gene.
Data structures.
idx_cds, idx_utr, idx_exon, idx_tscript:
as described above, regidx structures for fast lookup of exons/transcripts
overlapping a region, the payload is a pointer to tscript.cds
*/
#include <stdio.h>
#include <stdlib.h>
#include <assert.h>
#include <getopt.h>
#include <math.h>
#include <inttypes.h>
#include <htslib/hts.h>
#include <htslib/vcf.h>
#include <htslib/synced_bcf_reader.h>
#include <htslib/khash.h>
#include <htslib/khash_str2int.h>
#include <htslib/kseq.h>
#include <htslib/faidx.h>
#include <htslib/bgzf.h>
#include <errno.h>
#include <unistd.h>
#include <ctype.h>
#include <strings.h>
#include "bcftools.h"
#include "filter.h"
#include "regidx.h"
#include "kheap.h"
#include "smpl_ilist.h"
#include "rbuf.h"
#include "gff.h"
#ifndef __FUNCTION__
# define __FUNCTION__ __func__
#endif
// Logic of the filters: include or exclude sites which match the filters?
#define FLT_INCLUDE 1
#define FLT_EXCLUDE 2
#define N_REF_PAD 10 // number of bases to avoid boundary effects
// How to treat phased/unphased genotypes
#define PHASE_REQUIRE 0 // --phase r
#define PHASE_MERGE 1 // --phase m
#define PHASE_AS_IS 2 // --phase a
#define PHASE_SKIP 3 // --phase s
#define PHASE_NON_REF 4 // --phase R
#define PHASE_DROP_GT 5 // --samples -
// Node types in the haplotype tree
#define HAP_CDS 0
#define HAP_ROOT 1
#define HAP_SSS 2 // start/stop/splice
#define CSQ_PRINTED_UPSTREAM (1<<0)
#define CSQ_SYNONYMOUS_VARIANT (1<<1)
#define CSQ_MISSENSE_VARIANT (1<<2)
#define CSQ_STOP_LOST (1<<3)
#define CSQ_STOP_GAINED (1<<4)
#define CSQ_INFRAME_DELETION (1<<5)
#define CSQ_INFRAME_INSERTION (1<<6)
#define CSQ_FRAMESHIFT_VARIANT (1<<7)
#define CSQ_SPLICE_ACCEPTOR (1<<8)
#define CSQ_SPLICE_DONOR (1<<9)
#define CSQ_START_LOST (1<<10)
#define CSQ_SPLICE_REGION (1<<11)
#define CSQ_STOP_RETAINED (1<<12)
#define CSQ_UTR5 (1<<13)
#define CSQ_UTR3 (1<<14)
#define CSQ_NON_CODING (1<<15)
#define CSQ_INTRON (1<<16)
//#define CSQ_INTERGENIC (1<<17)
#define CSQ_INFRAME_ALTERING (1<<18)
#define CSQ_UPSTREAM_STOP (1<<19) // adds * in front of the csq string
#define CSQ_INCOMPLETE_CDS (1<<20) // to remove START/STOP in incomplete CDS, see ENSG00000173376/synon.vcf
#define CSQ_CODING_SEQUENCE (1<<21) // cannot tell exactly what it is, but it does affect the coding sequence
#define CSQ_ELONGATION (1<<22) // symbolic insertion
#define CSQ_START_RETAINED (1<<23)
// Haplotype-aware consequences, printed in one vcf record only, the rest has a reference @12345
#define CSQ_COMPOUND (CSQ_SYNONYMOUS_VARIANT|CSQ_MISSENSE_VARIANT|CSQ_STOP_LOST|CSQ_STOP_GAINED| \
CSQ_INFRAME_DELETION|CSQ_INFRAME_INSERTION|CSQ_FRAMESHIFT_VARIANT| \
CSQ_START_LOST|CSQ_STOP_RETAINED|CSQ_INFRAME_ALTERING|CSQ_INCOMPLETE_CDS| \
CSQ_UPSTREAM_STOP|CSQ_START_RETAINED)
#define CSQ_START_STOP (CSQ_STOP_LOST|CSQ_STOP_GAINED|CSQ_STOP_RETAINED|CSQ_START_LOST|CSQ_START_RETAINED)
#define CSQ_PRN_STRAND(csq) ((csq)&CSQ_COMPOUND && !((csq)&(CSQ_SPLICE_ACCEPTOR|CSQ_SPLICE_DONOR|CSQ_SPLICE_REGION)))
#define CSQ_PRN_TSCRIPT (~(CSQ_INTRON|CSQ_NON_CODING))
#define CSQ_PRN_NMD (~(CSQ_INTRON|CSQ_NON_CODING))
#define CSQ_PRN_BIOTYPE CSQ_NON_CODING
// see kput_vcsq()
const char *csq_strings[] =
{
NULL,
"synonymous",
"missense",
"stop_lost",
"stop_gained",
"inframe_deletion",
"inframe_insertion",
"frameshift",
"splice_acceptor",
"splice_donor",
"start_lost",
"splice_region",
"stop_retained",
"5_prime_utr",
"3_prime_utr",
"non_coding",
"intron",
"intergenic",
"inframe_altering",
NULL,
NULL,
"coding_sequence",
"feature_elongation",
"start_retained"
};
/*
Structures related to VCF output:
vcsq_t
information required to assemble consequence lines such as "inframe_deletion|XYZ|ENST01|+|5TY>5I|121ACG>A+124TA>T"
vrec_t
single VCF record and csq tied to this record. (Haplotype can have multiple
consequences in several VCF records. Each record can have multiple consequences
from multiple haplotypes.)
