extract.c

#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;
    }
    if(config.minMapq < 0) {
        fprintf(stderr, "-q %i is invalid. Resetting to 0, which is the lowest possible value.\n", config.minMapq);
        config.minMapq = 0;
    }
    if(config.keepDupes > 0 && (config.ignoreFlags & 0x400)) {
        config.ignoreFlags -= 0x400;
    }
    if(config.fraction+config.counts+config.logit+config.methylKit+config.cytosine_report > 1) {
        fprintf(stderr, "More than one of --fraction, --counts, --methylKit, --cytosine_report and --logit were specified. These are mutually exclusive.\n");
        extract_usage();
        return 1;
    }
    if(config.methylKit + config.merge == 2) {
        fprintf(stderr, "--mergeContext and --methylKit are mutually exclusive.\n");
        extract_usage();
        return 1;
    }
    if(config.cytosine_report + config.merge == 2) {
        fprintf(stderr, "--mergeContext and --cytosine_report are mutually exclusive.\n");
        extract_usage();
        return 1;
    }

    //Has more than one output format been requested?
    if(config.fraction + config.counts + config.logit > 1) {
        fprintf(stderr, "You may specify AT MOST one of -c/--counts, -f/--fraction, or -m/--logit.\n");
        return -6;
    }

    //Is there still a metric to output?
    if(!(config.keepCpG + config.keepCHG + config.keepCHH)) {
        fprintf(stderr, "You haven't specified any metrics to output!\nEither don't use the --noCpG option or specify --CHG and/or --CHH.\n");
        return -1;
    }

    //Open the files
    if(!config.noBAM)
    {
        config.FastaName = argv[optind];
        config.BAMName = argv[optind+1];
    }
    if(!config.noBAM)
    {
        if((config.fp = hts_open(argv[optind+1], "rb")) == NULL) {
            fprintf(stderr, "Couldn't open %s for reading!\n", argv[optind+1]);
            return -4;
        }
        if((config.bai = sam_index_load(config.fp, argv[optind+1])) == NULL) {
            fprintf(stderr, "Couldn't load the index for %s, will attempt to build it.\n", argv[optind+1]);
            if(bam_index_build(argv[optind+1], 0) < 0) {
                fprintf(stderr, "Couldn't build the index for %s! File corrupted?\n", argv[optind+1]);
                return -5;
            }
            if((config.bai = sam_index_load(config.fp, argv[optind+1])) == NULL) {
                fprintf(stderr, "Still couldn't load the index, quiting.\n");
                return -5;
            }
        }
        if(config.BBMName && (config.BBM_ptr = fopen(config.BBMName, "rb")) == NULL) {
            fprintf(stderr, "Couldn't open %s for reading!\n", config.BBMName);
            return -8;
        }
    }
    
    
    if(config.BWName && (config.BW_ptr = bwOpen(config.BWName, NULL, "r")) == NULL) {
        fprintf(stderr, "Couldn't open %s for reading!\n", config.BWName);
        return -4;
    }
    
    if(config.BWName) //has a bigWig file
    {
        config.filterMappability = 1; //set flag to do filtering
        config.chromCount = config.BW_ptr->cl->nKeys; //same chromosome count
        config.chromNames = malloc(config.chromCount*sizeof(char*));
        config.chromLengths = malloc(config.chromCount*sizeof(uint32_t));
        FILE* f = NULL; //this file pointer is only used when writing a BBM file, declared here to make sure it is in scope where it is needed
        uint16_t runlen; //length of a run of the same value, declared here for the same reasons as the above file pointer
        uint16_t chromNameLen; //length of the name of a chromosome, declared here for the same reasons as the above file pointer
        unsigned char lastval; //the previous value read
        unsigned char nullterm = 0; //this just stores a null terminator which is used in writing a BBM file
        if(config.outBBMName) //are we writing a BBM?
        {
            f = fopen(config.outBBMName, "wb"); //open the BBM file for writing
            if(f == NULL)
            {
                fprintf(stderr, "Couldn't open %s for writing! Insufficient permissions?\n", config.outBBMName);
                return -7;
            }
            unsigned char bbm_version = BBM_VERSION;
            fwrite(&bbm_version, sizeof(char), 1, f); //write version
        }
        config.bw_data = malloc(config.BW_ptr->cl->nKeys*sizeof(char*)); //init outer array
        fprintf(stderr, "loading mappability data from %s\n", config.BWName);
        if(config.outBBMName) //are we writing a BBM?
        {
            fwrite(&config.BW_ptr->cl->nKeys, sizeof(uint32_t), 1, f); //write chrom count
            fprintf(stderr, "writing .bbm file to %s\n", config.outBBMName);
        }
        for(i = 0; i<config.BW_ptr->cl->nKeys; i++) //loop over chroms
        {
            config.chromNames[i] = strdup(config.BW_ptr->cl->chrom[i]); //store chrom name
            config.chromLengths[i] = config.BW_ptr->cl->len[i]; //store chrom length
            if(config.outBBMName) //are we writing a BBM?
            {
                lastval = 255; //255 here is an placeholder value since there is no last value yet
                runlen = 0;
                chromNameLen = (uint16_t)(strlen(config.BW_ptr->cl->chrom[i])); //get length of chrom name (excluding null terminator)
                fwrite(&chromNameLen, sizeof(chromNameLen), 1, f); //write length of name to file
                fwrite(config.BW_ptr->cl->chrom[i], sizeof(char), chromNameLen, f); //write name to file
                fwrite(&nullterm, sizeof(unsigned char), 1, f); //write null terminator to file
                uint32_t chromLen = (uint32_t)(config.BW_ptr->cl->len[i]); //get length of actual chromosome
                fwrite(&chromLen, sizeof(uint32_t), 1, f); //write chromosome length to file
            }
            
