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#include "samtools.pysam.h"
/* bam_mate.c -- fix mate pairing information and clean up flags.
Copyright (C) 2009, 2011-2017, 2019, 2022, 2024 Genome Research Ltd.
Portions copyright (C) 2011 Broad Institute.
Portions copyright (C) 2012 Peter Cock, The James Hutton Institute.
Author: Heng Li <lh3@sanger.ac.uk>
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 notices 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 <ctype.h>
#include "htslib/thread_pool.h"
#include "sam_opts.h"
#include "htslib/kstring.h"
#include "htslib/sam.h"
#include "samtools.h"
#define MD_MIN_QUALITY 15
/*
* This function calculates ct tag for two bams, it assumes they are from the same template and
* writes the tag to the first read in position terms.
*/
static void bam_template_cigar(bam1_t *b1, bam1_t *b2, kstring_t *str)
{
bam1_t *swap;
int i;
hts_pos_t end;
uint32_t *cigar;
str->l = 0;
if (b1->core.tid != b2->core.tid || b1->core.tid < 0 || b1->core.pos < 0 || b2->core.pos < 0 || b1->core.flag&BAM_FUNMAP || b2->core.flag&BAM_FUNMAP) return; // coordinateless or not on the same chr; skip
if (b1->core.pos > b2->core.pos) swap = b1, b1 = b2, b2 = swap; // make sure b1 has a smaller coordinate
kputc((b1->core.flag & BAM_FREAD1)? '1' : '2', str); // segment index
kputc((b1->core.flag & BAM_FREVERSE)? 'R' : 'F', str); // strand
for (i = 0, cigar = bam_get_cigar(b1); i < b1->core.n_cigar; ++i) {
kputw(bam_cigar_oplen(cigar[i]), str);
kputc(bam_cigar_opchr(cigar[i]), str);
}
end = bam_endpos(b1);
kputw(b2->core.pos - end, str);
kputc('T', str);
kputc((b2->core.flag & BAM_FREAD1)? '1' : '2', str); // segment index
kputc((b2->core.flag & BAM_FREVERSE)? 'R' : 'F', str); // strand
for (i = 0, cigar = bam_get_cigar(b2); i < b2->core.n_cigar; ++i) {
kputw(bam_cigar_oplen(cigar[i]), str);
kputc(bam_cigar_opchr(cigar[i]), str);
}
uint8_t* data;
if ((data = bam_aux_get(b1,"ct")) != NULL) bam_aux_del(b1, data);
if ((data = bam_aux_get(b2,"ct")) != NULL) bam_aux_del(b2, data);
bam_aux_append(b1, "ct", 'Z', str->l+1, (uint8_t*)str->s);
}
/*
* What This Program is Supposed To Do:
* Fill in mate coordinates, ISIZE and mate related flags from a name-sorted
* alignment.
*
* How We Handle Input
*
* Secondary and supplementary Reads:
* -write to output unchanged
* All Reads:
* -if pos == 0 (1 based), tid == -1 set UNMAPPED flag
* single Reads:
* -if pos == 0 (1 based), tid == -1, or UNMAPPED then set UNMAPPED, pos = 0,
* tid = -1
* -clear bad flags (MREVERSE, PROPER_PAIR)
* -set mpos = 0 (1 based), mtid = -1 and isize = 0
* -write to output
* Paired Reads:
* -if read is unmapped and mate is not, set pos and tid to equal that of mate
* -sync mate flags (MREVERSE, MUNMAPPED), mpos, mtid
* -recalculate ISIZE if possible, otherwise set it to 0
* -optionally clear PROPER_PAIR flag from reads where mapping or orientation
* indicate this is not possible (Illumina orientation only)
* -calculate ct and apply to lowest positioned read
* -write to output
* Limitations
* -Does not handle tandem reads
* -Should mark supplementary reads the same as primary.
* Notes
* -CT definition appears to be something else in spec, this was in here before
* I started tampering with it, anyone know what is going on here? To work
* around this I have demoted the CT this tool generates to ct.
*/
static void sync_unmapped_pos_inner(bam1_t* src, bam1_t* dest) {
if ((dest->core.flag & BAM_FUNMAP) && !(src->core.flag & BAM_FUNMAP)) {
// Set unmapped read's RNAME and POS to those of its mapped mate
// (recommended best practice, ensures if coord sort will be together)
dest->core.tid = src->core.tid;
dest->core.pos = src->core.pos;
}
}
static void sync_mate_inner(bam1_t* src, bam1_t* dest)
{
// sync mate pos information
dest->core.mtid = src->core.tid; dest->core.mpos = src->core.pos;
// sync flag info
if (src->core.flag&BAM_FREVERSE)
dest->core.flag |= BAM_FMREVERSE;
else
dest->core.flag &= ~BAM_FMREVERSE;
if (src->core.flag & BAM_FUNMAP) {
dest->core.flag |= BAM_FMUNMAP;
}
}
// Is it plausible that these reads are properly paired?