csq_t
a top-level consequence tied to a haplotype
vbuf_t
pos2vbuf
VCF records with the same position clustered together for a fast lookup via pos2vbuf
*/
typedef struct _vbuf_t vbuf_t;
typedef struct _vcsq_t vcsq_t;
struct _vcsq_t
{
uint32_t strand:1,
type:31; // one of CSQ_* types
uint32_t trid;
uint32_t vcf_ial;
uint32_t biotype; // one of GF_* types
char *gene; // gene name
bcf1_t *ref; // if type&CSQ_PRINTED_UPSTREAM, ref consequence "@1234"
kstring_t vstr; // variant string, eg 5TY>5I|121ACG>A+124TA>T
};
typedef struct
{
bcf1_t *line;
uint32_t *fmt_bm; // bitmask of sample consequences with first/second haplotype interleaved
uint32_t nfmt:4, // the bitmask size (the number of integers per sample)
nvcsq:28, mvcsq;
vcsq_t *vcsq; // there can be multiple consequences for a single VCF record
}
vrec_t;
typedef struct
{
uint32_t pos;
vrec_t *vrec; // vcf line that this csq is tied to; needed when printing haplotypes (hap_stage_vcf)
int idx; // 0-based index of the csq at the VCF line, for FMT/BCSQ
vcsq_t type;
}
csq_t;
struct _vbuf_t
{
vrec_t **vrec; // buffer of VCF lines with the same position
int n, m;
uint32_t keep_until; // the maximum transcript end position
};
KHASH_MAP_INIT_INT(pos2vbuf, vbuf_t*)
/*
Structures related to haplotype-aware consequences in coding regions
hap_node_t
node of a haplotype tree. Each transcript has one tree
tscript_t
despite its general name, it is intended for coding transcripts only
hap_t
hstack_t
for traversal of the haplotype tree and braking combined
consequences into independent parts
*/
typedef struct _hap_node_t hap_node_t;
struct _hap_node_t
{
char *seq; // cds segment [parent_node,this_node)
char *var; // variant "ref>alt"
uint32_t type:2, // HAP_ROOT or HAP_CDS
csq:30; // this node's consequence
int dlen; // alt minus ref length: <0 del, >0 ins, 0 substitution
uint32_t rbeg; // variant's VCF position (0-based, inclusive)
int32_t rlen; // variant's rlen; alen=rlen+dlen; fake for non CDS types
uint32_t sbeg; // variant's position on the spliced reference transcript (0-based, inclusive, N_REF_PAD not included)
uint32_t icds; // which exon does this node's variant overlaps
hap_node_t **child, *prev; // children haplotypes and previous coding node
int nchild, mchild;
bcf1_t *cur_rec, *rec; // current VCF record and node's VCF record
int vcf_ial; // which VCF allele generated this node
uint32_t nend; // number of haplotypes ending in this node
int *cur_child, mcur_child; // mapping from the allele to the currently active child
csq_t *csq_list; // list of haplotype's consequences, broken by position (each corresponds to a VCF record)
int ncsq_list, mcsq_list;
};
#define TSCRIPT_AUX(x) ((tscript_t*)(x)->aux)
typedef struct
{
char *ref; // reference sequence, padded with N_REF_PAD bases on both ends
char *sref; // spliced reference sequence, padded with N_REF_PAD bases on both ends
hap_node_t *root; // root of the haplotype tree
hap_node_t **hap; // pointer to haplotype leaves, two for each sample
int nhap, nsref; // number of haplotypes and length of sref, including 2*N_REF_PAD
}
tscript_t;
static inline int cmp_tscript(gf_tscript_t **a, gf_tscript_t **b)
{
return ( (*a)->end < (*b)->end ) ? 1 : 0;
}
KHEAP_INIT(trhp, gf_tscript_t*, cmp_tscript)
typedef khp_trhp_t tr_heap_t;
typedef struct
{
hap_node_t *node; // current node
int ichild; // current child in the active node
int dlen; // total dlen, from the root to the active node
size_t slen; // total sequence length, from the root to the active node
}
hstack_t;
typedef struct
{
int mstack;
hstack_t *stack;
gf_tscript_t *tr; // tr->ref: spliced transcript on ref strand
kstring_t sseq; // spliced haplotype sequence on ref strand