            int arrlen; //variable to store the length of the array used for the data (this is not the same as the chromosome length, as each value is one bit and this is an array of characters, i.e. bytes)
            arrlen = config.BW_ptr->cl->len[i]/8; //array length is chromosome length over 8 (number of bits to number of bytes)
            if(config.BW_ptr->cl->len[i]%8 > 0) //if there is a remainder that didn't divide evenly
            {
                arrlen++; //add an extra byte to store it
            }
            config.bw_data[i] = malloc(arrlen*sizeof(char)); //init inner array
            bwOverlappingIntervals_t *vals = bwGetValues(config.BW_ptr, config.BW_ptr->cl->chrom[i], 0, config.BW_ptr->cl->len[i], 1); //get data from bigWig
            for(j = 0; j<config.BW_ptr->cl->len[i]; j++) //loop over data
            {
                int index; //index in array
                char offset; //offset in byte at index
                char aboveCutoff;
                double val_raw; //value from bigWig, goes from 0.00 thru 1.00
                unsigned char val; //integer form of the value, goes from 1 thru 100
                index = j/8;
                offset = j%8;
                if(offset == 0) //starting new byte
                {
                    config.bw_data[i][index] = 0; //init new byte
                }
                val_raw = vals->value[j];
                val = (char)((val_raw*100)+0.5); //0.5 is to prevent roundoff error issues
                if(isnan(val_raw))
                {
                    //convert NaN to 0
                    val = 0;
                    val_raw = 0;
                }
                if(config.outBBMName) //are we writing a BBM?
                {
                    if(val == lastval && runlen < 65535) //in a run and haven't maxed out the run length
                    {
                        runlen++;
                        
                    }
                    else
                    {
                        if(runlen > 1) //a run just ended
                        {
                            if(runlen < 156) //short run (2 byte format)
                            {
                                unsigned char runlen_towrite = (unsigned char)(runlen)+RUNOFFSET;
                                fwrite(&runlen_towrite, sizeof(char), 1, f); //write run length + RUNOFFSET
                                fwrite(&lastval, sizeof(char), 1, f); //write run value
                            }
                            else //long run (4 byte format)
                            {
                                unsigned char flag = 255; //flag for long run
                                fwrite(&flag, sizeof(char), 1, f); //write 255 to file
                                fwrite(&runlen, sizeof(uint16_t), 1, f); //write run length
                                fwrite(&lastval, sizeof(char), 1, f); //write run value
                            }
                            runlen = 0;
                        }
                        if(j<((config.BW_ptr->cl->len[i])-1) && (char)(((vals->value[j+1])*100)+0.5) == val) //starting a run
                        {
                            lastval = val;
                            runlen = 1;
                        }
                        else //individual value
                        {
                            fwrite(&val, sizeof(char), 1, f);
                            lastval = val;
                            runlen = 0;
                        }
                    }
                }
                else
                {
                    lastval = val;
                }
                aboveCutoff = (char)(val >= config.mappabilityCutoff*100.0); //check if above cutoff
                config.bw_data[i][index] = config.bw_data[i][index] | (aboveCutoff << offset); //set bit
            }
            if(config.outBBMName)
            {
                if(runlen > 1) //a run just ended
                {
                    if(runlen < 155) //short run (2 byte format)
                    {
                        unsigned char runval = (unsigned char)(runlen)+RUNOFFSET;
                        fwrite(&runval, sizeof(char), 1, f); //write run length + RUNOFFSET
                        fwrite(&lastval, sizeof(char), 1, f); //write run value
                    }
                    else //long run (4 byte format)
                    {
                        unsigned char flag = 255; //flag for long run
                        fwrite(&flag, sizeof(char), 1, f); //write 255 to file
                        fwrite(&runlen, sizeof(uint16_t), 1, f); //write run length
                        fwrite(&lastval, sizeof(char), 1, f); //write run value
                    }
                    runlen = 0;
                }
            }
            bwDestroyOverlappingIntervals(vals); //free memory from bigWig read
            