// Can't really give definitive answer without checking isize
static bool plausibly_properly_paired(bam1_t* a, bam1_t* b)
{
if ((a->core.flag & BAM_FUNMAP) || (b->core.flag & BAM_FUNMAP)) return false;
assert(a->core.tid >= 0); // This should never happen if FUNMAP is set correctly
if (a->core.tid != b->core.tid) return false;
bam1_t* first = a;
bam1_t* second = b;
hts_pos_t a_pos = a->core.flag&BAM_FREVERSE ? bam_endpos(a) : a->core.pos;
hts_pos_t b_pos = b->core.flag&BAM_FREVERSE ? bam_endpos(b) : b->core.pos;
if (a_pos > b_pos) {
first = b;
second = a;
} else {
first = a;
second = b;
}
if (!(first->core.flag&BAM_FREVERSE) && (second->core.flag&BAM_FREVERSE))
return true;
else
return false;
}
// Returns 0 on success, -1 on failure.
static int bam_format_cigar(const bam1_t* b, kstring_t* str)
{
// An empty cigar is a special case return "*" rather than ""
if (b->core.n_cigar == 0) {
return (kputc('*', str) == EOF) ? -1 : 0;
}
const uint32_t *cigar = bam_get_cigar(b);
uint32_t i;
for (i = 0; i < b->core.n_cigar; ++i) {
if (kputw(bam_cigar_oplen(cigar[i]), str) == EOF) return -1;
if (kputc(bam_cigar_opchr(cigar[i]), str) == EOF) return -1;
}
return 0;
}
// Returns 0 on success, -1 on failure.
static int sync_mq_mc(bam1_t* src, bam1_t* dest)
{
if ( (src->core.flag & BAM_FUNMAP) == 0 ) { // If mapped
// Copy Mate Mapping Quality
uint32_t mq = src->core.qual;
uint8_t* data;
if ((data = bam_aux_get(dest,"MQ")) != NULL) {
bam_aux_del(dest, data);
}
bam_aux_append(dest, "MQ", 'i', sizeof(uint32_t), (uint8_t*)&mq);
}
// Copy mate cigar if either read is mapped
if ( (src->core.flag & BAM_FUNMAP) == 0 || (dest->core.flag & BAM_FUNMAP) == 0 ) {
uint8_t* data_mc;
if ((data_mc = bam_aux_get(dest,"MC")) != NULL) {
bam_aux_del(dest, data_mc);
}
// Convert cigar to string
kstring_t mc = { 0, 0, NULL };
if (bam_format_cigar(src, &mc) < 0) return -1;
bam_aux_append(dest, "MC", 'Z', ks_len(&mc)+1, (uint8_t*)ks_str(&mc));
free(mc.s);
}
return 0;
}
// Copy flags.
// Returns 0 on success, -1 on failure.
static int sync_mate(bam1_t* a, bam1_t* b)
{
sync_unmapped_pos_inner(a,b);
sync_unmapped_pos_inner(b,a);
sync_mate_inner(a,b);
sync_mate_inner(b,a);
if (sync_mq_mc(a,b) < 0) return -1;
if (sync_mq_mc(b,a) < 0) return -1;
return 0;
}
static uint32_t calc_mate_score(bam1_t *b)
{
uint32_t score = 0;
uint8_t *qual = bam_get_qual(b);
int i;
for (i = 0; i < b->core.l_qseq; i++) {
if (qual[i] >= MD_MIN_QUALITY) score += qual[i];
}
return score;
}
static int add_mate_score(bam1_t *src, bam1_t *dest)
{
uint8_t *data_ms;
uint32_t mate_score = calc_mate_score(src);
if ((data_ms = bam_aux_get(dest, "ms")) != NULL) {
bam_aux_del(dest, data_ms);
}
if (bam_aux_append(dest, "ms", 'i', sizeof(uint32_t), (uint8_t*)&mate_score) == -1) {
return -1;
}
return 0;
}
// Completely delete the CIGAR field
static void clear_cigar(bam1_t *b) {
memmove(bam_get_cigar(b), bam_get_seq(b),
b->data + b->l_data - bam_get_seq(b));
b->l_data -= 4*b->core.n_cigar;
b->core.n_cigar = 0;
}
// Trim a CIGAR field to end on reference position "end". Remaining bases
// are turned to soft clips.
static int bam_trim(bam1_t *b, hts_pos_t end) {
hts_pos_t pos = b->core.pos;
int n_cigar = b->core.n_cigar, i;
uint32_t new_cigar_a[1024];
uint32_t *new_cigar = new_cigar_a;
uint32_t *cigar = bam_get_cigar(b);
// Find end of alignment or end of ref
int op = 0, oplen = 0;
for (i = 0; i < n_cigar; i++) {
op = bam_cigar_op(cigar[i]);
oplen = bam_cigar_oplen(cigar[i]);
if (!(bam_cigar_type(op) & 2))
continue;
pos += oplen;
if (pos > end)
break;
}
if (i == n_cigar)
// looks fine already
return 0;
int old_i = i, j = 0;
// At worst we grow by 1 element (eg 100M -> 70M30S)
if (n_cigar-i >= 1024-1) {
new_cigar = malloc(4*(n_cigar-i+1));
if (!new_cigar)
return -1;
}
// We fill out to new_cigar from here on.
if (pos-oplen < end) {
// Partial CIGAR op? Split existing tag.
cigar[old_i++] = bam_cigar_gen(end - (pos-oplen), op);
new_cigar[j++] = bam_cigar_gen(pos-end, BAM_CSOFT_CLIP);
} else if (pos > end) {
// entirely off the chromosome; this will trigger CIGAR *, MQUAL 0
b->core.flag |= BAM_FUNMAP;
b->core.flag &= ~BAM_FPROPER_PAIR;
} else {
// CIGAR op started on the trim junction
new_cigar[j++] = bam_cigar_gen(oplen, BAM_CSOFT_CLIP);
}
// Replace trailing elements.