kstring_t tseq; // the variable part of translated haplotype transcript, coding strand
kstring_t tref; // the variable part of translated reference transcript, coding strand
uint32_t sbeg; // stack's sbeg, for cases first node's type is HAP_SSS
int upstream_stop;
}
hap_t;
typedef struct _args_t
{
// the main regidx lookups, from chr:beg-end to overlapping features and
// index iterator
gff_t *gff;
regidx_t *idx_cds, *idx_utr, *idx_exon, *idx_tscript;
regitr_t *itr;
// text tab-delimited output (out) or vcf/bcf output (out_fh)
FILE *out;
htsFile *out_fh;
char *index_fn;
int write_index;
char *dump_gff;
// vcf
bcf_srs_t *sr;
bcf_hdr_t *hdr;
int hdr_nsmpl; // actual number of samples in the vcf, for bcf_update_format_values()
// include or exclude sites which match the filters
filter_t *filter;
char *filter_str;
int filter_logic; // FLT_INCLUDE or FLT_EXCLUDE
// samples to process
int sample_is_file;
char *sample_list;
smpl_ilist_t *smpl;
char *outdir, **argv, *fa_fname, *gff_fname, *output_fname;
char *bcsq_tag;
int argc, output_type, clevel;
int phase, verbosity, local_csq, record_cmd_line;
int ncsq2_max, nfmt_bcsq; // maximum number of csq per site that can be accessed from FORMAT/BCSQ (*2 and 1 bit skipped to avoid BCF missing values)
int ncsq2_small_warned;
int brief_predictions;
int unify_chr_names;
char *chr_name;
int mchr_name;
struct {
int unknown_chr,unknown_tscript_biotype,unknown_strand,unknown_phase,duplicate_id;
int unknown_cds_phase,incomplete_cds,wrong_phase,overlapping_cds;
} warned;
int rid; // current chromosome
tr_heap_t *active_tr; // heap of active transcripts for quick flushing
hap_t *hap; // transcript haplotype recursion
vbuf_t **vcf_buf; // buffered VCF lines to annotate with CSQ and flush
rbuf_t vcf_rbuf; // round buffer indexes to vcf_buf
kh_pos2vbuf_t *pos2vbuf; // fast lookup of buffered lines by position
gf_tscript_t **rm_tr; // buffer of transcripts to clean
int nrm_tr, mrm_tr;
csq_t *csq_buf; // pool of csq not managed by hap_node_t, i.e. non-CDS csqs
int ncsq_buf, mcsq_buf;
int force; // force run under various conditions. Currently only to skip out-of-phase transcripts
int n_threads; // extra compression/decompression threads
faidx_t *fai;
kstring_t str, str2;
int32_t *gt_arr, mgt_arr;
}
args_t;
// AAA, AAC, ...
const char *gencode = "KNKNTTTTRSRSIIMIQHQHPPPPRRRRLLLLEDEDAAAAGGGGVVVV*Y*YSSSS*CWCLFLF";
const uint8_t nt4[] =
{
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,
4,0,4,1, 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
};
const uint8_t cnt4[] =
{
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,
4,3,4,2, 4,4,4,1, 4,4,4,4, 4,4,4,4,
4,4,4,4, 0,4,4,4, 4,4,4,4, 4,4,4,4,
4,3,4,2, 4,4,4,1, 4,4,4,4, 4,4,4,4,
4,4,4,4, 0
};
#define dna2aa(x) gencode[ nt4[(uint8_t)(x)[0]]<<4 | nt4[(uint8_t)(x)[1]]<<2 | nt4[(uint8_t)(x)[2]] ]
#define cdna2aa(x) gencode[ cnt4[(uint8_t)(x)[2]]<<4 | cnt4[(uint8_t)(x)[1]]<<2 | cnt4[(uint8_t)(x)[0]] ]
static inline int ncsq2_to_nfmt(int ncsq2)
{
return 1 + (ncsq2 - 1) / 30;
}
static inline void icsq2_to_bit(int icsq2, int *ival, int *ibit)
{
*ival = icsq2 / 30;
*ibit = icsq2 % 30;
}
void init_data(args_t *args)
{
args->nfmt_bcsq = ncsq2_to_nfmt(args->ncsq2_max);
args->fai = fai_load(args->fa_fname);
if ( !args->fai ) error("Failed to load the fai index: %s\n", args->fa_fname);
args->gff = gff_init(args->gff_fname);
gff_set(args->gff,verbosity,args->verbosity);
gff_set(args->gff,strip_chr_names,args->unify_chr_names);
gff_set(args->gff,force_out_of_phase,args->force);
gff_set(args->gff,dump_fname,args->dump_gff);
gff_parse(args->gff);
args->idx_cds = gff_get(args->gff,idx_cds);
args->idx_utr = gff_get(args->gff,idx_utr);