        }
        if(config.outBBMName) //are we writing a BBM?
        {
            fclose(f); //close the BBM file
            if(config.noBAM)
            {
                for(i = 0; i<config.chromCount; i++)
                {
                    free(config.bw_data[i]);
                    free(config.chromNames[i]);
                }
                free(config.chromLengths);
                free(config.chromNames);
                free(config.bw_data);
                bwClose(config.BW_ptr);
                free(config.outBBMName);
                return 0;
            }
        }
        
    }
    
    
    if(config.BBM_ptr) //reading a BBM
    {
        config.filterMappability = 1; //set flag to filter mappability
        fprintf(stderr, "loading mappability data from %s\n", config.BBMName);
        char readlen; //used to store the length of data read from the file. Could be used in the future to detect unexpected EOF throughout reading the file, but it is currently only used to check for blank files and incorrect chrom name null terminators
        unsigned char bbm_version;
        readlen = fread(&bbm_version, sizeof(char), 1, config.BBM_ptr); //get BBM version
        if(bbm_version != BBM_VERSION)
        {
            fprintf(stderr, "fatal: %s has wrong BBM version or is malformed\n", config.BBMName);
            return -10;
        }
        readlen = fread(&config.chromCount, sizeof(config.chromCount), 1, config.BBM_ptr); //get chrom count
        config.chromNames = malloc(config.chromCount*sizeof(char*));
        config.chromLengths = malloc(config.chromCount*sizeof(uint32_t));
        if(readlen <= 0) //if the data from the file came back blank
        {
            return error(); //the file is empty, fail
        }
        int chromID = 0; //init chrom id
        config.bw_data = malloc(config.chromCount*sizeof(char*)); //init outer array
        while(chromID < config.chromCount) //loop over chroms
        {
            uint16_t nameLen;
            readlen = fread(&nameLen, sizeof(uint16_t), 1, config.BBM_ptr); //read in length of name
            config.chromNames[chromID] = malloc((nameLen+1)*sizeof(char)); //one more than length to make space for null terminator
            for(i = 0; i<nameLen; i++)
            {
                readlen = fread(&(config.chromNames[chromID][i]), sizeof(char), 1, config.BBM_ptr); //read in character of name
            }
            config.chromNames[chromID][nameLen] = 0; //null-terminate string
            char nullterm;
            readlen = fread(&nullterm, sizeof(char), 1, config.BBM_ptr); //read in null terminator from file
            if(nullterm) //file null terminator wasn't \0
            {
                return error(); //malformed file, fail
            }
            
            readlen = fread(&(config.chromLengths[chromID]), sizeof(uint32_t), 1, config.BBM_ptr); //get chromosome length
            uint32_t pos = 0;
            int arrlen; //variable to store the length of the array used for the data (this is not the same as the chromosome length, as each value is one bit and this is an array of characters, i.e. bytes)
            arrlen = config.chromLengths[chromID]/8; //array length is chromosome length over 8 (number of bits to number of bytes)
            if(config.chromLengths[chromID]%8 > 0) //if there is a remainder that didn't divide evenly
            {
                arrlen++; //add an extra byte to store it
            }
            config.bw_data[chromID] = malloc(arrlen*sizeof(char)); //init inner array
            while(pos<(config.chromLengths[chromID])) //loop over chrom
            {
                
                int index; //index in array
                char offset; //offset in byte at index
                char aboveCutoff;
                index = pos/8;
                offset = pos%8;
                if(offset == 0) //starting new byte
                {
                    config.bw_data[chromID][index] = 0; //init new byte
                }

