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extract.c
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extract.c
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#include "htslib/sam.h"
#include "htslib/hts.h"
#include "htslib/faidx.h"
#include "htslib/kstring.h"
#include <getopt.h>
#include <stdio.h>
#include <stdlib.h>
#include <string.h>
#include <inttypes.h>
#include <assert.h>
#include <errno.h>
#include <limits.h>
#include <math.h>
#include <pthread.h>
#include "MethylDackel.h"
#define RUNOFFSET 99 //used to calculate the run length value to store in a BBM file, as value-to-store = actual-run-length + RUNOFFSET. Placed up here as it's a constant.
#define BBM_VERSION 1 //the version of the BBM file format read/written here
void print_version(void);
static inline double logit(double p) {
return(log(p) - log(1 - p));
}
//N.B., a tid of -1 means that the lastCall was written
struct lastCall{
int32_t tid, pos;
uint32_t nmethyl, nunmethyl;
};
const char *TriNucleotideContexts[25] = {"CAA", "CAC", "CAG", "CAT", "CAN", \
"CCA", "CCC", "CCG", "CCT", "CCN", \
"CGA", "CGC", "CGG", "CGT", "CGN", \
"CTA", "CTC", "CTG", "CTT", "CTN", \
"CNA", "CNC", "CNG", "CNT", "CNN"};
void writeCall(kstring_t *ks, Config *config, char *chrom, int32_t pos, int32_t width, uint32_t nmethyl, uint32_t nunmethyl, char base, char *context, const char *tnc) {
char str[10000]; // I don't really like hardcoding it, but given the probability that it ever won't suffice...
char strand = (base=='C' || base=='c') ? 'F' : 'R';
if(nmethyl+nunmethyl < config->minDepth && !config->cytosine_report) return;
if (!config->fraction && !config->logit && !config->counts && !config->methylKit && !config->cytosine_report) {
snprintf(str, 10000, \
"%s\t%i\t%i\t%i\t%" PRIu32 "\t%" PRIu32 "\n", \
chrom, \
pos, \
pos+width, \
(int) (100.0*((double) nmethyl)/(nmethyl+nunmethyl)),\
nmethyl, \
nunmethyl);
} else if(config->fraction) {
snprintf(str, 10000, \
"%s\t%i\t%i\t%f\n", \
chrom, \
pos, \
pos+width, \
((double) nmethyl)/(nmethyl+nunmethyl));
} else if(config->counts) {
snprintf(str, 10000, \
"%s\t%i\t%i\t%i\n", \
chrom, \
pos, \
pos+width, \
nmethyl+nunmethyl);
} else if(config->logit) {
snprintf(str, 10000, \
"%s\t%i\t%i\t%f\n", \
chrom, \
pos, \
pos+width, \
logit(((double) nmethyl)/(nmethyl+nunmethyl)));
} else if(config->methylKit) {
snprintf(str, 10000, \
"%s.%i\t%s\t%i\t%c\t%i\t%6.2f\t%6.2f\n", \
chrom, \
pos+1, \
chrom, \
pos+1, \
strand, \
nmethyl+nunmethyl, \
100.0 * ((double) nmethyl)/(nmethyl+nunmethyl), \
100.0 * ((double) nunmethyl)/(nmethyl+nunmethyl));
} else if(config->cytosine_report) {
strand = (base=='C' || base=='c') ? '+' : '-';
snprintf(str, 10000, \
"%s\t%i\t%c\t%"PRIu32"\t%"PRIu32"\tC%s\t%s\n", \
chrom, \
pos+1, \
strand, \
nmethyl, \
nunmethyl, \
context, \
tnc);
}
kputs(str, ks);
}
char revcomp(char b) {
switch(b) {
case 'A':
case 'a':
return 'T';
case 'C':
case 'c':
return 'G';
case 'G':
case 'g':
return 'C';
case 'T':
case 't':
return 'A';
default:
return 'N';
}
}
int getTriNucContext(char *seq, uint32_t offset, int seqlen, int direction) {
int rv = 0;
char base;
//Last base: column
if((direction > 0 && offset+2 >= seqlen) || (direction < 0 && offset <= 1)) rv = 4;
else {
base = *(seq+offset+2*direction);
if(direction < 0) base = revcomp(base);
switch(base) {
case 'A':
case 'a':
rv = 0;
break;
case 'C':
case 'c':
rv = 1;
break;
case 'G':
case 'g':
rv = 2;
break;
case 'T':
case 't':
rv = 3;
break;
default:
rv = 4;
break;
}
}
//Middle
if((direction > 0 && offset+1 >= seqlen) || (direction < 0 && offset == 0)) rv += 20;
else {
base = *(seq+offset+direction);
if(direction < 0) base = revcomp(base);