args->idx_exon = gff_get(args->gff,idx_exon);
args->idx_tscript = gff_get(args->gff,idx_tscript);
args->itr = regitr_init(NULL);
args->rid = -1;
if ( args->filter_str )
args->filter = filter_init(args->hdr, args->filter_str);
args->pos2vbuf = kh_init(pos2vbuf);
args->active_tr = khp_init(trhp);
args->hap = (hap_t*) calloc(1,sizeof(hap_t));
// init samples
if ( !bcf_hdr_nsamples(args->hdr) ) args->phase = PHASE_DROP_GT;
if ( args->sample_list && !strcmp("-",args->sample_list) )
{
// ignore all samples
if ( args->output_type==FT_TAB_TEXT )
{
// significant speedup for plain VCFs
if (bcf_hdr_set_samples(args->hdr,NULL,0) < 0)
error_errno("[%s] Couldn't build sample filter", __func__);
}
args->phase = PHASE_DROP_GT;
}
else
args->smpl = smpl_ilist_init(args->hdr, args->sample_list, args->sample_is_file, SMPL_STRICT);
args->hdr_nsmpl = args->phase==PHASE_DROP_GT ? 0 : bcf_hdr_nsamples(args->hdr);
if ( args->output_type==FT_TAB_TEXT )
{
args->out = args->output_fname ? fopen(args->output_fname,"w") : stdout;
if ( !args->out ) error("Failed to write to %s: %s\n", !strcmp("-",args->output_fname)?"standard output":args->output_fname,strerror(errno));
fprintf(args->out,"# This file was produced by: bcftools +csq(%s+htslib-%s)\n", bcftools_version(),hts_version());
fprintf(args->out,"# The command line was:\tbcftools +%s", args->argv[0]);
int i;
for (i=1; i<args->argc; i++)
fprintf(args->out," %s",args->argv[i]);
fprintf(args->out,"\n");
fprintf(args->out,"# LOG\t[2]Message\n");
fprintf(args->out,"# CSQ"); i = 1;
fprintf(args->out,"\t[%d]Sample", ++i);
fprintf(args->out,"\t[%d]Haplotype", ++i);
fprintf(args->out,"\t[%d]Chromosome", ++i);
fprintf(args->out,"\t[%d]Position", ++i);
fprintf(args->out,"\t[%d]Consequence", ++i);
fprintf(args->out,"\n");
}
else
{
char wmode[8];
set_wmode(wmode,args->output_type,args->output_fname,args->clevel);
args->out_fh = hts_open(args->output_fname ? args->output_fname : "-", wmode);
if ( args->out_fh == NULL ) error("[%s] Error: cannot write to %s: %s\n", __func__,args->output_fname? args->output_fname : "standard output", strerror(errno));
if ( args->n_threads > 0)
hts_set_opt(args->out_fh, HTS_OPT_THREAD_POOL, args->sr->p);
if ( args->record_cmd_line ) bcf_hdr_append_version(args->hdr,args->argc,args->argv,"bcftools/csq");
bcf_hdr_printf(args->hdr,"##INFO=<ID=%s,Number=.,Type=String,Description=\"%s consequence annotation from BCFtools/csq, see http://samtools.github.io/bcftools/howtos/csq-calling.html for details. Format: Consequence|gene|transcript|biotype|strand|amino_acid_change|dna_change\">",args->bcsq_tag, args->local_csq ? "Local" : "Haplotype-aware");
if ( args->hdr_nsmpl )
bcf_hdr_printf(args->hdr,"##FORMAT=<ID=%s,Number=.,Type=Integer,Description=\"Bitmask of indexes to INFO/BCSQ, with interleaved first/second haplotype. Use \\\"bcftools query -f'[%%CHROM\\t%%POS\\t%%SAMPLE\\t%%TBCSQ\\n]'\\\" to translate.\">",args->bcsq_tag);
if ( bcf_hdr_write(args->out_fh, args->hdr)!=0 ) error("[%s] Error: cannot write the header to %s\n", __func__,args->output_fname?args->output_fname:"standard output");
if ( init_index2(args->out_fh,args->hdr,args->output_fname,
&args->index_fn, args->write_index) < 0 )
error("Error: failed to initialise index for %s\n",args->output_fname);
}
if ( args->verbosity > 0 ) fprintf(stderr,"Calling...\n");
}
void destroy_data(args_t *args)
{
if ( args->ncsq2_small_warned )
fprintf(stderr,
"Note: Some samples had too many consequences to be represented in %d bytes. If you need to record them all,\n"
" the limit can be increased by running with `--ncsq %d`.\n",ncsq2_to_nfmt(args->ncsq2_max)/8,1+args->ncsq2_small_warned/2);