for (i++; i < n_cigar; i++) {
op = bam_cigar_op(cigar[i]);
oplen = bam_cigar_oplen(cigar[i]);
if (op == BAM_CHARD_CLIP) {
new_cigar[j++] = cigar[i];
} else {
new_cigar[j-1] =
bam_cigar_gen(bam_cigar_oplen(new_cigar[j-1]) + oplen,
BAM_CSOFT_CLIP);
}
}
// We now have cigar[0..old_i-1] for existing CIGAR
// and new_cigar[0..j-1] for new CIGAR trailing component.
if (old_i+j == n_cigar) {
// Fits and no data move needed
memcpy(&cigar[old_i], new_cigar, j*4);
} else {
uint8_t *seq_old = bam_get_seq(b);
uint8_t *aux_end = b->data + b->l_data;
int nshift;
if (old_i+j < n_cigar) {
// Smaller, and can move data down
nshift = -4*(n_cigar - (old_i+j));
} else {
// Bigger, so grow BAM and move data up
nshift = 4*(old_i+j - n_cigar);
// FIXME: make htslib's sam_realloc_bam_data public
if (b->l_data + nshift > b->m_data) {
uint8_t *new_data = realloc(b->data, b->l_data + nshift);
if (!new_data) {
if (new_cigar != new_cigar_a)
free(new_cigar);
return -1;
}
b->m_data = b->l_data + nshift;
if (b->data != new_data) {
b->data = new_data;
seq_old = bam_get_seq(b);
aux_end = b->data + b->l_data;
cigar = bam_get_cigar(b);
}
}
}
memmove(seq_old+nshift, seq_old, aux_end - seq_old);
b->l_data += nshift;
memcpy(&cigar[old_i], new_cigar, j*4);
b->core.n_cigar = old_i+j;
}
if (new_cigar != new_cigar_a)
free(new_cigar);
return 0;
}
// Parses a comma-separated list of "pos", "mqual", "unmap", "cigar", and "aux"
// keywords for the bam sanitizer.
int bam_sanitize_options(const char *str) {
int opt = 0;
while (str && *str) {
const char *str_start;
while(*str && *str == ',')
str++;
for (str_start = str; *str && *str != ','; str++);
int len = str - str_start;
if (strncmp(str_start, "all", 3) == 0 || *str_start == '*')
opt = FIX_ALL;
else if (strncmp(str_start, "none", 4) == 0 ||
strncmp(str_start, "off", 3) == 0)
opt = 0;
else if (strncmp(str_start, "on", 2) == 0)
// default for position sorted data
opt = FIX_MQUAL | FIX_UNMAP | FIX_CIGAR | FIX_AUX;
else if (strncmp(str_start, "pos", 3) == 0)
opt |= FIX_POS;
else if (strncmp(str_start, "mqual", 5) == 0)
opt |= FIX_MQUAL;
else if (strncmp(str_start, "unmap", 5) == 0)
opt |= FIX_UNMAP;
else if (strncmp(str_start, "cigar", 5) == 0)
opt |= FIX_CIGAR;
else if (strncmp(str_start, "aux", 3) == 0)
opt |= FIX_AUX;
else {
print_error("sanitize", "Unrecognised keyword %.*s\n",
len, str_start);
return -1;
}
}
return opt;
}
int bam_sanitize(sam_hdr_t *h, bam1_t *b, int flags) {
if ((flags & FIX_POS) && b->core.tid < 0) {
// RNAME * => pos 0. NB can break alignment chr/pos sort order
b->core.pos = -1;
if (flags & FIX_UNMAP)
b->core.flag |= BAM_FUNMAP;
}
if ((flags & FIX_CIGAR) && !(b->core.flag & BAM_FUNMAP)) {
// Mapped => unmapped correction
if (b->core.pos < 0 && (flags & FIX_UNMAP)) {
b->core.flag |= BAM_FUNMAP;
} else {
hts_pos_t cur_end, rlen = sam_hdr_tid2len(h, b->core.tid);
if (b->core.pos >= rlen && (flags & FIX_UNMAP)) {
b->core.flag |= BAM_FUNMAP;
if (flags & FIX_POS)
b->core.tid = b->core.pos = -1;
} else if ((cur_end = bam_endpos(b)) > rlen) {
if (bam_trim(b, rlen) < 0)
return -1;
}
}
}
if (b->core.flag & BAM_FUNMAP) {
// Unmapped -> cigar/qual correctoins
if ((flags & FIX_CIGAR) && b->core.n_cigar > 0)
clear_cigar(b);
if (flags & FIX_MQUAL)
b->core.qual = 0;
// Remove NM, MD, CG, SM tags.
if (flags & FIX_AUX) {
uint8_t *from = bam_aux_first(b);
uint8_t *end = b->data + b->l_data;
uint8_t *to = from ? from-2 : end;
#define XTAG(a) (((a)[0]<<8) + (a)[1])
while (from) {
uint8_t *next = bam_aux_next(b, from);
if (!next && errno != ENOENT)
return -1;
// Keep tag unless one of a specific set.