                
                unsigned char val; //data value from file
                uint16_t runlen; //length of a run of the same value
                readlen = fread(&val, sizeof(val), 1, config.BBM_ptr); //read value
                if(val > 100) //if the value is >100, this is a flag for a run, not a standard value
                {
                    if(val == 255) //long run
                    {
                        readlen = fread(&runlen, sizeof(uint16_t), 1, config.BBM_ptr); //read in run length
                        readlen = fread(&val, sizeof(val), 1, config.BBM_ptr); //read value
                    }
                    else //short run
                    {
                        runlen = val-RUNOFFSET; //calculate run length
                        readlen = fread(&val, sizeof(val), 1, config.BBM_ptr); //read value
                    }
                    aboveCutoff = (char)(val >= config.mappabilityCutoff*100.0); //check if above cutoff
                    for(i = 0; i<runlen; i++) //loop runlen times to store a run of the value
                    {
                        int tempindex;
                        char tempoffset;
                        tempindex = (pos+i)/8;
                        tempoffset = (pos+i)%8;
                        if(tempoffset == 0) //starting new byte
                        {
                            config.bw_data[chromID][tempindex] = 0; //init new byte
                        }
                        config.bw_data[chromID][tempindex] = config.bw_data[chromID][tempindex] | (aboveCutoff << tempoffset); //set bit
                    }
                    pos+=runlen; //move ahead to after the run
                }
                else //individual value
                {
                    aboveCutoff = (char)(val >= config.mappabilityCutoff*100.0); //check if above cutoff
                    config.bw_data[chromID][index] = config.bw_data[chromID][index] | (aboveCutoff << offset); //set bit
                    pos++;
                }
            }
            chromID++; //next chromosome
        }
        