regitr_destroy(args->itr);
gff_destroy(args->gff);
if ( args->filter )
filter_destroy(args->filter);
khp_destroy(trhp,args->active_tr);
kh_destroy(pos2vbuf,args->pos2vbuf);
if ( args->smpl ) smpl_ilist_destroy(args->smpl);
int i,j,ret;
if ( args->out_fh )
{
if ( args->write_index )
{
if ( bcf_idx_save(args->out_fh)<0 )
{
if ( hts_close(args->out_fh)!=0 ) error("Error: close failed .. %s\n", args->output_fname?args->output_fname:"stdout");
error("Error: cannot write to index %s\n", args->index_fn);
}
free(args->index_fn);
}
ret = hts_close(args->out_fh);
}
else
ret = fclose(args->out);
if ( ret ) error("Error: close failed .. %s\n", args->output_fname?args->output_fname:"stdout");
for (i=0; i<args->vcf_rbuf.m; i++)
{
vbuf_t *vbuf = args->vcf_buf[i];
if ( !vbuf ) continue;
for (j=0; j<vbuf->m; j++)
{
if ( !vbuf->vrec[j] ) continue;
if ( vbuf->vrec[j]->line ) bcf_destroy(vbuf->vrec[j]->line);
free(vbuf->vrec[j]->fmt_bm);
free(vbuf->vrec[j]->vcsq);
free(vbuf->vrec[j]);
}
free(vbuf->vrec);
free(vbuf);
}
free(args->vcf_buf);
free(args->rm_tr);
free(args->csq_buf);
free(args->hap->stack);
free(args->hap->sseq.s);
free(args->hap->tseq.s);
free(args->hap->tref.s);
free(args->hap);
fai_destroy(args->fai);
free(args->gt_arr);
free(args->str.s);
free(args->str2.s);
free(args->chr_name);
}
/*
The splice_* functions are for consequences around splice sites: start,stop,splice_*
*/
#define SPLICE_VAR_REF 0 // ref: ACGT>ACGT, csq not applicable, skip completely
#define SPLICE_OUTSIDE 1 // splice acceptor or similar; csq set and is done, does not overlap the region
#define SPLICE_INSIDE 2 // overlaps coding region; csq can be set but coding prediction is needed
#define SPLICE_OVERLAP 3 // indel overlaps region boundary, csq set but could not determine csq
typedef struct
{
gf_tscript_t *tr;
struct {
int32_t pos, rlen, alen, ial;
char *ref, *alt;
bcf1_t *rec;
} vcf;
uint16_t check_acceptor:1, // check distance from exon start (fwd) or end (rev)
check_start:1, // this is the first coding exon (relative to transcript orientation), check first (fwd) or last (rev) codon
check_stop:1, // this is the last coding exon (relative to transcript orientation), check last (fwd) or first (rev) codon
check_donor:1, // as with check_acceptor
check_region_beg:1, // do/don't check for splices at this end, eg. in the first or last exon
check_region_end:1, //
check_utr:1, // check splice sites (acceptor/donor/region_*) only if not in utr
set_refalt:1; // set kref,kalt, if set, check also for synonymous events
uint32_t csq;
int tbeg, tend; // number of trimmed bases from beg and end of ref,alt allele
uint32_t ref_beg, // ref coordinates with spurious bases removed, ACC>AC can become AC>A or CC>C, whichever gives
ref_end; // a more conservative csq (the first and last base in kref.s)
kstring_t kref, kalt; // trimmed alleles, set only with SPLICE_OLAP
}
splice_t;
void splice_init(splice_t *splice, bcf1_t *rec)
{
memset(splice,0,sizeof(*splice));
splice->vcf.rec = rec;
splice->vcf.pos = rec->pos;
splice->vcf.rlen = rec->rlen;
splice->vcf.ref = rec->d.allele[0];
splice->csq = 0;
}
static inline void splice_build_hap(splice_t *splice, uint32_t beg, int len)
{
// len>0 .. beg is the first base, del filled from right
// len<0 .. beg is the last base, del filled from left
int rlen, alen, rbeg, abeg; // first base to include (ref coordinates)
if ( len<0 )
{
rlen = alen = -len;
rbeg = beg - rlen + 1;
int dlen = splice->vcf.alen - splice->vcf.rlen;
if ( dlen<0 && beg < splice->ref_end ) // incomplete del, beg is in the middle
dlen += splice->ref_end - beg;
abeg = rbeg + dlen;
}
else
{
rbeg = abeg = beg;
rlen = alen = len;
// check for incomplete del as above??
