int aln_pair_align(FILE *fp1, FILE *fp2, AlnParam *ap, int type, int misc_flag)
{
seq_t seq1, seq2;
int len1, len2, n;
char name1[MAX_NAME_LEN], name2[MAX_NAME_LEN];
path_t *pt, *pp;
AlnAln *aa;
INIT_SEQ(seq1); INIT_SEQ(seq2);
for (n = 0; ; ++n) {
len1 = read_fasta(fp1, &seq1, name1, 0);
len2 = read_fasta(fp2, &seq2, name2, 0);
if (len1 < 0 || len2 < 0) break;
aa = aln_align((char*)seq1.s, (char*)seq2.s, ap, type);
pp = aa->path; pt = aa->path + aa->path_len - 1;
printf(">%s\t%d\t%d\t%d\t%s\t%d\t%d\t%d\t%d\n", name1, len1, pt->i, pp->i,
name2, len2, pt->j, pp->j, aa->score);
if (aa->out1) printf("%s\n", aa->out1);
if (aa->outm) printf("%s\n", aa->outm);
if (aa->out2) printf("%s\n", aa->out2);
if (type != ALN_BOUND_ALIGN) printf("//\n");
fflush(stdout);
if (misc_flag)
aln_output_segment((char*)seq1.s, (char*)seq2.s, aa->path, aa->path_len, name1, name2);
aln_free_AlnAln(aa);
}
MYFREE(seq1.s); MYFREE(seq2.s);
return n;
}
PWDB* pw_aln_contigs(CtgDB *db) {
uint32_t i, j, n;
int k, mn, mm, off0, off1, aln_len;
PWDB *pwdb;
Ctg *c0, *c1;
pwdb = (PWDB*)malloc(sizeof(PWDB));
pwdb->pwctgs = init_pwctglist(6);
pwdb->hp = init_heap(aln_cmp, pwdb);
pwdb->ctgv = db->ctgs;
AlnParam ap = {10, 2, 2, aln_sm_nt, 16, 75};
n = db->ctgnum;
for (i = 0; i < n-1; i++) {
c0 = ref_ctglist(db->ctgs, i);
for (j = i+1; j < n; j++) {
c1 = ref_ctglist(db->ctgs, j);
AlnAln *aa;
mn = mm = 0;
off0 = off1 = -1;
aa = aln_stdaln(c0->seq, c1->seq, &ap, 0, 1);
aln_len = strlen(aa->out1);
for (k = 0; k < aln_len; k++) {
if (aa->out1[k] == '-' || aa->out2[k] == '-') continue;
if (aa->out1[k] != aa->out2[k]) mm++;
mn++;
}
PWcontig *pwc = (PWcontig*)malloc(sizeof(PWcontig));
pwc->id0 = c0->cls_id;
pwc->id1 = c1->cls_id;
pwc->overlap = mn;
pwc->score = aa->score;
pwc->het = (float)mm/mn;
push_heap(pwdb->hp, pwc);
push_pwctglist(pwdb->pwctgs, pwc);
//fprintf(stdout, "%d\t%d\t%d\t%d\t%d\t%d\t%.3f\n", c0->cls_id, c1->cls_id, pwc->id0, pwc->id1, mn, mm, pwc->het);
//fprintf(stdout, "%s\n%s\n", c0->seq, c1->seq);
//fprintf(stdout, "%d\t%d\t%d\t%d\t%d\t%d\n%s\n%s\n%s\n\n", aa->start1, aa->end1,aa->start2, aa->end2, pwc->score, pwc->overlap, aa->out1, aa->outm, aa->out2);
//fprintf(stdout, "%s\n%s\n%s\n\n", aa->out1, aa->outm, aa->out2);
fflush(stdout);
aln_free_AlnAln(aa);
}
}
return pwdb;
}
static void aln_1seq(const seqs_t *ss, const char *name, int l, const char *s, char strand)
{
int i;
for (i = 0; i < ss->n_seqs; ++i) {
AlnAln *aa;
seq1_t *p = ss->seqs + i;
g_aln_param.band_width = l + p->l;
aa = aln_stdaln_aux(s, (const char*)p->s, &g_aln_param, g_is_global, g_thres, l, p->l);
if (aa->score >= g_thres || g_is_global) {
printf(">%s\t%d\t%d\t%s\t%c\t%d\t%d\t%d\t%d\t", p->n, aa->start1? aa->start1 : 1, aa->end1, name, strand,
aa->start2? aa->start2 : 1, aa->end2, aa->score, aa->subo);
// NB: I put the short sequence as the first sequence in SW, an insertion to
// the reference becomes a deletion from the short sequence. Therefore, I use
// "MDI" here rather than "MID", and print ->out2 first rather than ->out1.
