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bam_ampliconclip.c
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bam_ampliconclip.c
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/* bam_ampliconclip.c -- loads amplicon primers from a BED file and cuts reads
from the 5' end.
Copyright (C) 2020-2024 Genome Research Ltd.
Authors: Andrew Whitwham <[email protected]>
Rob Davies <[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 <config.h>
#include <assert.h>
#include <stdbool.h>
#include <stdlib.h>
#include <string.h>
#include <unistd.h>
#include <errno.h>
#include "htslib/thread_pool.h"
#include "sam_opts.h"
#include <htslib/hts.h>
#include "htslib/hfile.h"
#include "htslib/kstring.h"
#include "htslib/sam.h"
#include "samtools.h"
#include "bam_ampliconclip.h"
typedef enum {
soft_clip,
hard_clip
} clipping_type;
typedef struct {
int add_pg;
int use_strand;
int write_clipped;
int mark_fail;
int both;
int fail_len;
int filter_len;
int unmapped;
int oa_tag;
int del_tag;
int tol;
int unmap_len;
char *arg_list;
char *stats_file;
char *primer_counts_file;
char *rejects_file;
} cl_param_t;
static int bed_entry_sort(const void *av, const void *bv) {
bed_entry_t *a = (bed_entry_t *) av;
bed_entry_t *b = (bed_entry_t *) bv;
return a->right < b->right ? -1 : (a->right == b->right ? 0 : 1);
}
int load_bed_file_multi_ref(char *infile, int get_strand, int sort_by_pos, khash_t(bed_list_hash) *bed_lists) {
hFILE *fp;
int line_count = 0, num_columns = 0, ret;
//variables to store the bed file data for each record
char ref[1024] = "";
int64_t left, right;
char name[1024] = "", score[1024] = "";
char strand;
//hash table to store clipping results and bed file data
kstring_t line = KS_INITIALIZE;
bed_entry_list_t *list;
khiter_t bed_itr;
//scanf components to parse the bed file, depending on the number of columns in the bed file only some may be used
char *scanf_components[] = {
"%1023s", // ref
"%"PRId64, // left
"%"PRId64, // right
"%1023s", // name
"%1023s", // score
"%c" // strand
};
if ((fp = hopen(infile, "r")) == NULL) {
print_error_errno("amplicon", "unable to open file %s.", infile);
return 1;
}
while (line.l = 0, kgetline(&line, (kgets_func *)hgets, fp) >= 0) {
line_count++;
int hret;
if (line.l == 0 || *line.s == '#') continue;
if (strncmp(line.s, "track ", 6) == 0) continue;
if (strncmp(line.s, "browser ", 8) == 0) continue;
num_columns = 0;
// count the number of line columns in line.s, and use that to determine the number of sscanf arguments to use
for (char *c = line.s; (c = strchr(c, '\t')) != NULL; c++) {
num_columns++;
}
num_columns++; // Add one to account for the last column
// num columns must be less than or equal to the number of scanf components
if (num_columns > sizeof(scanf_components)) {
fprintf(stderr, "[amplicon] error: too many columns in line %d of %s.\n"
"Found %d columns, but only up to %zu are expected.\n",
line_count, infile, num_columns, sizeof(scanf_components) / sizeof(scanf_components[0]));
ret = 1;
goto error;
}
// construct a scanf string using the scanf_components array elements used to parse the line
char scanf_str[1024] = "";
for (int i = 0; i < num_columns; i++) {
snprintf(scanf_str + strlen(scanf_str), sizeof(scanf_str) - strlen(scanf_str) - 1, "%s ", scanf_components[i]);
