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Sampling.cpp
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Sampling.cpp
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#include <cstdio>
#include <cstring>
#include <cstdlib>
#include <ctime>
#include <cassert>
#include <cstdint>
#include <iostream>
#include <cmath>
#include <algorithm>
#include "Sampling.h"
#include <htslib/faidx.h>
#include <htslib/sam.h>
#include <htslib/vcf.h>
#include <htslib/bgzf.h>
#include <htslib/kstring.h>
#include <zlib.h>
//#include <htslib/thread_pool.h>
#include <pthread.h>
#include "mrand.h"
#include "Briggs.h"
#include "Briggs2.h"
#include "NtSubModels.h"
#include "RandSampling.h"
#include "getFragmentLength.h"
#include "ThreadGeneration.h"
#include "Sampling.h"
#include "sample_qscores.h"
#include "fasta_sampler.h"
#include "add_indels.h"
#include "NGSNGS_misc.h"
#define LENS 10000
#define MAXBINS 100
extern int refToInt[256];
extern char NtComp[5];
extern const char *bass;
pthread_mutex_t write_mutex = PTHREAD_MUTEX_INITIALIZER;
void* Sampling_threads(void *arg) {
/*
Sampling_threads - create sampling threads
*/
//casting my struct as arguments for the thread creation
Parsarg_for_Sampling_thread *struct_obj = (Parsarg_for_Sampling_thread*) arg;
// local thread seeed ensuring each thread have a local seed
unsigned int loc_seed = struct_obj->threadseed+struct_obj->threadno;
// allocate memory for random sampling structure used for sampling with Alias method
mrand_t *rand_alloc = mrand_alloc(struct_obj->rng_type,loc_seed);
// Create character array for sequence and quality scores for first and second read in paired-end reads
char READ_ID[512];
char *FragmentSequence = (char*) calloc(LENS,1);
char seq_r1[LENS] = {0};
char seq_r2[LENS] = {0};
char qual_r1[LENS] = "\0";
char qual_r2[LENS] = "\0";
// change quality offset depending on output format
int ErrProbTypeOffset = 0;
if (struct_obj->OutputFormat==fqT || struct_obj->OutputFormat==fqgzT){ErrProbTypeOffset=33;}
// buffer length used when storing sequencing reads in outoul files
size_t BufferLength = struct_obj -> BufferLength;
// Create my Kstrings used for saving the sequencing reads to the output files
kstring_t *fqs[2];
for(int i=0;i<2;i++){
fqs[i] =(kstring_t*) calloc(1,sizeof(kstring_t));
fqs[i]->s = NULL;
fqs[i]->l = fqs[i]->m = 0;
}
//Generating kstring for potential records of the stochastic indels
char INDEL_INFO[1024];
kstring_t *indel;
indel =(kstring_t*) calloc(1,sizeof(kstring_t));
indel->s = NULL;
indel->l = indel->m = 0;
// Desired sequencing reads
size_t reads = struct_obj -> reads;
// thread specific reads
size_t localread = 0;
// global number of sequencing reads ensuring it does increase above input reads (-r)
size_t current_reads_atom = 0;
// calculate the value of reads for every print statement
int modulovalue;
if (reads > 1000000){
modulovalue = 10;
}
else{
modulovalue = 1;
}
size_t moduloread = reads/modulovalue;
char *chr; //this is an unallocated pointer to a chromosome name, eg chr1, chrMT etc
extern int SIG_COND;
std::default_random_engine RndGen(loc_seed);
// sample fragments of the original molecule