for (i = 0; i != aa->n_cigar; ++i)
printf("%d%c", aa->cigar32[i]>>4, "MDI"[aa->cigar32[i]&0xf]);
printf("\n%s\n%s\n%s\n", aa->out2, aa->outm, aa->out1);
}
aln_free_AlnAln(aa);
}
bool SGSmoothingVisitor::visit(StringGraph* pGraph, Vertex* pVertex)
{
(void)pGraph;
if(pVertex->getColor() == GC_RED)
return false;
bool found = false;
for(size_t idx = 0; idx < ED_COUNT; idx++)
{
EdgeDir dir = EDGE_DIRECTIONS[idx];
EdgePtrVec edges = pVertex->getEdges(dir);
if(edges.size() <= 1)
continue;
for(size_t i = 0; i < edges.size(); ++i)
{
if(edges[i]->getEnd()->getColor() == GC_RED)
return false;
}
//std::cout << "Smoothing " << pVertex->getID() << "\n";
const int MAX_WALKS = 10;
const int MAX_DISTANCE = 5000;
bool bIsDegenerate = false;
bool bFailGapCheck = false;
bool bFailDivergenceCheck = false;
bool bFailIndelSizeCheck = false;
SGWalkVector variantWalks;
SGSearch::findVariantWalks(pVertex, dir, MAX_DISTANCE, MAX_WALKS, variantWalks);
if(variantWalks.size() > 0)
{
found = true;
size_t selectedIdx = -1;
size_t selectedCoverage = 0;
// Calculate the minimum amount overlapped on the start/end vertex.
// This is used to properly extract the sequences from walks that represent the variation.
int minOverlapX = std::numeric_limits<int>::max();
int minOverlapY = std::numeric_limits<int>::max();
for(size_t i = 0; i < variantWalks.size(); ++i)
{
if(variantWalks[i].getNumEdges() <= 1)
bIsDegenerate = true;
// Calculate the walk coverage using the internal vertices of the walk.
// The walk with the highest coverage will be retained
size_t walkCoverage = 0;
for(size_t j = 1; j < variantWalks[i].getNumVertices() - 1; ++j)
walkCoverage += variantWalks[i].getVertex(j)->getCoverage();
if(walkCoverage > selectedCoverage || selectedCoverage == 0)
{
selectedIdx = i;
selectedCoverage = walkCoverage;
}
Edge* pFirstEdge = variantWalks[i].getFirstEdge();
Edge* pLastEdge = variantWalks[i].getLastEdge();
if((int)pFirstEdge->getMatchLength() < minOverlapX)
minOverlapX = pFirstEdge->getMatchLength();
if((int)pLastEdge->getTwin()->getMatchLength() < minOverlapY)
minOverlapY = pLastEdge->getTwin()->getMatchLength();
}
// Calculate the strings for each walk that represent the region of variation
StringVector walkStrings;
for(size_t i = 0; i < variantWalks.size(); ++i)
{
Vertex* pStartVertex = variantWalks[i].getStartVertex();
Vertex* pLastVertex = variantWalks[i].getLastVertex();
assert(pStartVertex != NULL && pLastVertex != NULL);
std::string full = variantWalks[i].getString(SGWT_START_TO_END);
int posStart = 0;
int posEnd = 0;
if(dir == ED_ANTISENSE)
{
// pLast -----------
// pStart ------------
// full --------------------
// out ----
posStart = pLastVertex->getSeqLen() - minOverlapY;
posEnd = full.size() - (pStartVertex->getSeqLen() - minOverlapX);
}
else
{
// pStart --------------
// pLast -----------
// full ---------------------
// out ----
posStart = pStartVertex->getSeqLen() - minOverlapX; // match start position
posEnd = full.size() - (pLastVertex->getSeqLen() - minOverlapY); // match end position
}
std::string out;
if(posEnd > posStart)
out = full.substr(posStart, posEnd - posStart);
walkStrings.push_back(out);
}
assert(selectedIdx != (size_t)-1);
SGWalk& selectedWalk = variantWalks[selectedIdx];
assert(selectedWalk.isIndexed());
// Check the divergence of the other walks to this walk
StringVector cigarStrings;
std::vector<int> maxIndel;
std::vector<double> gapPercent; // percentage of matching that is gaps
std::vector<double> totalPercent; // percent of total alignment that is mismatch or gap
cigarStrings.resize(variantWalks.size());
gapPercent.resize(variantWalks.size());
totalPercent.resize(variantWalks.size());
maxIndel.resize(variantWalks.size());
for(size_t i = 0; i < variantWalks.size(); ++i)
{
if(i == selectedIdx)
continue;
// We want to compute the total gap length, total mismatches and percent
// divergence between the two paths.