switch(base) {
case 'A':
case 'a':
rv += 0;
break;
case 'C':
case 'c':
rv += 5;
break;
case 'G':
case 'g':
rv += 10;
break;
case 'T':
case 't':
rv += 15;
break;
default:
rv += 20;
break;
}
}
return rv;
}
void writeBlank(kstring_t **ks, Config *config, char *chrom, int32_t pos, uint32_t localPos2, uint32_t *lastPos, char *seq, int seqlen) {
int triNucContext = 0;
int direction = 0;
char context[3] = "HG";
if(pos == -1) return;
for(;*lastPos < pos; (*lastPos)++) {
if((direction = isCpG(seq, *lastPos-localPos2, seqlen)) != 0) {
if(!config->keepCpG) continue;
triNucContext = getTriNucContext(seq, *lastPos - localPos2, seqlen, direction);
context[0] = 'G'; context[1] = 0;
} else if((direction = isCHG(seq, *lastPos-localPos2, seqlen)) != 0) {
if(!config->keepCHG) continue;
triNucContext = getTriNucContext(seq, *lastPos - localPos2, seqlen, direction);
context[0] = 'H'; context[1] = 'G';
} else if((direction = isCHH(seq, *lastPos-localPos2, seqlen)) != 0) {
if(!config->keepCHH) continue;
triNucContext = getTriNucContext(seq, *lastPos - localPos2, seqlen, direction);
context[0] = 'H'; context[1] = 'H';
} else {
continue;
}
writeCall(ks[0], config, chrom, *lastPos, 1, 0, 0, (direction>0)?'C':'G', context, TriNucleotideContexts[triNucContext]);
}
}
void processLast(kstring_t *ks, Config *config, struct lastCall *last, bam_hdr_t *hdr, int32_t tid, int32_t pos, int width, uint32_t nmethyl, uint32_t nunmethyl, char base) {
if(last->tid == tid && last->pos == pos) {
nmethyl += last->nmethyl;
nunmethyl += last->nunmethyl;
writeCall(ks, config, hdr->target_name[tid], pos, width, nmethyl, nunmethyl, base, NULL, NULL);
last->tid = -1;
} else {
if(last->tid != -1) {
writeCall(ks, config, hdr->target_name[last->tid], last->pos, width, last->nmethyl, last->nunmethyl, base, NULL, NULL);
}
last->tid = tid;
last->pos = pos;
last->nmethyl = nmethyl;
last->nunmethyl = nunmethyl;
}
}
// The opposite strand of a C should be a G. Ns are ignored
int isVariant(Config *config, const bam_pileup1_t *plp, uint32_t *coverage, int strand) {
uint8_t base = bam_seqi(bam_get_seq(plp->b), plp->qpos);
//Is the phred score even high enough?
if(bam_get_qual(plp->b)[plp->qpos] < config->minPhred) return 0;
*coverage += 1;
if(strand & 1) { //OT or CTOT
if(base != 4 && base != 15) return 1;
else return 0;
} else { // OB or CTOB
if(base != 2 && base != 15) return 1;
else return 0;
}
}
int error()
{
printf("fatal: malformed BBM file\n");
return -9;
}
void *extractCalls(void *foo) {
Config *config = (Config*) foo;
bam_hdr_t *hdr;
bam_mplp_t iter;
int ret, tid, i, seqlen, type, rv, o = 0;
hts_pos_t pos;
int32_t bedIdx = 0;
int n_plp; //This will need to be modified for multiple input files
int strand, direction;
uint32_t nmethyl = 0, nunmethyl = 0, nOff = 0, nVariant = 0;
uint32_t localBin = 0, localPos = 0, localEnd = 0, localTid = 0, localPos2 = 0, lastPos = 0;
uint64_t nVariantPositions = 0;
const bam_pileup1_t **plp = NULL;
char *seq = NULL, base = 'A';
char context[3] = "HG";
int tnc;
mplp_data *data = NULL;
struct lastCall *lastCpG = NULL;
struct lastCall *lastCHG = NULL;
kstring_t **os = NULL, *os_CpG = NULL, *os_CHG = NULL, *os_CHH = NULL;
faidx_t *fai;
hts_idx_t *bai;
htsFile *fp;
os = calloc(3, sizeof(kstring_t*));
os_CpG = calloc(1, sizeof(kstring_t));
os_CHG = calloc(1, sizeof(kstring_t));
os_CHH = calloc(1, sizeof(kstring_t));
if(!os_CpG || !os_CHG || !os_CHH || !os) {
fprintf(stderr, "Couldn't allocate space for the kstring_t structures in extractCalls()!\n");
return NULL;
}
os[0] = os_CpG;
os[1] = os_CHG;
os[2] = os_CHH;
//Open the files
if((fai = fai_load(config->FastaName)) == NULL) {
fprintf(stderr, "Couldn't open the index for %s!\n", config->FastaName);