sim_fragment *sf;
if (struct_obj->LengthType==0)
sf = sim_fragment_alloc(struct_obj->LengthType,struct_obj->FixedSize,struct_obj->distparam2,struct_obj->No_Len_Val,struct_obj->FragFreq,struct_obj->FragLen,struct_obj->rng_type,loc_seed,RndGen);
else
sf = sim_fragment_alloc(struct_obj->LengthType,struct_obj->distparam1,struct_obj->distparam2,struct_obj->No_Len_Val,struct_obj->FragFreq,struct_obj->FragLen,struct_obj->rng_type,loc_seed,RndGen);
// PMD specific substitution counts
int C_to_T_counter = 0;int C_total = 0;
int G_to_A_counter = 0;int G_total = 0;
int refCp1 = 0;int refCTp1 = 0;int refCp2 = 0;int refCTp2 = 0;
int sampled_skipped = 0;
int whileiter=0;
// sampling DNA molecules and simulate sequencing reads
while (current_reads_atom < reads && SIG_COND){
whileiter++;
//sample fragmentlength
int fraglength = 0;
fraglength = getFragmentLength(sf);
if(fraglength < struct_obj->lowerlimit){
if (struct_obj->LengthType>=2){
fraglength = getFragmentLength(sf);
while (fraglength < struct_obj->lowerlimit) {
//printf("%d\n", fraglength);
fraglength = getFragmentLength(sf);
}
}
else{
fraglength = struct_obj->lowerlimit;
}
}
int fragmentLength;
//maxbases is the number of nucleotides we will work with. set this to minimum of bases sequenced or fragment length. if output is fasta then length is simply the entire fragment
int maxbases = 0;
int cyclelength = 0;
//fprintf(stderr,"Max length %d \t and cycle length %d \n",maxbases,cyclelength);
if (struct_obj->OutputFormat==faT ||struct_obj->OutputFormat==fagzT || (struct_obj->FixedQual_r1r2 > 0 && struct_obj->qsreadcycle==0)){
//fprintf(stderr,"ARE WE IN THE FA LOOP?\n");
cyclelength = fraglength;
maxbases = fraglength;
}
else{
cyclelength = struct_obj->maxreadlength;
maxbases = std::min(fraglength,cyclelength);
}
// Selecting genomic start position across the generated contiguous contigs for which to extract
int chr_idx = -1;
int posB = 0; int posE = 0;
//get shallow copy of chromosome, offset into, is defined by posB, and posE
size_t chr_end;
char *chrseq = sample(struct_obj->reffasta,rand_alloc,&chr,chr_idx,posB,posE,fraglength,chr_end,struct_obj->simmode);
//extracting a biological fragment of the reference genome
if(struct_obj->simmode == 1){
// circular simulations, where sequencing reads can extend beyond the end chromosome end coordinate to continue at the start coordinate
if(posE>chr_end){
//breakpoint reads
fragmentLength = fraglength;
assert(fragmentLength>20);
size_t segment1_start = posB;
size_t segment1_length = chr_end-posB;
size_t segment2_start = 0;
size_t segment2_length = posE-chr_end;
memset(FragmentSequence,0,strlen(FragmentSequence));
// Copy first segment - end of the chromosome
strncpy(FragmentSequence, chrseq + segment1_start, segment1_length);
// Copy second segment to the correct position - start of the chromosome
strncpy(FragmentSequence + segment1_length, chrseq + segment2_start, segment2_length);
}
else{
// linear fragment
fragmentLength=posE-posB;
assert(posE>=posB&&fragmentLength>20);
memset(FragmentSequence,0,strlen(FragmentSequence));
strncpy(FragmentSequence,chrseq+(posB),fraglength);
}
}