}
int cols_parsed = 0;
// extract the data from the line into the variables. Variables corresponding to any missing columns will remain uninitialised
if ((cols_parsed = sscanf(line.s, scanf_str, ref, &left, &right, name, score, &strand)) != num_columns || cols_parsed < 3) {
fprintf(stderr, "[amplicon] error: invalid bed file format in line %d of %s.\n"
"Found %d columns, parsed %d using %s on line:\n%s\n"
"(N.B. ref/chrom name limited to 1023 characters.)\n",
line_count, infile, num_columns, cols_parsed, scanf_str, line.s);
ret = 1;
goto error;
}
bed_itr = kh_get(bed_list_hash, bed_lists, ref);
if (bed_itr == kh_end(bed_lists)) { // new ref entry
char *ref_name = strdup(ref); // need a copy for the hash key
if (!ref_name) {
fprintf(stderr, "[amplicon] error: unable to allocate memory for ref name.\n");
ret = 1;
goto error;
}
bed_itr = kh_put(bed_list_hash, bed_lists, ref_name, &hret);
if (hret > 0) {
list = &kh_val(bed_lists, bed_itr);
// initialise the new hash entry
list->longest = 0;
list->size = 0;
list->length = 0;
list->bp = NULL;
} else {
fprintf(stderr, "[amplicon] error: ref hashing failure.\n");
ret = 1;
goto error;
}
} else { // existing ref
list = &kh_val(bed_lists, bed_itr);
}
// add the bed entry to the list, growing the list if necessary
if (list->length == list->size) {
bed_entry_t *tmp;
list->size += list->size / 2 + 256;
if ((tmp = realloc(list->bp, list->size * sizeof(bed_entry_t))) == NULL) {
fprintf(stderr, "[amplicon] error: unable to allocate more memory for bed data.\n");
ret = 1;
goto error;
}
list->bp = tmp;
}
list->bp[list->length].left = left;
list->bp[list->length].right = right;
if (num_columns >= 4) {
list->bp[list->length].name = strdup(name);
if (list->bp[list->length].name == NULL) {
fprintf(stderr, "[amplicon] error: unable to allocate memory for name in line %d of %s: %s.\n", line_count, infile, line.s);
ret = 1;
goto error;
}
}
if (num_columns >= 5) {
list->bp[list->length].score = strdup(score);
if (list->bp[list->length].score == NULL) {
fprintf(stderr, "[amplicon] error: unable to allocate memory for score in line %d of %s: %s\n", line_count, infile, line.s);
ret = 1;
goto error;
}
}
list->bp[list->length].num_reads = 0;
if (get_strand) {
if (strand == '+') {
list->bp[list->length].rev = 0;
} else if (strand == '-') {
list->bp[list->length].rev = 1;
} else {
fprintf(stderr, "[amplicon] error: bad strand value in line %d, expecting '+' or '-', found '%c'.\n",
line_count, strand);
ret = 1;
goto error;
}
}
if (right - left > list->longest)
list->longest = right - left;
list->length++;
}
if (sort_by_pos) {
for (bed_itr = kh_begin(bed_lists); bed_itr != kh_end(bed_lists); ++bed_itr) {
if (kh_exist(bed_lists, bed_itr)) {
list = &kh_val(bed_lists, bed_itr);
qsort(list->bp, list->length, sizeof(list->bp[0]), bed_entry_sort);
}
}
}
if (kh_size(bed_lists) > 0) {// any entries
ret = 0;
} else {
ret = 1;
}
error:
ks_free(&line);
if (hclose(fp) != 0) {
fprintf(stderr, "[amplicon] warning: failed to close %s", infile);
}
return ret;
}
void destroy_bed_hash(khash_t(bed_list_hash) *hash) {
khiter_t itr;
for (itr = kh_begin(hash); itr != kh_end(hash); ++itr) {
if (kh_exist(hash, itr)) {
bed_entry_list_t list = kh_val(hash, itr);