// NB "to" always points to an aux tag start, while
// "from" is after key.
from -= 2;
int key = (int)from[0]<<8 | from[1];
if (key != XTAG("NM") && key != XTAG("MD") &&
key != XTAG("CG") && key != XTAG("SM")) {
ptrdiff_t len = (next ? next-2 : end) - from;
if (from != to)
memmove(to, from, len);
to += len;
}
from = next;
}
b->l_data = to - b->data;
}
}
return 0;
}
// Look for 3 tags in one pass, for efficiencies sake.
// We also convert the draft tags Mm and Ml to MM and ML here.
static inline void find_tags(bam1_t *b,
char *t1, uint8_t **t1p,
char *t2, uint8_t **t2p,
char *t3, uint8_t **t3p) {
*t1p = *t2p = *t3p = NULL;
uint8_t *aux = bam_aux_first(b);
while (aux) {
if (aux[-2] == t1[0] && toupper(aux[-1]) == t1[1]) {
*t1p = aux;
if (islower(aux[-1]))
aux[-1] = t1[1];
} else if (aux[-2] == t2[0] && toupper(aux[-1]) == t2[1]) {
*t2p = aux;
if (islower(aux[-1]))
aux[-1] = t2[1];
} else if (aux[-2] == t3[0] && toupper(aux[-1]) == t3[1]) {
*t3p = aux;
if (islower(aux[-1]))
aux[-1] = t3[1];
}
aux = bam_aux_next(b, aux);
}
}
// Return 5' and 3' CIGAR hard-clip counts
static inline void hard_clips(bam1_t *b, int *end5, int *end3) {
uint32_t *cigar = bam_get_cigar(b);
int ncigar = b->core.n_cigar;
int endL = 0, endR = 0, nh = 0;
if (ncigar && bam_cigar_op(cigar[0]) == BAM_CHARD_CLIP)
endL = bam_cigar_oplen(cigar[0]), nh=1;
if (ncigar > nh && bam_cigar_op(cigar[ncigar-1]) == BAM_CHARD_CLIP)
endR = bam_cigar_oplen(cigar[ncigar-1]);
if (b->core.flag & BAM_FREVERSE) {
*end5 = endR;
*end3 = endL;
} else {
*end5 = endL;
*end3 = endR;
}
}
// Get MM, ML and MN tags, and 5' and 3' hard-clip lengths.
// MNi is integer copy of MN, or -1 if absent/invalid
void get_mod_info(bam1_t *b, uint8_t **MM, uint8_t **ML, uint8_t **MN,
int *MNi, int *end5, int *end3) {
find_tags(b, "MM", MM, "ML", ML, "MN", MN);
if (*MN) {
int save_errno = errno;
errno = 0;
*MNi = bam_aux2i(*MN);
if (errno == EINVAL)
*MNi = -1;
errno = save_errno;
} else {
*MNi = -1;
}
if (*MM)
hard_clips(b, end5, end3);
else
*end5 = *end3 = 0; // don't need if MM not found
}
typedef struct MM_state {
// tags found on "pre" BAM
uint8_t *MM, *ML, *MN;
} MM_state;
uint8_t *MN_enc(uint8_t *tag, uint32_t n) {
if (n > UINT16_MAX) {
tag[0] = 'I';
i32_to_le(n, tag+1);
tag += 5;
} else if (n > UINT8_MAX) {
tag[0] = 'S';
i16_to_le(n, tag+1);
tag += 3;
} else {
*tag++ = 'C';
*tag++ = n;
}
return tag;
}
// Trim 5'/3' bases off MM and ML tags, using a previous sequence as a guide.
int trim_MM(bam1_t *pre, bam1_t *cur, int end5, int end3,
uint8_t *MM, uint8_t *ML, uint8_t *MN) {
// Count number of bases
int counts5[16] = {0}, counts3[16] = {0};
uint8_t *seq = bam_get_seq(pre);
int i;
for (i = 0; i < end5; i++)
counts5[bam_seqi(seq, i)]++;
memcpy(counts3, counts5, 16 * sizeof(*counts3));
for (; i < pre->core.l_qseq - end3; i++)
counts3[bam_seqi(seq, i)]++;
// "p" is position in pre.
// "q" is position in cur.
// Hence move up "p" to start and copy from there to "q".
uint8_t *MMp, *MLp, *MMq = NULL, *MLq = NULL;
if (ML && ML[0] == 'B' && ML[1] == 'C') {
MLp = ML+6;
} else {
ML = MLp = NULL;
}
MMq = MM+1;
MLq = MLp;
for (MMp = MM+1; *MMp; ) {
int fundamental = seq_nt16_table[*MMp];
while (*MMp && *MMp != ',')
*MMq++ = *MMp++;
if (*MMp)
*MMq++ = *MMp++;
// Now on comma separated list for MM and BC array for ML. Skip
int n = 0;
while (*MMp != ';' && n < counts5[fundamental]) {
char *endptr;
long delta = strtol((char *)MMp, &endptr, 10);
if (counts5[fundamental] - n > delta) {
// Skip entire delta in MM and ML.
// Eg counts[]=10, MM=3,10 ML=<10><20> => MM=10 ML=<20>
n += delta+1;
if(ML) MLp++;
} else if (counts3[fundamental] > counts5[fundamental]) {
// Shrink delta, writing MM and ML is unchanged.