else{
//linear simulation
fragmentLength=posE-posB;
assert(posE>=posB&&fragmentLength>20);
memset(FragmentSequence,0,strlen(FragmentSequence));
strncpy(FragmentSequence,chrseq+(posB),fraglength); // same orientation as reference genome 5' -------> FWD -------> 3'
}
int skipread = 0; // Initialize skipread to 0
// assuming original fragments with N at first and last position is more likely to originate from heterochromatin regions fully consisting of N
if(FragmentSequence[0]=='N' && FragmentSequence[(int)strlen(FragmentSequence)-1]=='N'){
skipread = 1;
memset(FragmentSequence,0,strlen(FragmentSequence));
sampled_skipped++;
continue;
}
//Generating random ID unique for each read output
double rand_val_id = mrand_pop(rand_alloc);
int rand_id = (rand_val_id * fraglength-1)+(rand_val_id*current_reads_atom);
int strandR1 = mrand_pop(rand_alloc)>0.5?0:1;
// Sequence alteration integers
int FragMisMatch = 0;
int has_seqerr = 0;
int has_indels = 0;
// Stochastic structural variation model
if(struct_obj->DoIndel){
double pars[4] = {struct_obj->IndelFuncParam[0],struct_obj->IndelFuncParam[1],struct_obj->IndelFuncParam[2],struct_obj->IndelFuncParam[3]};
int ops[2] ={0,0};
add_indel(rand_alloc,FragmentSequence,struct_obj->maxreadlength,pars,INDEL_INFO,ops);
//create the flag illustrating the indels
if (ops[0] > 0 && ops[1] == 0){
has_indels = 1;
}
else if (ops[0] == 0 && ops[1] > 0){
has_indels = 2;
}
else if (ops[0] > 0 && ops[1] > 0){
has_indels = 3;
}
int IndelFragLen = strlen(FragmentSequence);
maxbases = std::min(IndelFragLen,struct_obj->maxreadlength);
}
// Mismatch matrix input file
int MisMatchMod = 0;
if(struct_obj->doMisMatchErr){
FragMisMatch = MisMatchFile(FragmentSequence,rand_alloc,struct_obj->MisMatch,struct_obj->MisLength);
}
if (strandR1 == 1){
// 5' -------> REV -------> 3'
ReversComplement(FragmentSequence);
}
char **FragRes;
//Simulate PMD nucleotide alteration method
int ReadDeam = 0;
int Groupshift = 0;
int FragTotal = 4;
int iter = 1; //iterating through all fragments
if(struct_obj->DoBriggs){
//For the none-biotin briggs model we need to store 4 fragments with slightly different deaminations patterns
FragRes = new char *[FragTotal];
for(int i=0;i<FragTotal;i++){
FragRes[i] = new char[1024];
memset(FragRes[i],'\0',1024);
}
// simulate PMD to the original fragment and return the boolean value
ReadDeam=SimBriggsModel2(FragmentSequence, fragmentLength,
struct_obj->BriggsParam[0],
struct_obj->BriggsParam[1],
struct_obj->BriggsParam[2],
struct_obj->BriggsParam[3],rand_alloc,FragRes,strandR1,
C_to_T_counter,G_to_A_counter,C_total,G_total);
double C_Deam = (double)C_to_T_counter/(double)C_total;
double G_Deam = (double)G_to_A_counter/(double)C_total;
//fprintf(stderr,"C>T freq %f and G>A freq %f\n",C_Deam,G_Deam);
if (struct_obj->Duplicates == 1){
//keep one fragment out of the 4 possible
Groupshift = mrand_pop_long(rand_alloc) % 4;
FragTotal = Groupshift+1;
}
else if (struct_obj->Duplicates == 2){
// keep one pair of the deaminated fragments following blunt-end repair
Groupshift = mrand_pop(rand_alloc)>0.5?0:1;
iter = 2;
}
}
else if(struct_obj->DoBriggsBiotin){