return NULL;
}
if((fp = hts_open(config->BAMName, "rb")) == NULL) {
fprintf(stderr, "Couldn't open %s for reading!\n", config->BAMName);
return NULL;
}
if((bai = sam_index_load(fp, config->BAMName)) == NULL) {
fprintf(stderr, "Couldn't load the index for %s\n", config->BAMName);
return NULL;
}
hdr = sam_hdr_read(fp);
if(config->merge) {
if(config->keepCpG) {
lastCpG = calloc(1, sizeof(struct lastCall));
assert(lastCpG);
lastCpG->tid = -1;
}
if(config->keepCHG) {
lastCHG = calloc(1, sizeof(struct lastCall));
assert(lastCHG);
lastCHG->tid = -1;
}
}
data = calloc(1,sizeof(mplp_data));
if(data == NULL) {
fprintf(stderr, "Couldn't allocate space for the data structure in extractCalls()!\n");
return NULL;
}
data->config = config;
data->hdr = hdr;
data->fp = fp;
data->bedIdx = bedIdx;
plp = calloc(1, sizeof(bam_pileup1_t *));
if(plp == NULL) {
fprintf(stderr, "Couldn't allocate space for the plp structure in extractCalls()!\n");
return NULL;
}
while(1) {
//Lock and unlock the mutex so we can get/update the tid/position
pthread_mutex_lock(&positionMutex);
localBin = bin++;
localTid = globalTid;
localPos = globalPos;
localEnd = localPos + config->chunkSize;
if(localTid >= hdr->n_targets) {
pthread_mutex_unlock(&positionMutex);
break;
}
if(globalEnd && localEnd > globalEnd) localEnd = globalEnd;
adjustBounds(config, hdr, fai, &localTid, &localPos, &localEnd);
globalPos = localEnd;
if(globalEnd > 0 && globalPos >= globalEnd) {
//If we've specified a region, then break once we're outside of it
globalTid = (uint32_t) -1;
}
if(localTid < hdr->n_targets && globalTid != (uint32_t) -1) {
if(globalPos >= hdr->target_len[localTid]) {
localEnd = hdr->target_len[localTid];
globalTid++;
globalPos = 0;
}
}
pthread_mutex_unlock(&positionMutex);
//If we have a BED file, then jump to the first overlapping region:
if(config->bed) {
if(spanOverlapsBED(localTid, localPos, localEnd, config->bed, &bedIdx) != 1) {
//Set the bin as written and loop
while(1) {
pthread_mutex_lock(&outputMutex);
if(outputBin != localBin) {
pthread_mutex_unlock(&outputMutex);
continue;
}
outputBin++;
break;
}
pthread_mutex_unlock(&outputMutex);
continue;
}
}
localPos2 = 0;
if(localPos > 1) {
localPos2 = localPos - 2;
}
lastPos = localPos;
//Break out of the loop if finished
if(localTid >= hdr->n_targets) break; //Finish looping
if(globalEnd && localPos >= globalEnd) break;
data->iter = sam_itr_queryi(bai, localTid, localPos, localEnd);
seq = faidx_fetch_seq(fai, hdr->target_name[localTid], localPos2, localEnd+10, &seqlen);
if(seqlen < 0) {
fprintf(stderr, "faidx_fetch_seq returned %i while trying to fetch the sequence for tid %s:%"PRIu32"-%"PRIu32"!\n",\
seqlen, hdr->target_name[localTid], localPos2, localEnd);
fprintf(stderr, "Note that the output will be truncated!\n");
continue;
}
data->seq = seq;
data->offset = localPos2;
data->lseq = seqlen;
//Start the pileup
data->ohash = initOlapHash();
iter = bam_mplp_init(1, filter_func, (void **) &data);
bam_mplp_set_maxcnt(iter, INT_MAX);
bam_mplp_constructor(iter, custom_overlap_constructor);
bam_mplp_destructor(iter, custom_overlap_destructor);
while((ret = bam_mplp64_auto(iter, &tid, &pos, &n_plp, plp)) > 0) {
if(pos < localPos || pos >= localEnd) continue; // out of the region requested
if(config->bed) { //Handle -l
while((o = posOverlapsBED(tid, pos, config->bed, bedIdx)) == -1) bedIdx++;
if(o == 0) continue; //Wrong strand
}
if((direction = isCpG(seq, pos-localPos2, seqlen))) {
if(!config->keepCpG) continue;
type = 0;
} else if((direction = isCHG(seq, pos-localPos2, seqlen))) {
if(!config->keepCHG) continue;
type = 1;
} else if((direction = isCHH(seq, pos-localPos2, seqlen))) {