for (int i = 0; i < list.length; i++) {
free(list.bp[i].name);
free(list.bp[i].score);
}
free(list.bp);
free((char *)kh_key(hash, itr));
kh_key(hash, itr) = NULL;
}
}
kh_destroy(bed_list_hash, hash);
}
static int matching_clip_site(bed_entry_list_t *sites, hts_pos_t pos,
int is_rev, int use_strand, int64_t longest,
cl_param_t *param) {
int i, size, used_i;
int tol = param->tol;
int l = 0, mid = sites->length / 2, r = sites->length;
int pos_tol = is_rev ? (pos > tol ? pos - tol : 0) : pos;
while (r - l > 1) {
if (sites->bp[mid].right <= pos_tol) {
l = mid;
} else {
r = mid;
}
mid = (l + r) / 2;
}
size = 0;
used_i = -1;
for (i = l; i < sites->length; i++) {
hts_pos_t mod_left, mod_right;
if (use_strand && is_rev != sites->bp[i].rev)
continue;
if (is_rev) {
mod_left = sites->bp[i].left;
mod_right = sites->bp[i].right + tol;
} else {
if (sites->bp[i].left > tol) {
mod_left = sites->bp[i].left - tol;
} else {
mod_left = 0;
}
mod_right = sites->bp[i].right;
}
if (pos + longest + tol < mod_right)
break;
if (pos >= mod_left && pos <= mod_right) {
if (is_rev) {
if (size < pos - sites->bp[i].left) {
size = pos - sites->bp[i].left;
used_i = i;
}
} else {
if (size < sites->bp[i].right - pos) {
size = sites->bp[i].right - pos;
used_i = i;
}
}
}
}
if (used_i >= 0) {
sites->bp[used_i].num_reads++;
}
return size;
}
static int bam_trim_left(bam1_t *rec, bam1_t *rec_out, uint32_t bases,
clipping_type clipping) {
uint32_t *orig_cigar = bam_get_cigar(rec);
uint8_t *orig_seq = bam_get_seq(rec);
uint8_t *orig_qual = bam_get_qual(rec);
uint8_t *orig_aux = bam_get_aux(rec);
uint32_t *new_cigar;
uint8_t *new_qual;
size_t orig_l_aux = bam_get_l_aux(rec);
uint32_t i, j;
uint32_t ref_remove = bases, qry_removed = 0, hardclip = 0;
hts_pos_t new_pos = rec->core.pos;
uint32_t cig_type, cig_op;
if (rec->l_data + 8 > rec_out->m_data) {
uint8_t *new_data = realloc(rec_out->data, rec->l_data + 8);
if (!new_data) {
fprintf(stderr, "[ampliconclip] error: could not allocate memoy for new bam record\n");
return 1;
}
rec_out->data = new_data;
rec_out->m_data = rec->l_data + 8;
}
// Copy core data & name
memcpy(&rec_out->core, &rec->core, sizeof(rec->core));
memcpy(rec_out->data, rec->data, rec->core.l_qname);
// Modify CIGAR
new_cigar = bam_get_cigar(rec_out);
for (i = 0; i < rec->core.n_cigar; i++) {
cig_op = bam_cigar_op(orig_cigar[i]);
cig_type = bam_cigar_type(cig_op);
if (cig_op == BAM_CHARD_CLIP) {
hardclip += bam_cigar_oplen(orig_cigar[i]);
} else {
if (cig_type & 2) {
if (bam_cigar_oplen(orig_cigar[i]) <= ref_remove) {
ref_remove -= bam_cigar_oplen(orig_cigar[i]);
} else {
break;
}
new_pos += bam_cigar_oplen(orig_cigar[i]);
}
if (cig_type & 1) {
qry_removed += bam_cigar_oplen(orig_cigar[i]);
}
}
}
if (i < rec->core.n_cigar) {
cig_type = bam_cigar_type(bam_cigar_op(orig_cigar[i]));
// account for the last operation
if (cig_type & 2) {
new_pos += ref_remove;
}
if (cig_type & 1) {
qry_removed += ref_remove;
}
} else {
if (clipping == hard_clip) {
rec_out->core.l_qseq = 0;
rec_out->core.n_cigar = 0;
if (orig_l_aux)
memcpy(bam_get_aux(rec_out), orig_aux, orig_l_aux);
rec_out->l_data = bam_get_aux(rec_out) - rec_out->data + orig_l_aux;
return 0;
}
qry_removed = rec->core.l_qseq;
}
j = 0;