// use deamination with the 454 roche sequencing platform
FragTotal = 1;
FragRes = new char *[FragTotal];
FragRes[0] = FragmentSequence;
ReadDeam=0;
ReadDeam = Biotin_ds_454Roche(FragRes[0],fragmentLength,struct_obj->BriggsParam[0],
struct_obj->BriggsParam[1],
struct_obj->BriggsParam[2],
struct_obj->BriggsParam[3],rand_alloc,
strandR1,C_to_T_counter,G_to_A_counter,C_total,G_total);
}
else{
// This is for the none-deaminated data if we want PCR duplicates. Consider extending this to be probability of duplicate - check it works
FragTotal = 1;//struct_obj->Duplicates;
FragRes = new char *[FragTotal];
for (int i = 0; i < FragTotal; i++){
FragRes[i] = FragmentSequence;
}
}
// copy part of the DNA molecule into sequencing reads for both single-end and paired-end
for (int FragNo = 0+Groupshift; FragNo < FragTotal; FragNo+=iter){
qual_r1[0] = qual_r2[0] = seq_r1[0] = seq_r2[0] = '\0';
if(SE==struct_obj->SeqType){
if(struct_obj->DoBriggs){
strncpy(seq_r1,FragRes[FragNo],maxbases);
}
else{
if (strandR1 == 0){
strncpy(seq_r1,FragRes[FragNo],maxbases);
}
else{
strncpy(seq_r1,FragRes[FragNo]+(fraglength-maxbases),maxbases);
}
}
}
else if(PE==struct_obj->SeqType){
if (strandR1 == 0){
strncpy(seq_r1,FragRes[FragNo],maxbases);
strncpy(seq_r2,FragRes[FragNo]+(fraglength-maxbases),maxbases);
}
else{
strncpy(seq_r1,FragRes[FragNo]+(fraglength-maxbases),maxbases);
strncpy(seq_r2,FragRes[FragNo],maxbases);
}
}
//For the copied sequencing reads from the original molecues, if reads are all N, we discard both sequencing read pair
for(int i=0;i<(int)strlen(seq_r1);i++){
if(seq_r1[i]=='N'){
skipread = 1;
}
}
if(PE==struct_obj->SeqType){
for(int i=0;i<(int)strlen(seq_r2);i++){
if(seq_r2[i]=='N'){
skipread = 1;
}
}
}
// reset the sequencing and quality strings
if(skipread==1){
memset(qual_r1, 0, sizeof qual_r1);
memset(qual_r2, 0, sizeof qual_r2);
memset(seq_r1, 0, sizeof seq_r1);
memset(seq_r2, 0, sizeof seq_r2);
continue;
}
// discard reads below low limit
if(strlen(seq_r1) < 20)
continue;
//generating sam output information
int SamFlags[2] = {-1,-1}; //flag[0] is for read1, flag[1] is for read2
if(struct_obj->DoBriggs){
//fprintf(stderr,"SAMPLING DO BRIGGS\t FRAG %d\n",FragNo);
if (FragNo==0||FragNo==2){
// the first duplicate pair for the PCR duplicates
//The sequences are equal to the reference
if (SE==struct_obj->SeqType){
// R1 5' |---R1-->|--FWD------------> 3'
// 3' |-----------REV------------> 3'
SamFlags[0] = 0; // Forward strand
}
else if (PE==struct_obj->SeqType){
// R1 5' |---R1-->|--FWD------------> 3'
// R2 3' ------------REV---|<--R2---| 5'
SamFlags[0] = 97; // Read paired, mate reverse strand, first in pair 99
SamFlags[1] = 145; // Read paired, read reverse strand, second in pair 147
ReversComplement(seq_r2);
}
}
else if (FragNo==1||FragNo==3){
// the second duplicate pair for the PCR duplicates
//The sequences are reverse complementary of the original reference orientation
if (SE==struct_obj->SeqType){
// R1 5' |-----------FWD------------> 3'
// 3' |---R1-->|--REV------------> 3'
SamFlags[0] = 16;
}
else if (PE==struct_obj->SeqType){
//R2 5' ------------FWD---|<--R2---| 3'