if(!config->keepCHH) continue;
type = 2;
} else {
continue;
}
nmethyl = nunmethyl = nVariant = nOff = 0;
base = *(seq+pos-localPos2);
for(i=0; i<n_plp; i++) {
if(plp[0][i].is_del) continue;
if(plp[0][i].is_refskip) continue;
if(config->bed) if(!readStrandOverlapsBED(plp[0][i].b, config->bed->region[bedIdx])) continue;
strand = getStrand((plp[0]+i)->b);
if(strand & 1) {
if(base != 'C' && base != 'c') {
nVariant += isVariant(config, plp[0]+i, &nOff, strand);
continue;
}
} else {
if(base != 'G' && base != 'g') {
nVariant += isVariant(config, plp[0]+i, &nOff, strand);
continue;
}
}
rv = updateMetrics(config, plp[0]+i);
if(rv > 0) nmethyl++;
else if(rv<0) nunmethyl++;
}
// Skip likely variant positions
if(config->minOppositeDepth > 0 && \
nOff >= config->minOppositeDepth && \
((double) nVariant) / ((double) nOff) >= config->maxVariantFrac) {
nVariantPositions++;
//If we're merging context, then skip an entire merged site
if(config->merge) {
if(type == 0 && lastCpG->tid == tid && lastCpG->pos == pos - 1 && (base == 'G' || base == 'g')) {
lastCpG->nmethyl = 0;
lastCpG->nunmethyl = 0;
} else if(type == 1 && lastCHG->tid == tid && lastCHG->pos == pos - 2 && (base == 'G' || base == 'g')) {
lastCHG->nmethyl = 0;
lastCHG->nunmethyl = 0;
}
}
continue;
}
if(nmethyl+nunmethyl==0 && config->cytosine_report == 0) continue;
if(!config->merge || type==2) { //Also, cytosine report
if(config->cytosine_report) {
writeBlank(os, config, hdr->target_name[localTid], pos, localPos2, &lastPos, seq, seqlen);
//Set the C-context
if(type == 0) {
context[0] = 'G'; context[1] = 0;
} else if(type == 1) {
context[0] = 'H'; context[1] = 'G';
} else {
context[0] = 'H'; context[1] = 'H';
}
//Set the trinucleotide context
tnc = getTriNucContext(seq, pos - localPos2, seqlen, direction);
writeCall(os[0], config, hdr->target_name[tid], pos, 1, nmethyl, nunmethyl, base, context, TriNucleotideContexts[tnc]);
} else {
writeCall(os[type], config, hdr->target_name[tid], pos, 1, nmethyl, nunmethyl, base, NULL, NULL);
}
} else {
//Merge into per-CpG/CHG metrics
if(type==0) {
if(base=='G' || base=='g') pos--;
processLast(os_CpG, config, lastCpG, hdr, tid, pos, 2, nmethyl, nunmethyl, base);
} else {
if(base=='G' || base=='g') pos-=2;
processLast(os_CHG, config, lastCHG, hdr, tid, pos, 3, nmethyl, nunmethyl, base);
}
}
lastPos = pos+1;
}
bam_mplp_destroy(iter);
//Don't forget the last CpG/CHG
nmethyl = 0;
nunmethyl = 0;
if(config->merge) {
if(config->keepCpG && lastCpG->tid != -1) {
processLast(os_CpG, config, lastCpG, hdr, tid, pos, 2, nmethyl, nunmethyl, base);
lastCpG->tid = -1;
}
if(config->keepCHG && lastCHG->tid != -1) {
processLast(os_CHG, config, lastCHG, hdr, tid, pos, 3, nmethyl, nunmethyl, base);
lastCHG->tid = -1;
}
} else if(config->cytosine_report) {
writeBlank(os, config, hdr->target_name[localTid], localEnd, localPos2, &lastPos, seq, seqlen);
}
hts_itr_destroy(data->iter);
free(seq);
while(1) {
pthread_mutex_lock(&outputMutex);
if(outputBin != localBin) {
pthread_mutex_unlock(&outputMutex);
continue;
}
if(config->keepCpG && os_CpG->l) {
fputs(os_CpG->s, config->output_fp[0]);
os_CpG->l = 0;
}
if(config->keepCHG && os_CHG->l) {
fputs(os_CHG->s, config->output_fp[1]);
os_CHG->l = 0;
}
if(config->keepCHH && os_CHH->l) {
fputs(os_CHH->s, config->output_fp[2]);
os_CHH->l = 0;
}
outputBin++;
pthread_mutex_unlock(&outputMutex);
break;
}
destroyOlapHash(data->ohash);
}
free(os_CpG->s); free(os_CpG);
free(os_CHG->s); free(os_CHG);
free(os_CHH->s); free(os_CHH);
free(os);
bam_hdr_destroy(hdr);
fai_destroy(fai);
hts_close(fp);
hts_idx_destroy(bai);
free(data);
free(plp);
if(config->merge) {
if(config->keepCpG) free(lastCpG);