}
#define XDBG 0
#if XDBG
fprintf(stderr,"build_hap: rbeg=%d + %d abeg=%d \n",rbeg,rlen,abeg);
#endif
splice->kref.l = 0;
splice->kalt.l = 0;
// add the part before vcf.ref, in the vcf.ref and after vcf.ref
int roff; // how many vcf.ref bases already used
if ( rbeg < splice->vcf.pos )
{
assert( splice->tr->beg <= rbeg ); // this can be extended thanks to N_REF_PAD
kputsn(TSCRIPT_AUX(splice->tr)->ref + N_REF_PAD + rbeg - splice->tr->beg, splice->vcf.pos - rbeg, &splice->kref);
roff = 0;
}
else
roff = rbeg - splice->vcf.pos;
#if XDBG
fprintf(stderr,"r1: %s roff=%d\n",splice->kref.s,roff);
#endif
if ( roff < splice->vcf.rlen && splice->kref.l < rlen )
{
int len = splice->vcf.rlen - roff; // len still available in vcf.ref
if ( len > rlen - splice->kref.l ) len = rlen - splice->kref.l; // how much of ref allele is still needed
kputsn(splice->vcf.ref + roff, len, &splice->kref);
}
#if XDBG
fprintf(stderr,"r2: %s\n",splice->kref.s);
#endif
uint32_t end = splice->vcf.pos + splice->vcf.rlen; // position just after the ref allele
if ( splice->kref.l < rlen )
{
if ( end + rlen - splice->kref.l - 1 > splice->tr->end ) // trim, the requested sequence is too long (could be extended, see N_REF_PAD)
rlen -= end + rlen - splice->kref.l - 1 - splice->tr->end;
if ( splice->kref.l < rlen )
kputsn(TSCRIPT_AUX(splice->tr)->ref + N_REF_PAD + end - splice->tr->beg, rlen - splice->kref.l, &splice->kref);
}
#if XDBG
fprintf(stderr,"r3: %s\n",splice->kref.s);
#endif
int aoff;
if ( abeg < splice->vcf.pos )
{
assert( splice->tr->beg <= abeg );
kputsn(TSCRIPT_AUX(splice->tr)->ref + N_REF_PAD + abeg - splice->tr->beg, splice->vcf.pos - abeg, &splice->kalt);
aoff = 0;
}
else
aoff = abeg - splice->vcf.pos;
#if XDBG
fprintf(stderr,"a1: %s aoff=%d\n",splice->kalt.s,aoff);
#endif
if ( aoff < splice->vcf.alen && splice->kalt.l < alen )
{
int len = splice->vcf.alen - aoff; // len still available in vcf.alt
if ( len > alen - splice->kalt.l ) len = alen - splice->kalt.l; // how much of alt allele is still needed
kputsn(splice->vcf.alt + aoff, len, &splice->kalt);
aoff -= len;
}
if ( aoff < 0 ) aoff = 0;
else aoff--;
#if XDBG
fprintf(stderr,"a2: %s aoff=%d\n",splice->kalt.s,aoff);
#endif
end = splice->vcf.pos + splice->vcf.rlen; // position just after the ref allele
if ( splice->kalt.l < alen )
{
if ( end + alen + aoff - splice->kalt.l - 1 > splice->tr->end ) // trim, the requested sequence is too long
alen -= end + alen + aoff - splice->kalt.l - 1 - splice->tr->end;
if ( alen > 0 && alen > splice->kalt.l )
kputsn(TSCRIPT_AUX(splice->tr)->ref + aoff + N_REF_PAD + end - splice->tr->beg, alen - splice->kalt.l, &splice->kalt);
}
#if XDBG
fprintf(stderr,"a3: %s\n",splice->kalt.s);
fprintf(stderr," [%s]\n [%s]\n\n",splice->kref.s,splice->kalt.s);
#endif
}
void csq_stage(args_t *args, csq_t *csq, bcf1_t *rec);
static inline int csq_stage_utr(args_t *args, regitr_t *itr, bcf1_t *rec, uint32_t trid, uint32_t type, int ial)
{
while ( regitr_overlap(itr) )
{
gf_utr_t *utr = regitr_payload(itr, gf_utr_t*);
gf_tscript_t *tr = utr->tr;
if ( tr->id != trid ) continue;
csq_t csq;
memset(&csq, 0, sizeof(csq_t));
csq.pos = rec->pos;
csq.type.type = (utr->which==prime5 ? CSQ_UTR5 : CSQ_UTR3) | type;
csq.type.biotype = tr->type;
csq.type.strand = tr->strand;
csq.type.trid = tr->id;
csq.type.vcf_ial = ial;
csq.type.gene = tr->gene->name;
csq_stage(args, &csq, rec);
return csq.type.type;
}
return 0;
}
static inline void csq_stage_splice(args_t *args, bcf1_t *rec, gf_tscript_t *tr, uint32_t type, int ial)
{
#if XDBG
fprintf(stderr,"csq_stage_splice %d: type=%d\n",(int)rec->pos+1,type);
#endif
if ( !type ) return;
csq_t csq;
memset(&csq, 0, sizeof(csq_t));
csq.pos = rec->pos;
csq.type.type = type;
csq.type.biotype = tr->type;
csq.type.strand = tr->strand;
csq.type.trid = tr->id;
csq.type.vcf_ial = ial;
csq.type.gene = tr->gene->name;
csq_stage(args, &csq, rec);
}
static inline const char *drop_chr_prefix(args_t *args, const char *chr)
{
if ( !args->unify_chr_names ) return chr;
if ( !strncasecmp("chr",chr,3) ) return chr+3;
return chr;
}
static inline const char *add_chr_prefix(args_t *args, const char *chr)
{
if ( !args->unify_chr_names ) return chr;
int len = strlen(chr);
hts_expand(char,len+4,args->mchr_name,args->chr_name);