if (clipping == hard_clip && hardclip + qry_removed > 0) {
new_cigar[j++] = bam_cigar_gen(hardclip + qry_removed, BAM_CHARD_CLIP);
}
if (clipping == soft_clip) {
if (hardclip > 0) {
new_cigar[j++] = bam_cigar_gen(hardclip, BAM_CHARD_CLIP);
}
if (qry_removed > 0) {
new_cigar[j++] = bam_cigar_gen(qry_removed, BAM_CSOFT_CLIP);
}
}
if (i < rec->core.n_cigar
&& bam_cigar_oplen(orig_cigar[i]) > ref_remove) {
new_cigar[j++] = bam_cigar_gen(bam_cigar_oplen(orig_cigar[i]) - ref_remove, bam_cigar_op(orig_cigar[i]));
// fill in the rest of the cigar
i++;
for (; i < rec->core.n_cigar; i++) {
new_cigar[j++] = orig_cigar[i];
}
}
rec_out->core.n_cigar = j;
if (clipping == soft_clip) {
qry_removed = 0; // Copy all the sequence and confidence values
}
new_qual = bam_get_seq(rec_out) + (rec->core.l_qseq - qry_removed + 1) / 2;
// Copy remaining SEQ
if ((qry_removed & 1) == 0) {
memcpy(bam_get_seq(rec_out), orig_seq + (qry_removed / 2),
(rec->core.l_qseq - qry_removed + 1) / 2); // +1 to account for odd numbers
} else {
uint8_t *in = orig_seq + qry_removed / 2;
uint8_t *out = bam_get_seq(rec_out);
uint32_t i;
for (i = qry_removed; i < rec->core.l_qseq - 1; i += 2) {
*out++ = ((in[0] & 0x0f) << 4) | ((in[1] & 0xf0) >> 4);
in++;
}
if (i < rec->core.l_qseq) {
*out++ = (in[0] & 0x0f) << 4;
}
assert(out == new_qual);
}
// Copy remaining QUAL
memmove(new_qual, orig_qual + qry_removed, rec->core.l_qseq - qry_removed);
// Set new l_qseq
rec_out->core.l_qseq -= qry_removed;
// Move AUX
if (orig_l_aux)
memcpy(bam_get_aux(rec_out), orig_aux, orig_l_aux);
// Set new l_data
rec_out->l_data = bam_get_aux(rec_out) - rec_out->data + orig_l_aux;
// put in new pos
rec_out->core.pos = new_pos;
return 0;
}
static int bam_trim_right(bam1_t *rec, bam1_t *rec_out, uint32_t bases,
clipping_type clipping) {
uint32_t *orig_cigar = bam_get_cigar(rec);
uint8_t *orig_seq = bam_get_seq(rec);
uint8_t *orig_qual = bam_get_qual(rec);
uint8_t *orig_aux = bam_get_aux(rec);
uint32_t *new_cigar;
uint32_t new_n_cigar = 0;
uint8_t *new_qual;
size_t orig_l_aux = bam_get_l_aux(rec);
int32_t i;
int32_t j;
uint32_t ref_remove = bases, qry_removed = 0, hardclip = 0;
uint32_t cig_type, cig_op;
if (rec->l_data + 8 > rec_out->m_data) {
uint8_t *new_data = realloc(rec_out->data, rec->l_data + 8);
if (!new_data) {
fprintf(stderr, "[ampliconclip] error: could not allocate memoy for new bam record\n");
return 1;
}
rec_out->data = new_data;
rec_out->m_data = rec->l_data + 8;
}
// Copy core data & name
memcpy(&rec_out->core, &rec->core, sizeof(rec->core));
memcpy(rec_out->data, rec->data, rec->core.l_qname);
// Modify CIGAR here
new_cigar = bam_get_cigar(rec_out);
for (i = rec->core.n_cigar - 1; i >= 0; --i) {
cig_op = bam_cigar_op(orig_cigar[i]);
cig_type = bam_cigar_type(cig_op);
if (cig_op == BAM_CHARD_CLIP) {
hardclip += bam_cigar_oplen(orig_cigar[i]);
} else {
if (cig_type & 2) {
if (bam_cigar_oplen(orig_cigar[i]) <= ref_remove) {
ref_remove -= bam_cigar_oplen(orig_cigar[i]);
} else {
break;
}
}
if (cig_type & 1) {
qry_removed += bam_cigar_oplen(orig_cigar[i]);
}
}
}
if (i >= 0) {
cig_type = bam_cigar_type(bam_cigar_op(orig_cigar[i]));
if (cig_type & 1) {
qry_removed += ref_remove;
}
j = i;
if (qry_removed > 0) j++;