if(config->keepCHG) free(lastCHG);
}
if(nVariantPositions > 0) {
pthread_mutex_lock(&outputMutex);
globalnVariantPositions += nVariantPositions;
pthread_mutex_unlock(&outputMutex);
}
return NULL;
}
void printHeader(FILE *of, char *context, char *opref, Config config) {
fprintf(of, "track type=\"bedGraph\" description=\"%s %s", opref, context);
if(config.merge) fprintf(of, " merged");
if(config.fraction) fprintf(of, " methylation fractions\"\n");
else if(config.counts) fprintf(of, " methylation counts\"\n");
else if(config.logit) fprintf(of, " logit transformed methylation fractions\"\n");
else fprintf(of, " methylation levels\"\n");
}
void extract_usage() {
fprintf(stderr, "\nUsage: MethylDackel extract [OPTIONS] <ref.fa> <sorted_alignments.bam>\n");
fprintf(stderr,
"\n"
"Options:\n"
" -q INT Minimum MAPQ threshold to include an alignment (default 10)\n"
" -p INT Minimum Phred threshold to include a base (default 5). This\n"
" must be >0.\n"
" -D INT Ignored, kept only for backward compatibility.\n"
" -d INT Minimum per-base depth for reporting output. If you use\n"
" --mergeContext, this then applies to the merged CpG/CHG.\n"
" (default 1)\n"
" -r STR Region string in which to extract methylation\n"
" -l FILE A BED file listing regions for inclusion.\n"
" --keepStrand If a BED file is specified, then this option will cause the\n"
" strand column (column 6) to be utilized, if present. Thus, if\n"
" a region has a '+' in this column, then only metrics from the\n"
" top strand will be output. Note that the -r option can be used\n"
" to limit the regions of -l.\n"
" -M, --mappability FILE A bigWig file containing mappability data for\n"
" filtering reads.\n"
" -t, --mappabilityThreshold FLOAT If a bigWig file is provided, this sets the\n"
" threshold mappability value above which a base is considered\n"
" mappable (default 0.01).\n"
" -b, --minMappableBases INT If a bigWig file is provided, this sets the\n"
" number of mappable bases needed for a read to be considered\n"
" mappable (default 15).\n"
" -O, --outputBBMFile If this is specified, a Binary Bismap (.bbm) file will\n"
" be written with the same base name as the provided bigWig file,\n"
" but with the .bbm extension. Neither this option nor -N have any\n"
" effect if a bigWig is not specified with -M.\n"
" -N, --outputBBMFileName FILE If this is specified, a Binary Bismap (.bbm) file will\n"
" be written at the provided filename. Neither this option nor -O\n"
" have any effect if a bigWig is not specified with -M.\n"
" -B, --mappabilityBB FILE A .bbm file containing mappability data for\n"
" filtering reads.\n"
" -@ nThreads The number of threads to use, the default 1\n"
" --chunkSize INT The size of the genome processed by a single thread at a time.\n"
" The default is 1000000 bases. This value MUST be at least 1.\n"
" --mergeContext Merge per-Cytosine metrics from CpG and CHG contexts into\n"
" per-CPG or per-CHG metrics.\n"
" -o, --opref STR Output filename prefix. CpG/CHG/CHH context metrics will be\n"
" output to STR_CpG.bedGraph and so on.\n"
" --keepDupes By default, any alignment marked as a duplicate is ignored.\n"
" This option causes them to be incorporated. This will unset\n"
" bit 0x400 in --ignoreFlags.\n"
" --keepSingleton By default, if only one read in a pair aligns (a singleton)\n"
" then it's ignored.\n"
" --keepDiscordant By default, paired-end alignments with the properly-paired bit\n"
" unset in the FLAG field are ignored. Note that the definition\n"
" of concordant and discordant is based on your aligner\n"
" settings.\n"
" -F, --ignoreFlags By default, any alignment marked as secondary (bit 0x100),\n"