//R1 3' |---R1-->|--REV------------> 5'
SamFlags[0] = 81; // 83
SamFlags[1] = 161; // 161
ReversComplement(seq_r2);
}
}
}
else{
// without any duplicates less reverse complement is necessary
if (SE==struct_obj->SeqType){
if (strandR1 == 0)
SamFlags[0] = 0;
else if (strandR1 == 1){
SamFlags[0] = 16;
}
}
if (PE==struct_obj->SeqType){
if (strandR1 == 0){
SamFlags[0] = 97;
SamFlags[1] = 145;
}
else if (strandR1 == 1){
SamFlags[0] = 81;
SamFlags[1] = 161;
}
ReversComplement(seq_r2);
}
}
//so now everything is on 5->3 and some of them will be reverse complement to referene
//now everything is the same strand as reference, which we call plus/+
// Create the sequencing read ID - and it depends on the simulate mode
if(struct_obj->bedfilesample == 1){
snprintf(READ_ID,512,"T%d_RID%d_S%d_%s:%d-%d_length:%d_mod%d%d%d", struct_obj->threadno, rand_id,strandR1,
struct_obj->reffasta->BedReferenceEntries[chr_idx].chromosome,
struct_obj->reffasta->BedReferenceEntries[chr_idx].start+posB,
struct_obj->reffasta->BedReferenceEntries[chr_idx].start+posE-1,
fraglength,ReadDeam,FragMisMatch,has_indels);
}
else if(struct_obj->VCFcapture == 1){
snprintf(READ_ID,512,"T%d_RID%d_S%d_%s:%d-%d_length:%d_mod%d%d%d", struct_obj->threadno, rand_id,strandR1,
struct_obj->reffasta->seqs_names[chr_idx],posB+1,posE,
fraglength,ReadDeam,FragMisMatch,has_indels);
}
else{
snprintf(READ_ID,512,"T%d_RID%d_S%d_%s:%d-%d_length:%d_mod%d%d%d", struct_obj->threadno, rand_id,strandR1,chr,posB+1,posE,fraglength,ReadDeam,FragMisMatch,has_indels);
}
int nsofts[2] = {0,0}; //this will contain the softclip information to be used by sam/bam/cram output format for adapter and polytail
//below will contain the number of bases for R1 and R2 that should align to reference before adding adapters and polytail
int naligned[2] = {(int)strlen(seq_r1),-1};
if(PE==struct_obj->SeqType)
naligned[1] = strlen(seq_r2);
//add adapters
if(struct_obj->AddAdapt){
//Because i have reverse complemented the correct sequences and adapters depending on the strand origin (or flags), i know all adapters will be in 3' end
nsofts[0] = std::min(struct_obj->maxreadlength-strlen(seq_r1),std::string(struct_obj->Adapter_1).length());
strncpy(seq_r1+strlen(seq_r1),struct_obj->Adapter_1,nsofts[0]);
if(PE==struct_obj->SeqType){
nsofts[1] = std::min(struct_obj->maxreadlength-strlen(seq_r2),std::string(struct_obj->Adapter_2).length());
strncpy(seq_r2+strlen(seq_r2),struct_obj->Adapter_2,nsofts[1]);
}
}
//add polytail, like Poly-G appended after the adapater if sequence+adapter is below sequencing cycle length
if (struct_obj->PolyNt != 'F') {
int nitems = struct_obj->maxreadlength-strlen(seq_r1);
memset(seq_r1+strlen(seq_r1),struct_obj->PolyNt,nitems);
nsofts[0] += nitems;
if(PE==struct_obj->SeqType){
nitems = struct_obj->maxreadlength-strlen(seq_r2);
memset(seq_r2+strlen(seq_r2),struct_obj->PolyNt,nitems);
nsofts[1] += nitems;
}
}
if(struct_obj->SAMout){
if((int)strlen(seq_r1)!=(naligned[0]+nsofts[0])){
fprintf(stderr,"Number of aligned bases + number of adap + poly does not match\n");
exit(1);
}
//below only runs for PE that is when nalign[1] is not -1