if (hardclip > 0 && (clipping == soft_clip || qry_removed == 0)) j++;
} else {
if (clipping == hard_clip) {
rec_out->core.l_qseq = 0;
rec_out->core.n_cigar = 0;
if (orig_l_aux)
memcpy(bam_get_aux(rec_out), orig_aux, orig_l_aux);
rec_out->l_data = bam_get_aux(rec_out) - rec_out->data + orig_l_aux;
return 0;
}
qry_removed = rec->core.l_qseq;
j = 0;
if (hardclip > 0 && clipping == soft_clip) j++;
}
if (clipping == hard_clip && hardclip + qry_removed > 0) {
new_cigar[j] = bam_cigar_gen(hardclip + qry_removed, BAM_CHARD_CLIP);
new_n_cigar++;
}
if (clipping == soft_clip) {
if (hardclip > 0) {
new_cigar[j] = bam_cigar_gen(hardclip, BAM_CHARD_CLIP);
new_n_cigar++;
if (qry_removed > 0) --j;
}
if (qry_removed > 0) {
new_cigar[j] = bam_cigar_gen(qry_removed, BAM_CSOFT_CLIP);
new_n_cigar++;
}
}
if (j > 0) {
new_cigar[--j] = bam_cigar_gen(bam_cigar_oplen(orig_cigar[i]) - ref_remove, bam_cigar_op(orig_cigar[i]));
new_n_cigar++;
}
// fill in the rest of the cigar
while (j > 0) {
new_cigar[--j] = orig_cigar[--i];
new_n_cigar++;
}
rec_out->core.n_cigar = new_n_cigar;
if (clipping == soft_clip)
qry_removed = 0; // Copy all the sequence and confidence values
new_qual = bam_get_seq(rec_out) + (rec->core.l_qseq - qry_removed + 1) / 2;
// Copy remaining SEQ
memcpy(bam_get_seq(rec_out), orig_seq, (rec->core.l_qseq - qry_removed + 1) / 2);
// Copy remaining QUAL
memcpy(new_qual, orig_qual, rec->core.l_qseq - qry_removed);
// Set new l_qseq
rec_out->core.l_qseq -= qry_removed;
// Copy AUX
if (orig_l_aux)
memcpy(bam_get_aux(rec_out), orig_aux, orig_l_aux);
// Set new l_data
rec_out->l_data = bam_get_aux(rec_out) - rec_out->data + orig_l_aux;
return 0;
}
static hts_pos_t active_query_len(bam1_t *b) {
uint32_t *cigar = bam_get_cigar(b);
uint32_t cig_type, cig_op;
hts_pos_t len = 0;
int i;
for (i = 0; i < b->core.n_cigar; i++) {
cig_op = bam_cigar_op(cigar[i]);
cig_type = bam_cigar_type(cig_op);
if ((cig_type & 1) && (cig_op != BAM_CSOFT_CLIP)) {
len += bam_cigar_oplen(cigar[i]);
}
}
return len;
}
static inline void swap_bams(bam1_t **a, bam1_t **b) {
bam1_t *tmp = *a;
*a = *b;
*b = tmp;
}
// Format OA:Z:(RNAME,POS,strand,CIGAR,MAPQ,NM;
static inline int tag_original_data(bam1_t *orig, kstring_t *oa_tag) {
char strand;
uint8_t *nm_tag, *old_oa_tag;
uint32_t *cigar;
int64_t nm = 0;
int i, res = 0;
ks_clear(oa_tag);
// if there is an existing OA tag the new one gets appended to it
if ((old_oa_tag = bam_aux_get(orig, "OA"))) {
res |= ksprintf(oa_tag, "%s", bam_aux2Z(old_oa_tag)) < 0;
}
if (orig->core.flag & BAM_FREVERSE)
strand = '-';
else
strand = '+';
if ((nm_tag = bam_aux_get(orig, "NM"))) {
nm = bam_aux2i(nm_tag);
}
res |= ksprintf(oa_tag, "%s,%"PRIhts_pos",%c,", bam_get_qname(orig), orig->core.pos + 1, strand) < 0;
for (i = 0, cigar = bam_get_cigar(orig); i < orig->core.n_cigar && res == 0; ++i) {
res |= kputw(bam_cigar_oplen(cigar[i]), oa_tag) < 0;
res |= kputc(bam_cigar_opchr(cigar[i]), oa_tag) < 0;
}
if (nm_tag) {
res |= ksprintf(oa_tag, ",%d,%"PRId64";", orig->core.qual, nm) < 0;
} else {
res |= ksprintf(oa_tag, "%d,;", orig->core.qual) < 0;
}
return res;
}
static int bam_clip(samFile *in, samFile *out, samFile *reject, char *bedfile,
clipping_type clipping, cl_param_t *param) {
int ret = 1, r, file_open = 0;
bam_hdr_t *header = NULL;