" failing QC (bit 0x200), a PCR/optical duplicate (0x400) or\n"
" supplemental (0x800) is ignored. This equates to a value of\n"
" 0xF00 or 3840 in decimal. If you would like to change that,\n"
" you can specify a new value here.\n"
" ignored. Specifying this causes them to be included.\n"
" -R, --requireFlags Require each alignment to have all bits in this value\n"
" present, or else the alignment is ignored. This is equivalent\n"
" to the -f option in samtools. The default is 0, which\n"
" includes all alignments.\n"
" --noCpG Do not output CpG context methylation metrics\n"
" --CHG Output CHG context methylation metrics\n"
" --CHH Output CHH context methylation metrics\n"
" --fraction Extract fractional methylation (only) at each position. This\n"
" produces a file with a .meth.bedGraph extension.\n"
" --counts Extract base counts (only) at each position. This produces a\n"
" file with a .counts.bedGraph extension.\n"
" --logit Extract logit(M/(M+U)) (only) at each position. This produces\n"
" a file with a .logit.bedGraph extension.\n"
" --ignoreNH Ignore NH auxiliary tags. By default, if an NH tag is present\n"
" and its value is >1 then an entry is ignored as a\n"
" multimapper.\n"
" --minOppositeDepth If you would like to exclude sites that likely contain\n"
" SNPs, then specifying a value greater than 0 here will\n"
" indicate the minimum depth required on the strand opposite of\n"
" a C to look for A/T/C bases. The fraction of these necessary\n"
" to exclude a position from methylation extraction is specified\n"
" by the --maxVariantFrac parameter. The default is 0, which\n"
" means that no positions will be excluded. Note that the -p and\n"
" -q apply here as well. Note further that if you use\n"
" --mergeContext that a merged site will be excluded if either\n"
" of its individual Cs would be excluded.\n"
" --minConversionEfficiency The minimum non-CpG conversion efficiency observed\n"
" in a read to include it in the output. The default is 0.0 and\n"
" the maximum is 1.0 (100%% conversion). You are strongly\n"
" encouraged to NOT use this option without an EXTREMELY\n"
" compelling reason!\n"
" --maxVariantFrac The maximum fraction of A/T/C base calls on the strand\n"
" opposite of a C to allow before a position is declared a\n"
" variant (thereby being excluded from output). The default is\n"
" 0.0. See also --minOppositeDepth.\n"
" --methylKit Output in the format required by methylKit. Note that this is\n"
" incompatible with --mergeContext, --fraction and --counts.\n"
" --cytosine_report A per-base exhaustive report comparable to that produced\n"
" with the same option in Bismark's methylation extractor. The\n"
" output is a tab-separated file with the following columns:\n"
" chromosome, position (1-based), strand, number of alignments\n"
" supporting methylation, number of alignments supporting\n"
" unmethylation, CG/CHG/CHH, trinucleotide context. This is not\n"
" compatible with --fraction, --counts, --methylKit, or\n"
" --mergeContext. The produces a single file with a\n"
" .cytosine_report.txt extension. Note that even bases with no\n"
" coverage will be included in the output.\n"
" --OT INT,INT,INT,INT Inclusion bounds for methylation calls from reads/pairs\n"
" originating from the original top strand. Suggested values can\n"
" be obtained from the MBias program. Each integer represents a\n"
" 1-based position on a read. For example --OT A,B,C,D\n"
" translates to, \"Include calls at positions from A through B\n"
" on read #1 and C through D on read #2\". If a 0 is used a any\n"
" position then that is translated to mean start/end of the\n"