memcpy(args->chr_name,"chr",3);
memcpy(args->chr_name+3,chr,len+1);
return args->chr_name;
}
static inline int splice_csq_ins(args_t *args, splice_t *splice, uint32_t ex_beg, uint32_t ex_end)
{
// coordinates that matter for consequences, eg AC>ACG trimmed to C>CG, 1bp
// before and after the inserted bases
if ( splice->tbeg || splice->vcf.ref[0]!=splice->vcf.alt[0] )
{
splice->ref_beg = splice->vcf.pos + splice->tbeg - 1;
splice->ref_end = splice->vcf.pos + splice->vcf.rlen - splice->tend;
}
else
{
if ( splice->tend ) splice->tend--;
splice->ref_beg = splice->vcf.pos;
splice->ref_end = splice->vcf.pos + splice->vcf.rlen - splice->tend;
}
#if XDBG
fprintf(stderr,"ins: %s>%s .. ex=%d,%d beg,end=%d,%d tbeg,tend=%d,%d check_utr=%d start,stop,beg,end=%d,%d,%d,%d\n", splice->vcf.ref,splice->vcf.alt,ex_beg,ex_end,splice->ref_beg,splice->ref_end,splice->tbeg,splice->tend,splice->check_utr,splice->check_start,splice->check_stop,splice->check_region_beg,splice->check_region_end);
#endif
int ret;
if ( splice->ref_beg >= ex_end ) // fully outside, beyond the exon
{
if ( splice->check_utr )
{
regitr_t *itr = regitr_init(NULL);
const char *chr = drop_chr_prefix(args, bcf_seqname(args->hdr,splice->vcf.rec));
if ( regidx_overlap(args->idx_utr,chr,splice->ref_beg+1,splice->ref_beg+1, itr) ) // adjacent utr
{
ret = csq_stage_utr(args, itr, splice->vcf.rec, splice->tr->id, splice->csq, splice->vcf.ial);
if ( ret!=0 )
{
regitr_destroy(itr);
return SPLICE_OUTSIDE; // overlaps utr
}
}
regitr_destroy(itr);
}
if ( !splice->check_region_end ) return SPLICE_OUTSIDE;
char *ref = NULL, *alt = NULL;
if ( splice->set_refalt ) // seq identity is checked only when tr->ref is available
{
splice_build_hap(splice, ex_end+1, N_SPLICE_REGION_INTRON);
ref = splice->kref.s, alt = splice->kalt.s;
}
if ( splice->ref_beg < ex_end + N_SPLICE_REGION_INTRON && splice->ref_end > ex_end + N_SPLICE_DONOR )
{
splice->csq |= CSQ_SPLICE_REGION;
if ( ref && !strncmp(ref,alt,N_SPLICE_REGION_INTRON) ) splice->csq |= CSQ_SYNONYMOUS_VARIANT;
}
if ( splice->ref_beg < ex_end + N_SPLICE_DONOR )
{
if ( splice->check_donor && splice->tr->strand==STRAND_FWD ) splice->csq |= CSQ_SPLICE_DONOR;
if ( splice->check_acceptor && splice->tr->strand==STRAND_REV ) splice->csq |= CSQ_SPLICE_ACCEPTOR;
if ( ref && !strncmp(ref,alt,N_SPLICE_DONOR) ) splice->csq |= CSQ_SYNONYMOUS_VARIANT;
}
csq_stage_splice(args, splice->vcf.rec, splice->tr, splice->csq, splice->vcf.ial);
return SPLICE_OUTSIDE;
}
if ( splice->ref_end < ex_beg || (splice->ref_end == ex_beg && !splice->check_region_beg) ) // fully outside, before the exon
{
if ( splice->check_utr )
{
regitr_t *itr = regitr_init(NULL);
const char *chr = drop_chr_prefix(args, bcf_seqname(args->hdr,splice->vcf.rec));
if ( regidx_overlap(args->idx_utr,chr,splice->ref_end-1,splice->ref_end-1, itr) ) // adjacent utr
{
ret = csq_stage_utr(args, itr, splice->vcf.rec, splice->tr->id, splice->csq, splice->vcf.ial);
if ( ret!=0 )
{
regitr_destroy(itr);
return SPLICE_OUTSIDE; // overlaps utr
}
}
regitr_destroy(itr);
}
if ( !splice->check_region_beg ) return SPLICE_OUTSIDE;
char *ref = NULL, *alt = NULL;
if ( splice->set_refalt ) // seq identity is checked only when tr->ref is available
{
splice_build_hap(splice, ex_beg - N_SPLICE_REGION_INTRON, N_SPLICE_REGION_INTRON);
ref = splice->kref.s, alt = splice->kalt.s;
}
if ( splice->ref_end > ex_beg - N_SPLICE_REGION_INTRON && splice->ref_beg < ex_beg - N_SPLICE_DONOR )
{
splice->csq |= CSQ_SPLICE_REGION;
if ( ref && !strncmp(ref,alt,N_SPLICE_REGION_INTRON) ) splice->csq |= CSQ_SYNONYMOUS_VARIANT;
}
if ( splice->ref_end > ex_beg - N_SPLICE_DONOR )
{
if ( splice->check_donor && splice->tr->strand==STRAND_REV ) splice->csq |= CSQ_SPLICE_DONOR;
if ( splice->check_acceptor && splice->tr->strand==STRAND_FWD ) splice->csq |= CSQ_SPLICE_ACCEPTOR;
if ( ref && !strncmp(ref+N_SPLICE_REGION_INTRON-N_SPLICE_DONOR,alt+N_SPLICE_REGION_INTRON-N_SPLICE_DONOR,N_SPLICE_DONOR) ) splice->csq |= CSQ_SYNONYMOUS_VARIANT;
}
csq_stage_splice(args, splice->vcf.rec, splice->tr, splice->csq, splice->vcf.ial);
return SPLICE_OUTSIDE;
}
// overlaps the exon or inside the exon
// possible todo: find better alignment for frameshifting variants?