bam1_t *b = NULL, *b_tmp = NULL;
long f_count = 0, r_count = 0, n_count = 0, l_count = 0, l_exclude = 0, b_count = 0;
long filtered = 0, written = 0, failed = 0;
kstring_t str = KS_INITIALIZE;
kstring_t oat = KS_INITIALIZE;
kstring_t seq = KS_INITIALIZE;
bed_entry_list_t *sites;
FILE *stats_fp = stderr;
FILE *bed_count_summary_fp = stderr;
khash_t(bed_list_hash) *bed_hash = kh_init(bed_list_hash);
if (load_bed_file_multi_ref(bedfile, param->use_strand, 1, bed_hash)) {
fprintf(stderr, "[ampliconclip] error: unable to load bed file.\n");
goto fail;
}
if ((header = sam_hdr_read(in)) == NULL) {
fprintf(stderr, "[ampliconclip] error: could not read header\n");
goto fail;
}
// changing pos can ruin coordinate sort order
if (sam_hdr_find_tag_hd(header, "SO", &str) == 0 && str.s && strcmp(str.s, "coordinate") == 0) {
const char *new_order = "unknown";
if (sam_hdr_update_hd(header, "SO", new_order) == -1) {
fprintf(stderr, "[ampliconclip] error: unable to change sort order to 'SO:%s'\n", new_order);
goto fail;
}
}
ks_free(&str);
if (param->add_pg && sam_hdr_add_pg(header, "samtools", "VN", samtools_version(),
param->arg_list ? "CL" : NULL,
param->arg_list ? param->arg_list : NULL,
NULL) != 0) {
fprintf(stderr, "[ampliconclip] warning: unable to add @PG line to header.\n");
}
if (sam_hdr_write(out, header) < 0) {
fprintf(stderr, "[ampliconclip] error: could not write header.\n");
goto fail;
}
if (reject) {
if (sam_hdr_write(reject, header) < 0) {
fprintf(stderr, "[ampliconclip] error: could not write header to rejects file.\n");
goto fail;
}
}
b = bam_init1();
b_tmp = bam_init1();
if (!b || !b_tmp) {
fprintf(stderr, "[ampliconclip] error: out of memory when trying to create record.\n");
goto fail;
}
int32_t last_tid = -1;
int ref_found = 0;
while ((r = sam_read1(in, header, b)) >= 0) {
hts_pos_t pos;
int is_rev;
int p_size;
int been_clipped = 0, filter = 0;
int exclude = (BAM_FUNMAP | BAM_FQCFAIL);
khiter_t itr;
l_count++;
if (b->core.tid != last_tid) {
const char *ref_name;
ref_found = 0;
last_tid = b->core.tid;
if ((ref_name = sam_hdr_tid2name(header, b->core.tid)) != NULL) {
itr = kh_get(bed_list_hash, bed_hash, ref_name);
if (itr != kh_end(bed_hash)) {
sites = &kh_val(bed_hash, itr);
ref_found = 1;
}
}
}
if (!(b->core.flag & exclude) && ref_found) {
if (param->oa_tag)
if (tag_original_data(b, &oat))
goto fail;
if (!param->both) {
if (bam_is_rev(b)) {
pos = bam_endpos(b);
is_rev = 1;
} else {
pos = b->core.pos;
is_rev = 0;
}
if ((p_size = matching_clip_site(sites, pos, is_rev, param->use_strand, sites->longest, param))) {
if (is_rev) {
if (bam_trim_right(b, b_tmp, p_size, clipping) != 0)
goto fail;
swap_bams(&b, &b_tmp);
r_count++;
} else {
if (bam_trim_left(b, b_tmp, p_size, clipping) != 0)
goto fail;
swap_bams(&b, &b_tmp);
f_count++;
}
if (param->oa_tag) {
if (bam_aux_update_str(b, "OA", oat.l + 1, (const char *)oat.s))
goto fail;
}
if (param->del_tag) {
uint8_t *tag;
if ((tag = bam_aux_get(b, "NM")))
bam_aux_del(b, tag);
if ((tag = bam_aux_get(b, "MD")))
bam_aux_del(b, tag);
}
been_clipped = 1;
} else {
if (param->mark_fail) {
b->core.flag |= BAM_FQCFAIL;
}
n_count++;
}
} else {
int left = 0, right = 0;
// left first
pos = b->core.pos;
is_rev = 0;