int matchLen = 0;
int totalDiff = 0;
int gapLength = 0;
int maxGapLength = 0;
// We have to handle the degenerate case where one internal string has zero length
// this can happen when there is an isolated insertion/deletion and the walks are like:
// x -> y -> z
// x -> z
if(walkStrings[selectedIdx].empty() || walkStrings[i].empty())
{
matchLen = std::max(walkStrings[selectedIdx].size(), walkStrings[i].size());
totalDiff = matchLen;
gapLength = matchLen;
}
else
{
AlnAln *aln_global;
aln_global = aln_stdaln(walkStrings[selectedIdx].c_str(), walkStrings[i].c_str(), &aln_param_blast, 1, 1);
// Calculate the alignment parameters
while(aln_global->outm[matchLen] != '\0')
{
if(aln_global->outm[matchLen] == ' ')
totalDiff += 1;
matchLen += 1;
}
std::stringstream cigarSS;
for (int j = 0; j != aln_global->n_cigar; ++j)
{
char cigarOp = "MID"[aln_global->cigar32[j]&0xf];
int cigarLen = aln_global->cigar32[j]>>4;
if(cigarOp == 'I' || cigarOp == 'D')
{
gapLength += cigarLen;
if(gapLength > maxGapLength)
maxGapLength = gapLength;
}
cigarSS << cigarLen;
cigarSS << cigarOp;
}
cigarStrings[i] = cigarSS.str();
aln_free_AlnAln(aln_global);
}
double percentDiff = (double)totalDiff / matchLen;
double percentGap = (double)gapLength / matchLen;
if(percentDiff > m_maxTotalDivergence)
bFailDivergenceCheck = true;
if(percentGap > m_maxGapDivergence)
bFailGapCheck = true;
if(maxGapLength > m_maxIndelLength)
bFailIndelSizeCheck = true;
gapPercent[i] = percentGap;
totalPercent[i] = percentDiff;
maxIndel[i] = maxGapLength;
}
if(bIsDegenerate || bFailGapCheck || bFailDivergenceCheck || bFailIndelSizeCheck)
continue;
// Write the selected path to the variants file as variant 0
int variantIdx = 0;
std::string selectedSequence = selectedWalk.getString(SGWT_START_TO_END);
std::stringstream ss;
ss << "variant-" << m_numRemovedTotal << "/" << variantIdx++;
writeFastaRecord(&m_outFile, ss.str(), selectedSequence);
// The vertex set for each walk is not necessarily disjoint,
// the selected walk may contain vertices that are part
// of other paths. We handle this be initially marking all
// vertices of the
for(size_t i = 0; i < variantWalks.size(); ++i)
{
if(i == selectedIdx)
continue;
SGWalk& currWalk = variantWalks[i];
for(size_t j = 0; j < currWalk.getNumEdges() - 1; ++j)
{
Edge* currEdge = currWalk.getEdge(j);
// If the vertex is also on the selected path, do not mark it
Vertex* currVertex = currEdge->getEnd();
if(!selectedWalk.containsVertex(currVertex->getID()))
{
currEdge->getEnd()->setColor(GC_RED);
}
}
// Write the variant to a file
std::string variantSequence = currWalk.getString(SGWT_START_TO_END);
std::stringstream variantID;
std::stringstream ss;
ss << "variant-" << m_numRemovedTotal << "/" << variantIdx++;
ss << " IGD:" << (double)gapPercent[i] << " ITD:" << totalPercent[i] << " MID: " << maxIndel[i] << " InternalCigar:" << cigarStrings[i];
writeFastaRecord(&m_outFile, ss.str(), variantSequence);
}
if(variantWalks.size() == 2)
m_simpleBubblesRemoved += 1;
else
m_complexBubblesRemoved += 1;
++m_numRemovedTotal;
}
}
int main( int argc, char* argv[] ) {
unsigned long long num_pairs;
unsigned long long num_merged;
unsigned long long num_adapter;
unsigned long long num_discarded;
unsigned long long num_too_ambiguous_to_merge;
unsigned long long max_pretty_print = DEF_MAX_PRETTY_PRINT;