if(naligned[1]!=-1 && (int)strlen(seq_r2)!=naligned[1]+nsofts[1]){
fprintf(stderr,"Number of aligned bases + number of adap + poly does not match\n");
exit(1);
}
}
//now seq_r1 and seq_r2 is completely done, we can generate the quality score if the format is different for Fasta
if (struct_obj->OutputFormat==faT ||struct_obj->OutputFormat==fagzT){
snprintf(READ_ID+strlen(READ_ID),512-strlen(READ_ID),"%d F%d",0,FragNo);
ksprintf(fqs[0],">%s R1\n%s\n",READ_ID,seq_r1);//make this into read
if (PE==struct_obj->SeqType)
ksprintf(fqs[1],">%s R2\n%s\n",READ_ID,seq_r2);
}
else{
//Fastq and Sam needs quality scores
if(struct_obj->FixedQual_r1r2 > 0){
has_seqerr = sample_qscores_fix(seq_r1,qual_r1,struct_obj->FixedQual_r1r2,strlen(seq_r1),rand_alloc,struct_obj->DoSeqErr,ErrProbTypeOffset);
if (PE==struct_obj->SeqType)
has_seqerr = sample_qscores_fix(seq_r2,qual_r2,struct_obj->FixedQual_r1r2,strlen(seq_r2),rand_alloc,struct_obj->DoSeqErr,ErrProbTypeOffset);
}
else{
has_seqerr = sample_qscores(seq_r1,qual_r1,strlen(seq_r1),struct_obj->QualDist_r1,struct_obj->NtQual_r1,rand_alloc,struct_obj->DoSeqErr,ErrProbTypeOffset);
if (PE==struct_obj->SeqType)
has_seqerr = sample_qscores(seq_r2,qual_r2,strlen(seq_r2),struct_obj->QualDist_r1,struct_obj->NtQual_r1,rand_alloc,struct_obj->DoSeqErr,ErrProbTypeOffset);
}
// add boolean flag and duplicate number
snprintf(READ_ID+strlen(READ_ID),512-strlen(READ_ID),"%d F%d",has_seqerr,FragNo);
//write fq if requested
if (struct_obj->OutputFormat==fqT || struct_obj->OutputFormat==fqgzT){
ksprintf(fqs[0],"@%s R1\n%s\n+\n%s\n",READ_ID,seq_r1,qual_r1);
if (PE==struct_obj->SeqType)
ksprintf(fqs[1],"@%s R2\n%s\n+\n%s\n",READ_ID,seq_r2,qual_r2);
}
//If Sequence Alignment/Map format is desired all sequencing reads needs to be reverted back to reference orientation (+ strand)
if(struct_obj->SAMout){
// generate CIGAR string with different operations
uint32_t AlignCigar[2][10];
size_t n_cigar[2] = {1,1};
if (struct_obj->Align){
AlignCigar[0][0] = bam_cigar_gen(naligned[0], BAM_CMATCH);
if(nsofts[0]>0){
AlignCigar[0][1] = bam_cigar_gen(nsofts[0], BAM_CSOFT_CLIP);
n_cigar[0] = 2;
}
if(SamFlags[0]==16||SamFlags[0]==81){
// change reads orientation and change alignment flag
ReversComplement(seq_r1);
reverseChar(qual_r1,strlen(seq_r1));
if(n_cigar[0]>1){
//swap softclip and match
uint32_t tmp= AlignCigar[0][0];
AlignCigar[0][0] = AlignCigar[0][1];
AlignCigar[0][1] = tmp;
}
}
if (PE==struct_obj->SeqType){
// create operations for second read within a pair
AlignCigar[1][0] = bam_cigar_gen(naligned[1], BAM_CMATCH);
if(nsofts[1]>0){
AlignCigar[1][1] = bam_cigar_gen(nsofts[1], BAM_CSOFT_CLIP);
n_cigar[1] = 2;
}
if(SamFlags[0] == 97){
ReversComplement(seq_r2);
reverseChar(qual_r2,strlen(seq_r2));
if(n_cigar[1]>1){
//swap softclip and match
uint32_t tmp= AlignCigar[1][0];
AlignCigar[1][0] = AlignCigar[1][1];
AlignCigar[1][1] = tmp;
}
}
}
}
else{
//unaligned part
AlignCigar[0][0] = bam_cigar_gen(strlen(seq_r1), BAM_CSOFT_CLIP);
AlignCigar[1][0] = bam_cigar_gen(strlen(seq_r2), BAM_CSOFT_CLIP);
}
//now reads, cigards and quals are correct
//generating sam field information - id, position etc.