// Eg counts[]=3, MM=10,4 ML=<10><20> => MM=7,4 ML=<10><20>
char num[50];
int l = sprintf(num, "%ld",
delta - (counts5[fundamental]-n));
memcpy((char *)MMq, num, l);
MMq += l;
*MMq++ = *endptr;
n += delta+1;
if (ML)
*MLq++ = *MLp++;
} else {
// next base mod is on boundary of 3' clip point
break;
}
MMp = (uint8_t *)endptr;
if (*MMp != ',')
// error? if not ; also?
break;
MMp++;
}
// Copy
while (*MMp != ';' && n < counts3[fundamental]) {
char *endptr;
long delta = strtol((char *)MMp, &endptr, 10);
if (counts3[fundamental] - n > delta) {
// Copy entire delta in MM and ML including [,;]
memmove(MMq, MMp, (uint8_t *)endptr - MMp + 1);
MMq += (uint8_t *)endptr - MMp + 1;
n += delta+1;
if (ML)
*MLq++ = *MLp++;
} else {
// Next mod is into 3' cutoff, so can terminate MM/ML now
n = counts3[fundamental];
if (ML)
MLp++;
}
MMp = (uint8_t *)endptr;
if (*MMp != ',')
break;
MMp++;
}
// Skip
while (*MMp && *MMp != ';') {
while (*MMp && *MMp != ',' && *MMp != ';')
MMp++;
if (*MMp == ',')
MMp++;
if (ML)
MLp++;
}
MMq[-1] = ';'; // replaces , with ; if clipping right
if (*MMp)
MMp++;
}
MMp++; // skip nul
*MMq++ = 0;
// Adjust ML B array length
if (ML)
u32_to_le(MLq-(ML+6), ML+2);
// Move MM and ML down to include their MM:Z and ML:B bits
if (MM) MM-=2;
if (ML) ML-=2;
// Now MM/ML are start of tags, MMq/MLq are ends of edited tags,
// and MMp/MLp are ends of original tags. Walk through tags taking up
// any gaps
//
// Eg XXXXXXmmmmm--YYYlllll-ZZ (m and l are edited MM and ML tags)
// => XXXXXXmmmmmYYYlllllZZ
uint8_t *tag = bam_get_aux(cur), *tag_end = cur->data + cur->l_data;
uint8_t *to = tag;
while (tag && tag < tag_end) {
if (tag[0] == 'M' && (tag[1] == 'M' || tag[1] == 'm')) {
// Slow but easy
memmove(to, MM, MMq-MM); // length of new tag
to += MMq-MM;
tag = MMp; // size of old tag
} else if (tag[0] == 'M' && (tag[1] == 'L' || tag[1] == 'l')) {
memmove(to, ML, MLq-ML);
to += MLq-ML;
tag = MLp;
} else if (tag[0] == 'M' && tag[1] == 'N') {
tag = bam_aux_next(cur, tag+2);
// Skip it as we'll overwrite this later, although this
// does change the tag order. Instead we could do:
//
// *to++ = 'M';
// *to++ = 'N';
// to = MN_enc(to, cur->core.l_qseq);
} else {
// Want aux_skip, but it's private.
// So we use bam_aux_next with work-arounds. :(
uint8_t *from = tag;
tag = bam_aux_next(cur, tag+2);
tag = tag ? tag-2 : tag_end;
memmove(to, from, tag-from);
to += tag-from;
}
}
cur->l_data = to - cur->data;
return 0;
}
// Removes base modification tags: MM, ML and MN.
// This is more efficient than a series of bam_aux_remove and
// bam_aux_find calls, as the previous removes shuffle the tags we've
// previously found. However it's still not optimal.
void delete_mod_tags(bam1_t *b) {
uint8_t *tag = bam_aux_first(b), *next;
uint8_t *to = tag;
while (tag) {
next = bam_aux_next(b, tag);
if (tag[-2] == 'M' &&
(tag[-1] == 'M' || tag[-1] == 'm' ||
tag[-1] == 'L' || tag[-1] == 'l' ||
tag[-1] == 'N')) {
// Skip. Equivalent to bam_aux_remove without multiple passes
} else {
// Copy. All these +/-2s are an annoyance caused by the
// tag iterator pointing to the byte after the 2-letter code
uint8_t *end = next ? next : b->data + b->l_data + 2;
if (tag != to)
memmove(to-2, tag-2, end-tag);
to += end-tag;
}
tag = next;
}
b->l_data = (to-2) - b->data;
}
int validate_MM(bam1_t *b, hts_base_mod_state *state) {
hts_base_mod mods[10];
int n, pos;
while ((n = bam_next_basemod(b, state, mods, 10, &pos)) > 0) {
// bam_next_basemod will trigger MM out-of-bound checks
}
return n;
}
// Fix base modification tags MM, ML and MN.
// For supplementary-style alignments we may have hard-clipped the sequence
// and just duplicated the MM/ML tags. Use the primary alignment to get the
// clipped sequence so we can trim MM/ML accordingly.
//
// We call this first on primary reads with pre == NULL. This caches
// MM and ML data into MM_state.
//
// We then call it again on secondary and/or supplementary data with
// pre == the primary record and pass in the associated state. This then
// validates MM/ML/MN match, and if not adjusts them if they have hard-clips
// which yields consistent data.