" alignment, as appropriate. For example, --OT 5,0,0,0 would\n"
" include all but the first 4 bases on read #1. Users are\n"
" strongly advised to consult a methylation bias plot, for\n"
" example by using the MBias program.\n"
" --OB INT,INT,INT,INT\n"
" --CTOT INT,INT,INT,INT\n"
" --CTOB INT,INT,INT,INT As with --OT, but for the original bottom, complementary\n"
" to the original top, and complementary to the original bottom\n"
" strands, respectively.\n"
" --nOT INT,INT,INT,INT Like --OT, but always exclude INT bases from a given end\n"
" from inclusion,regardless of the length of an alignment. This\n"
" is useful in cases where reads may have already been trimmed\n"
" to different lengths, but still none-the-less contain a\n"
" certain length bias at one or more ends.\n"
" --nOB INT,INT,INT,INT\n"
" --nCTOT INT,INT,INT,INT\n"
" --nCTOB INT,INT,INT,INT As with --nOT, but for the original bottom,\n"
" complementary to the original top, and complementary to the\n"
" original bottom strands, respectively.\n"
" --version Print version and then quit.\n"
"\nNote that --fraction, --counts, and --logit are mutually exclusive!\n");
}
int extract_main(int argc, char *argv[]) {
char *opref = NULL, *oname, *p;
int c, i, j, keepStrand = 0;
Config config;
bam_hdr_t *hdr = NULL;
globalTid = globalPos = globalEnd = bin = globalnVariantPositions = 0;
//Defaults
config.outputBB = 0;
config.chromCount = 0;
config.BWName = NULL;
config.BBMName = NULL;
config.outBBMName = NULL;
config.BW_ptr = NULL;
config.BBM_ptr = NULL;
config.filterMappability = 0;
config.mappabilityCutoff = 0.01;
config.minMappableBases = 15;
config.keepCpG = 1; config.keepCHG = 0; config.keepCHH = 0;
config.minMapq = 10; config.minPhred = 5; config.keepDupes = 0;
config.keepSingleton = 0, config.keepDiscordant = 0;
config.ignoreNH = 0;
config.minDepth = 1;
config.methylKit = 0;
config.merge = 0;
config.minOppositeDepth = 0;
config.maxVariantFrac = 0.0;
config.chromNames = NULL;
config.chromLengths = NULL;
config.chromCount = 0;
config.fp = NULL;
config.bai = NULL;
config.reg = NULL;
config.bedName = NULL;
config.bed = NULL;
config.fraction = 0;
config.counts = 0;
config.logit = 0;
config.ignoreFlags = 0xF00;
config.requireFlags = 0;
config.nThreads = 1;
config.chunkSize = 1000000;
config.cytosine_report = 0;
config.noBAM = 0;
config.minConversionEfficiency = 0.0;
for(i=0; i<16; i++) config.bounds[i] = 0;
for(i=0; i<16; i++) config.absoluteBounds[i] = 0;
static struct option lopts[] = {
{"opref", 1, NULL, 'o'},
{"fraction", 0, NULL, 'f'},
{"counts", 0, NULL, 'c'},
{"logit", 0, NULL, 'm'},
{"minDepth", 1, NULL, 'd'},
{"noCpG", 0, NULL, 1},
{"CHG", 0, NULL, 2},
{"CHH", 0, NULL, 3},
{"keepDupes", 0, NULL, 4},
{"keepSingleton",0, NULL, 5},
{"keepDiscordant",0,NULL, 6},
{"OT", 1, NULL, 7},
{"OB", 1, NULL, 8},
{"CTOT", 1, NULL, 9},
{"CTOB", 1, NULL, 10},
{"mergeContext", 0, NULL, 11},
{"methylKit", 0, NULL, 12},
{"nOT", 1, NULL, 13},
{"nOB", 1, NULL, 14},
{"nCTOT", 1, NULL, 15},
{"nCTOB", 1, NULL, 16},
{"minOppositeDepth", 1, NULL, 17},
{"maxVariantFrac", 1, NULL, 18},
{"chunkSize", 1, NULL, 19},
{"keepStrand", 0, NULL, 20},
{"cytosine_report", 0, NULL, 21},
{"minConversionEfficiency", 1, NULL, 22},
{"ignoreNH", 0, NULL, 23},
{"ignoreFlags", 1, NULL, 'F'},
{"requireFlags", 1, NULL, 'R'},
{"help", 0, NULL, 'h'},
{"version", 0, NULL, 'v'},
{"mappability", 1, NULL, 'M'},
{"mappabilityThreshold", 1, NULL, 't'},
{"minMappableBases", 1, NULL, 'b'},
{"outputBBMFile", 1, NULL, 'O'},
{"outputBBMFileName", 1, NULL, 'N'},
{"mappabilityBBM", 1, NULL, 'B'},
{0, 0, NULL, 0}
};
while((c = getopt_long(argc, argv, "hvq:p:r:l:o:D:f:c:m:d:F:R:@:M:t:b:ON:B:", lopts,NULL)) >=0){
switch(c) {