unsigned long long num_pretty_print = 0;
int adapter_thresh = DEF_ADAPTER_SCORE_THRES;
int read_thresh = DEF_READ_SCORE_THRES;
clock_t start, end;
//init to 0
num_pairs = num_merged = num_adapter = num_discarded = num_too_ambiguous_to_merge = 0;
extern char* optarg;
bool p64 = false;
char forward_fn[MAX_FN_LEN];
char reverse_fn[MAX_FN_LEN];
char forward_out_fn[MAX_FN_LEN];
char reverse_out_fn[MAX_FN_LEN];
char forward_discard_fn[MAX_FN_LEN];
char reverse_discard_fn[MAX_FN_LEN];
char merged_out_fn[MAX_FN_LEN];
bool do_read_merging = false;
bool print_overhang = false;
bool write_discard=false;
char forward_primer[MAX_SEQ_LEN+1];
strcpy(forward_primer, DEF_FORWARD_PRIMER); //set default
char forward_primer_dummy_qual[MAX_SEQ_LEN+1];
char reverse_primer[MAX_SEQ_LEN+1];
strcpy(reverse_primer, DEF_REVERSE_PRIMER); //set default
char reverse_primer_dummy_qual[MAX_SEQ_LEN+1];
int i;
for(i=0;i<MAX_SEQ_LEN+1;i++){
forward_primer_dummy_qual[i] = 'N';//phred score of 45
reverse_primer_dummy_qual[i] = 'N';
}
int ich;
int min_ol_adapter = DEF_OL2MERGE_ADAPTER;
int min_ol_reads = DEF_OL2MERGE_READS;
unsigned short int min_read_len =DEF_MIN_READ_LEN;
float min_match_adapter_frac = DEF_MIN_MATCH_ADAPTER;
float min_match_reads_frac = DEF_MIN_MATCH_READS;
float max_mismatch_adapter_frac = DEF_MAX_MISMATCH_ADAPTER;
float max_mismatch_reads_frac = DEF_MAX_MISMATCH_READS;
float read_frac_thresh = DEF_READ_GAP_FRAC_CUTOFF;
unsigned short max_mismatch_adapter[MAX_SEQ_LEN+1];
unsigned short max_mismatch_reads[MAX_SEQ_LEN+1];
unsigned short min_match_adapter[MAX_SEQ_LEN+1];
unsigned short min_match_reads[MAX_SEQ_LEN+1];
char qcut = (char)DEF_QCUT+33;
bool pretty_print = false;
char pretty_print_fn[MAX_FN_LEN+1];
SQP sqp = SQP_init();
char untrim_fseq[MAX_SEQ_LEN+1];
char untrim_fqual[MAX_SEQ_LEN+1];
char untrim_rseq[MAX_SEQ_LEN+1];
char untrim_rqual[MAX_SEQ_LEN+1];
/* No args - help! */
if ( argc == 1 ) {
help(argv[0]);
}
int req_args = 0;
while( (ich=getopt( argc, argv, "f:r:1:2:3:4:q:A:s:y:B:O:E:x:M:N:L:o:m:b:w:W:p:P:X:Q:t:e:Z:n:6gh" )) != -1 ) {
switch( ich ) {
//REQUIRED ARGUMENTS
case 'f' :
req_args ++;
strcpy( forward_fn, optarg );
break;
case 'r' :
req_args ++;
strcpy( reverse_fn, optarg );
break;
case '1' :
req_args ++;
strcpy(forward_out_fn, optarg);
break;
case '2' :
req_args ++;
strcpy(reverse_out_fn, optarg);
break;
//OPTIONAL GENERAL ARGUMENTS
case '3' :
write_discard=true;
strcpy(forward_discard_fn, optarg);
break;
case '4' :
write_discard=true;
strcpy(reverse_discard_fn, optarg);
break;
case 'h' :
help(argv[0]);
break;
case '6' :
p64 = true;
break;
case 'q' :
qcut = atoi(optarg)+33;
break;
case 'L' :
min_read_len = atoi(optarg);
break;
//OPTIONAL ADAPTER/PRIMER TRIMMING ARGUMENTS
case 'A':
strcpy(forward_primer, optarg);
break;
case 'B':
strcpy(reverse_primer, optarg);
break;
case 'O':
min_ol_adapter = atoi(optarg);
break;
case 'M':
max_mismatch_adapter_frac = atof(optarg);
break;
case 'N':
min_match_adapter_frac = atof(optarg);
break;
case 'b':
aln_param_nt2nt.band_width = atoi(optarg);
break;
case 'Q':
aln_param_nt2nt.gap_open = atoi(optarg);
break;
case 't':
aln_param_nt2nt.gap_ext = atoi(optarg);
break;
case 'e':
aln_param_nt2nt.gap_end = atoi(optarg);
break;
case 'Z':
adapter_thresh = atoi(optarg);
break;
case 'w':