size_t l_aux = 2; uint8_t mapq = 60;
hts_pos_t min_beg = posB;
hts_pos_t max_end = posE;
hts_pos_t insert = max_end - min_beg;
hts_pos_t mpos;
const char* suffR1 = " R1";
const char* suffR2 = " R2";
char READ_ID2[512];
strcpy(READ_ID2,READ_ID);
strcat(READ_ID,suffR1);
int chr_max_end_mate = 0; //PNEXT 0-> unavailable for SE
int insert_mate = 0; //TLEN
int chr_idx_read = -1;
if(PE==struct_obj->SeqType){
strcat(READ_ID2,suffR2);
if (struct_obj->Align == 0){
chr_max_end_mate = 0;
}
else{
chr_idx_read = chr_idx;
chr_max_end_mate = max_end;
insert_mate = insert;
}
}
if (struct_obj->Align == 0){
mapq = 255;
SamFlags[0] = SamFlags[1] = 4;
chr_idx = -1;
chr_idx_read = -1;
min_beg = -1;
max_end = 0;
}
/*
https://github.com/samtools/htslib/blob/develop/htslib/sam.h#L1040C1-L1047C1
int bam_set1(bam1_t *bam,
size_t l_qname, const char *qname,
uint16_t flag, int32_t tid, hts_pos_t pos, uint8_t mapq,
size_t n_cigar, const uint32_t *cigar,
int32_t mtid, hts_pos_t mpos, hts_pos_t isize,
size_t l_seq, const char *seq, const char *qual,
size_t l_aux);
*/
//we have set the parameters accordingly above for no align and PE
if (SE==struct_obj->SeqType){
mpos = -1;
if (struct_obj->DoBriggs){
if (FragNo==1||FragNo==3){
min_beg = max_end-strlen(seq_r1);
}
}
// store bam information
bam_set1(struct_obj->list_of_reads[struct_obj->LengthData++],
strlen(READ_ID),READ_ID,
SamFlags[0],chr_idx,min_beg,mapq,
n_cigar[0],AlignCigar[0],
chr_idx_read,mpos,insert_mate,
strlen(seq_r1),seq_r1,qual_r1,
l_aux);
}
//write PE also
if (PE==struct_obj->SeqType){
if (struct_obj->DoBriggs){
if (strandR1 == 0){
if (FragNo==1||FragNo==3){
// sam flags 81 and 161
//reverse the start and end positions
min_beg = max_end-strlen(seq_r1);
max_end = posB+strlen(seq_r2); //adding strlen(seq_r2) since its removed further down on line 641 in the case of read 1 being on opposite strand
}
}
else if (strandR1 == 1){
if (FragNo==0||FragNo==2){
// sam flags 81 and 161
//reverse the start and end positions
min_beg = max_end-strlen(seq_r1);
max_end = posB+strlen(seq_r2); //adding strlen(seq_r2) since its removed further down on line 641 in the case of read 1 being on opposite strand
}
}
}
bam_set1(struct_obj->list_of_reads[struct_obj->LengthData++],
strlen(READ_ID),READ_ID,
SamFlags[0],chr_idx,min_beg,mapq,
n_cigar[0],AlignCigar[0],
chr_idx_read,max_end-strlen(seq_r2),insert_mate,
strlen(seq_r1),seq_r1,qual_r1,
l_aux);
bam_set1(struct_obj->list_of_reads[struct_obj->LengthData++],
strlen(READ_ID2),READ_ID2,SamFlags[1],chr_idx,max_end-strlen(seq_r2),mapq,
n_cigar[1],AlignCigar[1],
chr_idx_read,min_beg,0-insert_mate,
strlen(seq_r2),seq_r2,qual_r2,l_aux);
}
if (struct_obj->LengthData < struct_obj->MaximumLength){
// write the simulated sequencing reads to sam format
pthread_mutex_lock(&write_mutex);
for (int k = 0; k < struct_obj->LengthData; k++){
assert(sam_write1(struct_obj->SAMout,struct_obj->SAMHeader,struct_obj->list_of_reads[k]) >=0 );