        fclose(config.BBM_ptr); //done with the file

    }


    //Output files
    config.output_fp = malloc(sizeof(FILE *) * 3);
    assert(config.output_fp);
    if(opref == NULL) {
        opref = strdup(argv[optind+1]);
        assert(opref);
        p = strrchr(opref, '.');
        if(p != NULL) *p = '\0';
        fprintf(stderr, "writing to prefix:'%s'\n", opref);
    }
    if(config.fraction) { 
        oname = malloc(sizeof(char) * (strlen(opref)+19));
    } else if(config.counts) {
        oname = malloc(sizeof(char) * (strlen(opref)+21));
    } else if(config.logit) {
        oname = malloc(sizeof(char) * (strlen(opref)+20));
    } else if(config.methylKit) {
        oname = malloc(sizeof(char) * (strlen(opref)+15));
    } else if(config.cytosine_report) {
        oname = malloc(sizeof(char) * (strlen(opref)+21));
        sprintf(oname, "%s.cytosine_report.txt", opref);
        config.output_fp[0] = fopen(oname, "w");
        config.output_fp[1] = config.output_fp[0];
        config.output_fp[2] = config.output_fp[0];
    } else { 
        oname = malloc(sizeof(char) * (strlen(opref)+14));
    }
    assert(oname);
    if(config.keepCpG && !config.cytosine_report) {
        if(config.fraction) { 
            sprintf(oname, "%s_CpG.meth.bedGraph", opref);
        } else if(config.counts) {
            sprintf(oname, "%s_CpG.counts.bedGraph", opref);
        } else if(config.logit) {
            sprintf(oname, "%s_CpG.logit.bedGraph", opref);
        } else if(config.methylKit) {
            sprintf(oname, "%s_CpG.methylKit", opref);
        } else { 
            sprintf(oname, "%s_CpG.bedGraph", opref);
        }
        config.output_fp[0] = fopen(oname, "w");
        if(config.output_fp[0] == NULL) {
            fprintf(stderr, "Couldn't open the output CpG metrics file for writing! Insufficient permissions?\n");
            return -3;
        }
        if(config.methylKit) {
            fprintf(config.output_fp[0], "chrBase\tchr\tbase\tstrand\tcoverage\tfreqC\tfreqT\n");
        } else {
            printHeader(config.output_fp[0], "CpG", opref, config);
        }
    }
    if(config.keepCHG && !config.cytosine_report) {
        if(config.fraction) { 
            sprintf(oname, "%s_CHG.meth.bedGraph", opref);
        } else if(config.counts) {
            sprintf(oname, "%s_CHG.counts.bedGraph", opref);
        } else if(config.logit) {
            sprintf(oname, "%s_CHG.logit.bedGraph", opref);
        } else if(config.methylKit) {
            sprintf(oname, "%s_CHG.methylKit", opref);
        } else { 
            sprintf(oname, "%s_CHG.bedGraph", opref);
        }
        config.output_fp[1] = fopen(oname, "w");
        if(config.output_fp[1] == NULL) {
            fprintf(stderr, "Couldn't open the output CHG metrics file for writing! Insufficient permissions?\n");
            return -3;
        }
        if(config.methylKit) {
            fprintf(config.output_fp[1], "chrBase\tchr\tbase\tstrand\tcoverage\tfreqC\tfreqT\n");
        } else {
            printHeader(config.output_fp[1], "CHG", opref, config);
        }
    }
    if(config.keepCHH && !config.cytosine_report) {
        if(config.fraction) { 
            sprintf(oname, "%s_CHH.meth.bedGraph", opref);
        } else if(config.counts) {
            sprintf(oname, "%s_CHH.counts.bedGraph", opref);
        } else if(config.logit) {
            sprintf(oname, "%s_CHH.logit.bedGraph", opref);
        } else if(config.methylKit) {
            sprintf(oname, "%s_CHH.methylKit", opref);
        } else { 
            sprintf(oname, "%s_CHH.bedGraph", opref);
        }
        config.output_fp[2] = fopen(oname, "w");
        if(config.output_fp[2] == NULL) {
            fprintf(stderr, "Couldn't open the output CHH metrics file for writing! Insufficient permissions?\n");
            return -3;
        }
        if(config.methylKit) {
            fprintf(config.output_fp[2], "chrBase\tchr\tbase\tstrand\tcoverage\tfreqC\tfreqT\n");
        } else {
            printHeader(config.output_fp[2], "CHH", opref, config);
        }
    }
    //parse the region, if needed
    if(config.reg) {
        const char *foo;
        char *bar = NULL;
        int s, e;
        hdr = sam_hdr_read(config.fp);
        foo = hts_parse_reg(config.reg, &s, &e);
        if(foo == NULL) {
            fprintf(stderr, "Could not parse the specified region!\n");
            return -4;
        }
        bar = malloc(foo - config.reg + 1);
        if(bar == NULL) {
            fprintf(stderr, "Could not allocate temporary space for parsing the requested region!\n");
            return -5;
        }
        strncpy(bar, config.reg, foo - config.reg);
        bar[foo - config.reg] = 0;
        globalTid = bam_name2id(hdr, bar);
        if(globalTid == (uint32_t) -1) {
            fprintf(stderr, "%s did not match a known chromosome/contig name!\n", config.reg);
            return -6;
        }
        if(s>0) globalPos = s;
        if(e>0) globalEnd = e;
        if(globalEnd > hdr->target_len[globalTid]) globalEnd = hdr->target_len[globalTid];
        free(bar);
        if(!config.bedName) bam_hdr_destroy(hdr);
    }
    if(config.bedName) {
        if(!hdr) hdr = sam_hdr_read(config.fp);
        config.bed = parseBED(config.bedName, hdr, keepStrand);
        bam_hdr_destroy(hdr);
        if(!config.bed) {
            fprintf(stderr, "There was an error while reading in your BED file!\n");
            return 1;
        }
    }
    //Run the pileup
    pthread_mutex_init(&positionMutex, NULL);
    pthread_mutex_init(&bwMutex, NULL);
    pthread_t *threads = calloc(config.nThreads, sizeof(pthread_t));
    for(i=0; i < config.nThreads; i++) pthread_create(threads+i, NULL, &extractCalls, &config);
    for(i=0; i < config.nThreads; i++) pthread_join(threads[i], NULL);
    free(threads);
    pthread_mutex_destroy(&bwMutex);
    pthread_mutex_destroy(&positionMutex);

    //If we've filtered out variant sites
    if(globalnVariantPositions) printf("%"PRIu64" positions were excluded due to likely being variants.\n", globalnVariantPositions);
    //Close things up
    hts_close(config.fp);
    if(config.cytosine_report) fclose(config.output_fp[0]);
    if(config.keepCpG && !config.cytosine_report) fclose(config.output_fp[0]);
    if(config.keepCHG && !config.cytosine_report) fclose(config.output_fp[1]);
    if(config.keepCHH && !config.cytosine_report) fclose(config.output_fp[2]);
    hts_idx_destroy(config.bai);
    free(opref);
    if(config.bed) destroyBED(config.bed);
    free(oname);
    free(config.output_fp);
    if(config.filterMappability)
    {
        for(i = 0; i<config.chromCount; i++)
        {
            free(config.bw_data[i]);
            free(config.chromNames[i]);
        }
        free(config.chromLengths);
        free(config.chromNames);
        free(config.bw_data);
    }
    bwClose(config.BW_ptr);
    return 0;
}