//
// TODO: add sanity check on counts of base types and MM tag to ensure it's
// possible. We can do this post-trimming, so we sanitize everything.
//
// Returns 0 on success,
// -1 on failure
int fix_MM(bam1_t *pre, bam1_t *cur, MM_state *state) {
int end5, end3;
int MNi = 0; // MN of -1 is used as indicator for no valid mods
if (!pre && state) {
// First time we've see this name.
// Look for base modification tags and sanity check.
get_mod_info(cur, &state->MM, &state->ML, &state->MN, &MNi,
&end5, &end3);
if (!state->MM) {
delete_mod_tags(cur);
return 0;
}
if (!end5 && !end3 && MNi <= 0) {
// No MN tag, but also no clipping. Assume MM is valid
if (cur->core.l_qseq)
if (bam_aux_update_int(cur, "MN", cur->core.l_qseq) < 0)
return -1;
} else if ((end3 || end5) && cur->core.l_qseq != MNi) {
// We have hard clips and MN tag, but the MN tag doesn't match
// observed sequence length so it appears the hard-clipping
// happened after base-mods called without updating.
// Fail as this is a primary read.
delete_mod_tags(cur);
}
// Otherwise we assume the base modifications are correct
} else if (state) {
// A supplementary or secondary alignment with known primary
uint8_t *cur_MM = NULL, *cur_ML = NULL, *cur_MN = NULL;
MNi = -1;
get_mod_info(cur, &cur_MM, &cur_ML, &cur_MN, &MNi, &end5, &end3);
if (!cur_MM) {
delete_mod_tags(cur);
return 0;
}
// Does MN match seq length? If so, we believe it's already valid
if (MNi == cur->core.l_qseq)
goto validate;
// Length mismatch and/or no known length, so check vs full seq.
if (pre->core.l_qseq != cur->core.l_qseq + end3 + end5) {
delete_mod_tags(cur);
return 0;
} else if (end5 || end3) {
if (MNi < 0 || MNi == pre->core.l_qseq)
trim_MM(pre, cur, end5, end3, cur_MM, cur_ML, cur_MN);
} // else no hard clips so MM is already valid
// Set MN so we've validated it, provided seq isn't "*".
// inefficient, but minimal compared to everything else
if (cur->core.l_qseq)
if (bam_aux_update_int(cur, "MN", cur->core.l_qseq) < 0)
return -1;
}
validate:
;
// Also validate MM length matches sequence length. This mirrors the
// logic in htslib/sam_mods.c.
// For now we take the inefficient approach of using bam_parse_basemod2.
// Inefficient, but robust.
hts_base_mod_state *mst = hts_base_mod_state_alloc();
if (!mst)
return -1;
enum htsLogLevel lvl = hts_get_log_level();
hts_set_log_level(HTS_LOG_OFF);
if (bam_parse_basemod(cur, mst) < 0)
// Maybe we want hts_log_warning still though?
delete_mod_tags(cur);
if (validate_MM(cur, mst) < 0)
delete_mod_tags(cur);
hts_set_log_level(lvl);
hts_base_mod_state_free(mst);
return 0;
}
// Ensure the b[] array is at least n.
// Returns 0 on success,
// -1 on failure
static int grow_b_array(bam1_t **b, int *ba, int n) {
if (n < *ba)
return 0;
bam1_t *bnew = realloc(*b, (n+=10) * sizeof(**b));
if (!bnew)
return -1;
*b = bnew;
// bam_init1 equivalent
int i;
for (i = *ba; i < n; i++)
memset(&(*b)[i], 0, sizeof(bam1_t));
*b = bnew;
*ba = n;
return 0;
}
// We have b[0]..b[bn-1] entries all from the same template (qname)
typedef struct {
bam1_t *b;
int n, ba; // number used and number allocated
int b_next; // b[b_next] for start of next set, -1 if unset
int eof; // marker for having seen eof
} bam_set;
// Fetches a new batch of BAM records all containing the same name.
// NB: we cache the last (non-matching) name in b[n], so we can use it to
// start the next batch.
// Returns the number of records on success,
// <0 on failure or EOF (sam_read1 return vals)
static int next_template(samFile *in, sam_hdr_t *header, bam_set *bs,
int sanitize_flags) {
int result;
if (bs->eof)
return -1;
// First time through, prime the template name
if (bs->b_next < 0) {
if (grow_b_array(&bs->b, &bs->ba, 1) < 0)
return -2;
result = sam_read1(in, header, &bs->b[0]);
if (result < 0)
return result;
if (bam_sanitize(header, &bs->b[0], sanitize_flags) < 0)
return -2;
} else {
// Otherwise use the previous template name read
bam1_t btmp = bs->b[0];
bs->b[0] = bs->b[bs->b_next];
bs->b[bs->b_next] = btmp; // For ->{,l_,m_}data
}
bs->n = 1;
// Now keep reading until we find a read that mismatches or we hit eof.