if ((p_size = matching_clip_site(sites, pos, is_rev, param->use_strand, sites->longest, param))) {
if (bam_trim_left(b, b_tmp, p_size, clipping) != 0)
goto fail;
swap_bams(&b, &b_tmp);
f_count++;
left = 1;
been_clipped = 1;
}
// the right
pos = bam_endpos(b);
is_rev = 1;
if ((p_size = matching_clip_site(sites, pos, is_rev, param->use_strand, sites->longest, param))) {
if (bam_trim_right(b, b_tmp, p_size, clipping) != 0)
goto fail;
swap_bams(&b, &b_tmp);
r_count++;
right = 1;
been_clipped = 1;
}
if (left || right) {
uint8_t *tag;
if (param->oa_tag) {
if (bam_aux_update_str(b, "OA", oat.l + 1, (const char *)oat.s))
goto fail;
}
if (param->del_tag) {
if ((tag = bam_aux_get(b, "NM")))
bam_aux_del(b, tag);
if ((tag = bam_aux_get(b, "MD")))
bam_aux_del(b, tag);
}
}
if (left && right) {
b_count++;
} else if (!left && !right) {
if (param->mark_fail) {
b->core.flag |= BAM_FQCFAIL;
}
n_count++;
}
}
if (param->fail_len >= 0 || param->filter_len >= 0 || param->unmap_len >= 0) {
hts_pos_t aql = active_query_len(b);
if (param->fail_len >= 0 && aql <= param->fail_len) {
b->core.flag |= BAM_FQCFAIL;
}
if (param->filter_len >= 0 && aql <= param->filter_len) {
filter = 1;
}
if (param->unmap_len >= 0 && aql <= param->unmap_len) {
if (ks_resize(&seq, b->core.l_qseq) < 0) {
fprintf(stderr, "[ampliconclip] error: allocate memory for sequence %s\n", bam_get_seq(b));
goto fail;
}
ks_clear(&seq);
char *sb = ks_str(&seq);
uint8_t *sequence = bam_get_seq(b);
int i;
for (i = 0; i < b->core.l_qseq ; ++i) {
*sb++ = seq_nt16_str[bam_seqi(sequence, i)];
}
if (bam_set1(b_tmp, b->core.l_qname - b->core.l_extranul - 1, bam_get_qname(b),
(b->core.flag | BAM_FUNMAP), b->core.tid, b->core.pos, 0,
0, NULL, b->core.mtid, b->core.mpos, b->core.isize,
b->core.l_qseq, seq.s, (const char *)bam_get_qual(b),
bam_get_l_aux(b)) < 0) {
fprintf(stderr, "[ampliconclip] error: could not unmap read %s\n", bam_get_seq(b));
goto fail;
}
memcpy(bam_get_aux(b_tmp), bam_get_aux(b), bam_get_l_aux(b));
b_tmp->l_data += bam_get_l_aux(b);
swap_bams(&b, &b_tmp);
}
}
if (b->core.flag & BAM_FQCFAIL) {
failed++;
}
if (param->write_clipped && !been_clipped) {
filter = 1;
}
} else {
l_exclude++;
if (param->unmapped) {
filter = 1;
}
}
if (!filter) {
if (sam_write1(out, header, b) < 0) {
fprintf(stderr, "[ampliconclip] error: could not write line %ld.\n", l_count);
goto fail;
}
written++;
} else {
if (reject) {
if (sam_write1(reject, header, b) < 0) {
fprintf(stderr, "[ampliconclip] error: could not write to reject file %s\n",
param->rejects_file);
goto fail;
}
}
filtered++;
}
}
if (r < -1) {
fprintf(stderr, "[ampliconclip] error: failed to read input.\n");
goto fail;
}
if (param->stats_file) {
if ((stats_fp = fopen(param->stats_file, "w")) == NULL) {
fprintf(stderr, "[ampliconclip] warning: cannot write stats to %s.\n", param->stats_file);
} else {
file_open = 1;
}
}
fprintf(stats_fp, "COMMAND: %s\n"
"TOTAL READS: %ld\n"
"TOTAL CLIPPED: %ld\n"
"FORWARD CLIPPED: %ld\n"
"REVERSE CLIPPED: %ld\n"
"BOTH CLIPPED: %ld\n"
"NOT CLIPPED: %ld\n"
"EXCLUDED: %ld\n"
"FILTERED: %ld\n"
"FAILED: %ld\n"
"WRITTEN: %ld\n", param->arg_list, l_count, f_count + r_count,
f_count, r_count, b_count, n_count, l_exclude,
filtered, failed, written);
if (file_open) {
fclose(stats_fp);
file_open = 0;
}