case 'h':
extract_usage();
return 0;
case 'v':
print_version();
return 0;
case 'o':
opref = strdup(optarg);
break;
case 'D':
//This is now ignored. It was --maxDepth
break;
case 'd':
config.minDepth = atoi(optarg);
if(config.minDepth < 1) {
fprintf(stderr, "Error, the minimum depth must be at least 1!\n");
return 1;
}
break;
case 'r':
config.reg = optarg;
break;
case 'l':
config.bedName = optarg;
break;
case 1:
config.keepCpG = 0;
break;
case 2:
config.keepCHG = 1;
break;
case 3:
config.keepCHH = 1;
break;
case 4:
config.keepDupes = 1;
break;
case 5:
config.keepSingleton = 1;
break;
case 6:
config.keepDiscordant = 1;
break;
case 7:
parseBounds(optarg, config.bounds, 0);
break;
case 8:
parseBounds(optarg, config.bounds, 1);
break;
case 9:
parseBounds(optarg, config.bounds, 2);
break;
case 10:
parseBounds(optarg, config.bounds, 3);
break;
case 11:
config.merge = 1;
break;
case 12:
config.methylKit = 1;
break;
case 13:
parseBounds(optarg, config.absoluteBounds, 0);
break;
case 14:
parseBounds(optarg, config.absoluteBounds, 1);
break;
case 15:
parseBounds(optarg, config.absoluteBounds, 2);
break;
case 16:
parseBounds(optarg, config.absoluteBounds, 3);
break;
case 17:
config.minOppositeDepth = atoi(optarg);
break;
case 18:
config.maxVariantFrac = atof(optarg);
break;
case 19:
config.chunkSize = strtoul(optarg, NULL, 10);
if(config.chunkSize < 1) {
fprintf(stderr, "Error: The chunk size must be at least 1!\n");
return 1;
}
break;
case 20:
keepStrand = 1;
break;
case 21:
config.cytosine_report = 1;
break;
case 22:
config.minConversionEfficiency = atof(optarg);
break;
case 23:
config.ignoreNH = 1;
break;
case 'M':
config.BWName = optarg;
break;
case 't':
config.mappabilityCutoff = atof(optarg);
break;
case 'b':
config.minMappableBases = atoi(optarg);
break;
case 'O':
config.outBBMName = NULL;
config.outputBB = 1;
break;
case 'N':
config.outBBMName = strcat(optarg, ".bbm");
config.outputBB = 1;
break;
case 'B':
config.BBMName = optarg;
break;
case 'F':
config.ignoreFlags = atoi(optarg);
break;
case 'R':
config.requireFlags = atoi(optarg);
break;
case 'q':
config.minMapq = atoi(optarg);
break;
case 'p':
config.minPhred = atoi(optarg);
break;
case 'm':
config.logit = 1;
break;
case 'f':
config.fraction = 1;
break;
case 'c':
config.counts = 1;
break;
case '@':
config.nThreads = atoi(optarg);
break;
case '?':
default :
fprintf(stderr, "Invalid option '%c'\n", c);
extract_usage();
return 1;
}
}
if(config.outputBB && (!config.outBBMName && config.BWName))
{
char* tmp = strdup(config.BWName);
int fullLen = strlen(config.BWName); //full length
char* p = strrchr(tmp, '.');
if(p != NULL) *p = '\0';
int nameLen = strlen(tmp); //length of string not including extension
if(nameLen+4 > fullLen) //if new name will be bigger than old name
{
char* tmp2 = realloc(tmp, nameLen+5); //expand str
if(tmp2 == NULL) //if realloc failed, manually copy over
{
char* tmp2 = malloc(nameLen+5);
strcpy(tmp2, tmp);
free(tmp);
}
tmp = tmp2; //assign tmp1 to point to new str
}
config.outBBMName = strcat(tmp, ".bbm");
}
if(config.outputBB && !config.BWName)
{
fprintf(stderr, "You must specify a bigWig file when attempting to create a BBM file!\n");
extract_usage();
return -1;
}
if(argc == 1) {
extract_usage();
return 0;
}
if(argc-optind < 2) {
if(config.outputBB)
{
config.noBAM = 1; //just write BBM, don't extract
}
else
{
fprintf(stderr, "You must supply a reference genome in fasta format and an input BAM file!!!\n");
extract_usage();
return -1;
}
}
//Are the options reasonable?
if(config.minPhred < 1) {
fprintf(stderr, "-p %i is invalid. resetting to 1, which is the lowest possible value.\n", config.minPhred);
config.minPhred = 1;
}