char *name = bam_get_qname(&bs->b[0]);
for (;;) {
if (grow_b_array(&bs->b, &bs->ba, bs->n+1) < 0)
return -2;
result = sam_read1(in, header, &bs->b[bs->n]);
if (result < -1)
return result;
if (result < 0) {
bs->eof = 1;
bs->b_next = -1;
break;
} else {
if (bam_sanitize(header, &bs->b[bs->n], sanitize_flags) < 0)
return -2;
bs->b_next = bs->n;
if (strcmp(name, bam_get_qname(&bs->b[bs->n])) != 0)
break;
}
bs->n++;
}
return bs->n;
}
// currently, this function ONLY works if each read has one hit
//
// Returns 0 on success,
// >0 on failure
static int bam_mating_core(samFile *in, samFile *out, int remove_reads,
int proper_pair_check, int add_ct,
int do_mate_scoring, char *arg_list, int no_pg,
int sanitize_flags, int base_mods)
{
sam_hdr_t *header;
int result, n;
kstring_t str = KS_INITIALIZE;
bam_set bs = {NULL, 0, 0, -1, 0};
header = sam_hdr_read(in);
if (header == NULL) {
fprintf(samtools_stderr, "[bam_mating_core] ERROR: Couldn't read header\n");
return 1;
}
// Accept unknown, unsorted, or queryname sort order, but error on coordinate sorted.
if (!sam_hdr_find_tag_hd(header, "SO", &str) && str.s && !strcmp(str.s, "coordinate")) {
fprintf(samtools_stderr, "[bam_mating_core] ERROR: Coordinate sorted, require grouped/sorted by queryname.\n");
goto fail;
}
ks_free(&str);
if (!no_pg && sam_hdr_add_pg(header, "samtools",
"VN", samtools_version(),
arg_list ? "CL": NULL,
arg_list ? arg_list : NULL,
NULL))
goto fail;
if (sam_hdr_write(out, header) < 0) goto write_fail;
// Iterate template by template fetching bs->n records at a time
while ((result = next_template(in, header, &bs, sanitize_flags)) >= 0) {
bam1_t *cur = NULL, *pre = NULL, *rnum[2] = {NULL, NULL};
int prev = -1, curr = -1;
hts_pos_t pre_end = 0, cur_end = 0;
// Find and fix up primary alignments
MM_state state[2];
for (n = 0; n < bs.n; n++) {
int is_r2 = (bs.b[n].core.flag & BAM_FREAD2) != 0;
if (bs.b[n].core.flag & (BAM_FSECONDARY | BAM_FSUPPLEMENTARY))
continue;
if (base_mods)
if (fix_MM(NULL, &bs.b[n], &state[is_r2]) < 0)
goto fail;
if (!pre) {
pre = &bs.b[prev = n];
rnum[(pre->core.flag & BAM_FREAD2) != 0] = pre;
pre_end = (pre->core.flag & BAM_FUNMAP) == 0
? bam_endpos(pre) : 0;
continue;
}
// Note, more than 2 primary alignments will use 'curr' as last
cur = &bs.b[curr = n];
rnum[(cur->core.flag & BAM_FREAD2) != 0] = cur;
cur_end = (cur->core.flag & BAM_FUNMAP) == 0
? bam_endpos(cur) : 0;
pre->core.flag |= BAM_FPAIRED;
cur->core.flag |= BAM_FPAIRED;
if (sync_mate(pre, cur))
goto fail;
// If safe set TLEN/ISIZE
if (pre->core.tid == cur->core.tid
&& !(cur->core.flag & (BAM_FUNMAP | BAM_FMUNMAP))
&& !(pre->core.flag & (BAM_FUNMAP | BAM_FMUNMAP))) {
hts_pos_t cur5, pre5;
cur5 = (cur->core.flag & BAM_FREVERSE)
? cur_end
: cur->core.pos;
pre5 = (pre->core.flag & BAM_FREVERSE)
? pre_end
: pre->core.pos;
cur->core.isize = pre5 - cur5;
pre->core.isize = cur5 - pre5;
} else {
cur->core.isize = pre->core.isize = 0;
}
if (add_ct)
bam_template_cigar(pre, cur, &str);
// TODO: Add code to properly check if read is in a proper
// pair based on ISIZE distribution
if (proper_pair_check && !plausibly_properly_paired(pre,cur)) {
pre->core.flag &= ~BAM_FPROPER_PAIR;
cur->core.flag &= ~BAM_FPROPER_PAIR;
}
if (do_mate_scoring) {
if ((add_mate_score(pre, cur) == -1) ||
(add_mate_score(cur, pre) == -1)) {
fprintf(samtools_stderr, "[bam_mating_core] ERROR: "
"unable to add mate score.\n");
goto fail;
}
}
// If we have to remove reads make sure we do it in a way that
// doesn't create orphans with bad flags
if (remove_reads) {
if (pre->core.flag&BAM_FUNMAP)
cur->core.flag &=
~(BAM_FMREVERSE|BAM_FPROPER_PAIR);
if (cur->core.flag&BAM_FUNMAP)
pre->core.flag &=
~(BAM_FMREVERSE|BAM_FPROPER_PAIR);
}
}
// Handle unpaired primary data
if (!cur && pre) {
pre->core.mtid = -1;
pre->core.mpos = -1;
pre->core.isize = 0;
pre->core.flag &= ~(BAM_FMREVERSE|BAM_FPROPER_PAIR);
}
// Now process secondary and supplementary alignments
for (n = 0; n < bs.n; n++) {
if (!(bs.b[n].core.flag & (BAM_FSECONDARY|BAM_FSUPPLEMENTARY))) {
// primary
continue;
}
// Secondary or supplementary
int is_r2 = (bs.b[n].core.flag & BAM_FREAD2) != 0;