}
pthread_mutex_unlock(&write_mutex);
struct_obj->LengthData = 0;
}
fqs[0]->l = fqs[1]->l = 0;
}
}
// store sequencing reads and indel information to internal stochastic indel file
if (struct_obj->DoIndel && struct_obj->IndelDumpFile != NULL){
ksprintf(indel,"%s\t%s\n",READ_ID,INDEL_INFO);
if (struct_obj->bgzf_fp[2]){
if (indel->l > 0){
pthread_mutex_lock(&write_mutex);
assert(bgzf_write(struct_obj->bgzf_fp[2],indel->s,indel->l)!=0);
pthread_mutex_unlock(&write_mutex);
indel->l = 0;
}
}
}
// write simulated sequencing reads to output file
if (struct_obj->bgzf_fp[0]){
if (fqs[0]->l > BufferLength){
pthread_mutex_lock(&write_mutex);
assert(bgzf_write(struct_obj->bgzf_fp[0],fqs[0]->s,fqs[0]->l)!=0);
if (PE==struct_obj->SeqType){
assert(bgzf_write(struct_obj->bgzf_fp[1],fqs[1]->s,fqs[1]->l)!=0);
}
pthread_mutex_unlock(&write_mutex);
fqs[0]->l = fqs[1]->l = 0;
}
}
// reset sequencing reads and quality strings
memset(qual_r1, 0, sizeof qual_r1);
memset(qual_r2, 0, sizeof qual_r2);
memset(seq_r1, 0, sizeof seq_r1);
memset(seq_r2, 0, sizeof seq_r2);
// increment simulate read count
localread++;
current_reads_atom++;
//printing out the number of simulated reads depending on the modulo value
if (current_reads_atom > 1 && current_reads_atom%moduloread == 0)
fprintf(stderr,"\t-> Thread %d produced %zu reads with a current total of %zu\n",struct_obj->threadno,moduloread,current_reads_atom);
}
// delete assigned memory for PCR duplicate
chr_idx = -1;
if(struct_obj->DoBriggs){
for(int i=0;i<4;i++)
delete[] FragRes[i];
}
delete[] FragRes;
}
// save the remaining reads
if (struct_obj->bgzf_fp[0]){
if (fqs[0]->l > 0){
pthread_mutex_lock(&write_mutex);
assert(bgzf_write(struct_obj->bgzf_fp[0],fqs[0]->s,fqs[0]->l)!=0);
if (PE==struct_obj->SeqType){
assert(bgzf_write(struct_obj->bgzf_fp[1],fqs[1]->s,fqs[1]->l)!=0);
}
pthread_mutex_unlock(&write_mutex);
fqs[0]->l = fqs[1]->l = 0;
}
}
if (struct_obj->bgzf_fp[2]){
if (indel->l > 0){
pthread_mutex_lock(&write_mutex);
assert(bgzf_write(struct_obj->bgzf_fp[0],indel->s,indel->l)!=0);
pthread_mutex_unlock(&write_mutex);
indel->l = 0;
}
}
if(struct_obj->DoBriggs){
double Pair1 = (double) refCTp1/(double)refCp1;
double Pair2 = (double) refCTp2/(double)refCp2;
fprintf(stderr,"\t-> Global deamination frequency of C>T and G>A deamination at posiiton 1 is %f and %f\n",Pair1,Pair2);
}
fprintf(stderr,"\t-> Extracted fragment but skipped due to 'N' %d \n",sampled_skipped);
// free all memory for created sequences
for(int j=0; j<struct_obj->MaximumLength;j++){
bam_destroy1(struct_obj->list_of_reads[j]);
}
free(sf->rand_alloc);
delete sf;
if(fqs[0]->s)
free(fqs[0]->s);
if(fqs[1]->s)
free(fqs[1]->s);
free(fqs[0]);
free(fqs[1]);
free(indel->s);
free(indel);
free(rand_alloc);
fprintf(stderr,"\t-> Number of reads generated by thread %d is %zu \n",struct_obj->threadno,localread);
free(FragmentSequence);
if(struct_obj->totalThreads>1)
pthread_exit(NULL);
return 0;
}