if ( splice->ref_beg <= ex_beg + 2 ) // in the first 3bp
{
if ( splice->check_region_beg ) splice->csq |= CSQ_SPLICE_REGION;
if ( splice->tr->strand==STRAND_FWD ) { if ( splice->check_start ) splice->csq |= CSQ_START_LOST; }
else { if ( splice->check_stop ) splice->csq |= CSQ_STOP_LOST; }
}
if ( splice->ref_end > ex_end - 2 )
{
if ( splice->check_region_end ) splice->csq |= CSQ_SPLICE_REGION;
if ( splice->tr->strand==STRAND_REV ) { if ( splice->check_start ) splice->csq |= CSQ_START_LOST; }
else { if ( splice->check_stop ) splice->csq |= CSQ_STOP_LOST; }
}
if ( splice->set_refalt )
{
// Make sure the variant will not end up left aligned to avoid overlapping vcf records
// splice_build_hap(splice, splice->ref_beg, splice->vcf.alen - splice->tend - splice->tbeg + 1);
// splice->vcf.rlen -= splice->tbeg + splice->tend - 1;
// if ( splice->kref.l > splice->vcf.rlen ) { splice->kref.l = splice->vcf.rlen; splice->kref.s[splice->kref.l] = 0; }
if ( splice->ref_beg < splice->vcf.pos ) // this must have been caused by too much trimming from right
{
int dlen = splice->vcf.pos - splice->ref_beg;
assert( dlen==1 );
splice->tbeg += dlen;
if ( splice->tbeg + splice->tend == splice->vcf.rlen ) splice->tend -= dlen;
splice->ref_beg = splice->vcf.pos;
}
if ( splice->ref_end==ex_beg ) splice->tend--; // prevent zero-length ref allele
splice_build_hap(splice, splice->ref_beg, splice->vcf.alen - splice->tend - splice->tbeg + 1);
splice->vcf.rlen -= splice->tbeg + splice->tend - 1;
if ( splice->kref.l > splice->vcf.rlen ) { splice->kref.l = splice->vcf.rlen; splice->kref.s[splice->kref.l] = 0; }
}
csq_stage_splice(args, splice->vcf.rec, splice->tr, splice->csq, splice->vcf.ial);
return SPLICE_INSIDE;
}
int shifted_del_synonymous(args_t *args, splice_t *splice, uint32_t ex_beg, uint32_t ex_end)
{
static int small_ref_padding_warned = 0;
gf_tscript_t *tr = splice->tr;
// We know the VCF record overlaps the exon, but does it overlap the start codon?
if ( tr->strand==STRAND_REV && splice->vcf.pos + splice->vcf.rlen + 2 <= ex_end ) return 0;
if ( tr->strand==STRAND_FWD && splice->vcf.pos >= ex_beg + 3 ) return 0;
#if XDBG
fprintf(stderr,"shifted_del_synonymous: %d-%d %s\n",ex_beg,ex_end, tr->strand==STRAND_FWD?"fwd":(tr->strand==STRAND_REV?"rev":"unk"));
fprintf(stderr," %d .. %s > %s\n",splice->vcf.pos+1,splice->vcf.ref,splice->vcf.alt);
#endif
// is there enough ref sequence for the extension? All coordinates are 0-based
int ref_len = strlen(splice->vcf.ref);
int alt_len = strlen(splice->vcf.alt);
assert( ref_len > alt_len );
int ndel = ref_len - alt_len;
if ( tr->strand==STRAND_REV )
{
int32_t vcf_ref_end = splice->vcf.pos + ref_len - 1; // end pos of the VCF REF allele
int32_t tr_ref_end = splice->tr->end + N_REF_PAD; // the end pos of accessible cached ref seq
if ( vcf_ref_end + ndel > tr_ref_end )
{
if ( !small_ref_padding_warned )
{
fprintf(stderr,"Warning: Could not verify synonymous start/stop at %s:%d due to small N_REF_PAD. (Improve me?)\n",bcf_seqname(args->hdr,splice->vcf.rec),splice->vcf.pos+1);
small_ref_padding_warned = 1;
}
return 0;
}
char *ptr_vcf = splice->vcf.ref + alt_len; // the first deleted base in the VCF REF allele
char *ptr_ref = TSCRIPT_AUX(splice->tr)->ref + N_REF_PAD + (vcf_ref_end + 1 - splice->tr->beg); // the first ref base after the ndel bases deleted
#if XDBG
fprintf(stderr,"vcf: %s\nref: %s\n",ptr_vcf,ptr_ref);
#endif
int i = 0;
while ( ptr_vcf[i] && ptr_vcf[i]==ptr_ref[i] ) i++;
if ( ptr_vcf[i] ) return 0; // the deleted sequence cannot be replaced