bam1_t *primary = rnum[is_r2];
if (primary) {
if (base_mods)
fix_MM(primary, &bs.b[n], &state[is_r2]);
} else {
// Record with base modifications but no known primary
//fprintf(samtools_stderr, "Unpaired secondary or supplementary\n");
if (base_mods)
fix_MM(NULL, &bs.b[n], NULL);
}
}
// Finally having curated everything, write out all records in their
// original ordering
for (n = 0; n < bs.n; n++) {
bam1_t *cur = &bs.b[n];
// We may remove unmapped and secondary alignments
if (remove_reads && (cur->core.flag & (BAM_FSECONDARY|BAM_FUNMAP)))
continue;
if (sam_write1(out, header, cur) < 0)
goto write_fail;
}
}
if (result < -1)
goto read_fail;
sam_hdr_destroy(header);
for (n = 0; n < bs.ba; n++)
free(bs.b[n].data);
free(bs.b);
ks_free(&str);
return 0;
read_fail:
print_error("fixmate", "Couldn't read from input file");
goto fail;
write_fail:
print_error_errno("fixmate", "Couldn't write to output file");
fail:
sam_hdr_destroy(header);
for (n = 0; n < bs.ba; n++)
free(bs.b[n].data);
free(bs.b);
ks_free(&str);
return 1;
}
void usage(FILE* where)
{
fprintf(where,
"Usage: samtools fixmate <in.nameSrt.bam> <out.nameSrt.bam>\n"
"Options:\n"
" -r Remove unmapped reads and secondary alignments\n"
" -p Disable FR proper pair check\n"
" -c Add template cigar ct tag\n"
" -m Add mate score tag\n"
" -u Uncompressed output\n"
" -z, --sanitize FLAG[,FLAG]\n"
" Sanitize alignment fields [defaults to all types]\n"
" -M Fix base modification tags (MM/ML/MN)\n"
" --no-PG do not add a PG line\n");
sam_global_opt_help(where, "-.O..@-.");
fprintf(where,
"\n"
"As elsewhere in samtools, use '-' as the filename for stdin/samtools_stdout. The input\n"
"file must be grouped by read name (e.g. sorted by name). Coordinated sorted\n"
"input is not accepted.\n");
}
int bam_mating(int argc, char *argv[])
{
htsThreadPool p = {NULL, 0};
samFile *in = NULL, *out = NULL;
int c, remove_reads = 0, proper_pair_check = 1, add_ct = 0, res = 1,
mate_score = 0, no_pg = 0, sanitize_flags = FIX_ALL, base_mods = 0;
sam_global_args ga = SAM_GLOBAL_ARGS_INIT;
char wmode[4] = {'w', 'b', 0, 0};
static const struct option lopts[] = {
SAM_OPT_GLOBAL_OPTIONS('-', 0, 'O', 0, 0, '@'),
{"no-PG", no_argument, NULL, 1},
{ NULL, 0, NULL, 0 }
};
char *arg_list = NULL;
// parse args
if (argc == 1) { usage(samtools_stdout); return 0; }
while ((c = getopt_long(argc, argv, "rpcmMO:@:uz:", lopts, NULL)) >= 0) {
switch (c) {
case 'r': remove_reads = 1; break;
case 'p': proper_pair_check = 0; break;
case 'c': add_ct = 1; break;
case 'm': mate_score = 1; break;
case 'M': base_mods = 1; break;
case 'u': wmode[2] = '0'; break;
case 1: no_pg = 1; break;
default: if (parse_sam_global_opt(c, optarg, lopts, &ga) == 0) break;
/* else fall-through */
case '?': usage(samtools_stderr); goto fail;
case 'z':
if ((sanitize_flags = bam_sanitize_options(optarg)) < 0)
samtools_exit(1);
break;
}
}
if (optind+1 >= argc) { usage(samtools_stderr); goto fail; }
if (!no_pg && !(arg_list = stringify_argv(argc+1, argv-1)))
goto fail;
// init
if ((in = sam_open_format(argv[optind], "rb", &ga.in)) == NULL) {
print_error_errno("fixmate", "cannot open input file");
goto fail;
}
sam_open_mode(wmode+1, argv[optind+1], NULL);
if ((out = sam_open_format(argv[optind+1], wmode, &ga.out)) == NULL) {
print_error_errno("fixmate", "cannot open output file");
goto fail;
}
if (ga.nthreads > 0) {
if (!(p.pool = hts_tpool_init(ga.nthreads))) {
fprintf(samtools_stderr, "Error creating thread pool\n");
goto fail;
}
hts_set_opt(in, HTS_OPT_THREAD_POOL, &p);
hts_set_opt(out, HTS_OPT_THREAD_POOL, &p);
}
// run
res = bam_mating_core(in, out, remove_reads, proper_pair_check, add_ct,
mate_score, arg_list, no_pg, sanitize_flags,
base_mods);
// cleanup
sam_close(in);
if (sam_close(out) < 0) {
fprintf(samtools_stderr, "[bam_mating] error while closing output file\n");
res = 1;
}
if (p.pool) hts_tpool_destroy(p.pool);
free(arg_list);
sam_global_args_free(&ga);
return res;
fail:
if (in) sam_close(in);
if (out) sam_close(out);
if (p.pool) hts_tpool_destroy(p.pool);
free(arg_list);
sam_global_args_free(&ga);
return 1;
}
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