aln_param_rd2rd.band_width = atoi(optarg);
break;
case 'W':
aln_param_rd2rd.gap_open = atoi(optarg);
break;
case 'p':
aln_param_rd2rd.gap_ext = atoi(optarg);
break;
case 'P':
aln_param_rd2rd.gap_end = atoi(optarg);
break;
case 'X':
read_frac_thresh = atof(optarg);
break;
//OPTIONAL MERGING ARGUMENTS
case 'y' :
maximum_quality = optarg[0];
break;
case 'g' :
print_overhang = true;
break;
case 's' :
do_read_merging = true;
strcpy( merged_out_fn, optarg );
break;
case 'o':
min_ol_reads = atoi(optarg);
break;
case 'm':
max_mismatch_reads_frac = atof(optarg);
break;
case 'n':
min_match_reads_frac = atof(optarg);
break;
case 'E':
pretty_print = true;
strcpy(pretty_print_fn,optarg);
break;
case 'x':
max_pretty_print = atol(optarg);
break;
default :
help(argv[0]);
}
}
if(req_args < 4){
fprintf(stderr, "Missing a required argument!\n");
help(argv[0]);
}
start = clock();
//allocate alignment memory
// int min_match = 8;
// int ngaps = 1;
// int maxglen = 3;
// AlnParam aln_param_adapter = { 5, 13, 19, aln_sm_read, 16, 75 };
//
//Calculate table matching overlap length to min matches and max mismatches
for(i=0;i<MAX_SEQ_LEN+1;i++){
max_mismatch_reads[i] = floor(((float)i)*max_mismatch_reads_frac);
max_mismatch_adapter[i] = floor(((float)i)*max_mismatch_adapter_frac);
min_match_reads[i] = ceil(((float)i)*min_match_reads_frac);
min_match_adapter[i] = ceil(((float)i)*min_match_adapter_frac);
}
//get length of forward and reverse primers
int forward_primer_len = strlen(forward_primer);
int reverse_primer_len = strlen(reverse_primer);
gzFile ffq = fileOpen(forward_fn, "r");
gzFile ffqw = fileOpen(forward_out_fn,"w");
gzFile rfq = fileOpen(reverse_fn, "r");
gzFile rfqw = fileOpen(reverse_out_fn,"w");
gzFile mfqw = NULL;
gzFile ppaw = NULL;
gzFile dffqw = NULL;
gzFile drfqw = NULL;
if(do_read_merging)
mfqw = fileOpen(merged_out_fn,"w");
if(pretty_print)
ppaw = fileOpen(pretty_print_fn,"w");
if(write_discard){
dffqw = fileOpen(forward_discard_fn,"w");
drfqw = fileOpen(reverse_discard_fn,"w");
}
/**
* Loop over all of the reads
*/
while(next_fastqs( ffq, rfq, sqp, p64 )){ //returns false when done
update_spinner(num_pairs++);
AlnAln *faaln, *raaln, *fraln;
//save a copy of the original sequences/qualities first
strcpy(untrim_fseq,sqp->fseq);
strcpy(untrim_fqual,sqp->fqual);
strcpy(untrim_rseq,sqp->rseq);
strcpy(untrim_rqual,sqp->rqual);
faaln = aln_stdaln_aux(sqp->fseq, forward_primer, &aln_param_nt2nt,
ALN_TYPE_LOCAL, adapter_thresh , sqp->flen, forward_primer_len);
raaln = aln_stdaln_aux(sqp->rseq, reverse_primer, &aln_param_nt2nt,
ALN_TYPE_LOCAL, adapter_thresh, sqp->rlen, reverse_primer_len);
//check for direct adapter match.
if(adapter_trim(sqp, min_ol_adapter,
forward_primer, forward_primer_dummy_qual,
forward_primer_len,
reverse_primer, reverse_primer_dummy_qual,
reverse_primer_len,
min_match_adapter,
max_mismatch_adapter,
min_match_reads,
max_mismatch_reads,
qcut) ||
faaln->score >= adapter_thresh ||
raaln->score >= adapter_thresh){
num_adapter++; //adapter present
//print it if user wants
if(pretty_print && num_pretty_print < max_pretty_print){
//void pretty_print_alignment_stdaln(gzFile out, SQP sqp, AlnAln *aln, bool first_adapter, bool second_adapter)
if(faaln->score >= adapter_thresh){
num_pretty_print++;
pretty_print_alignment_stdaln(ppaw,sqp,faaln,true,false,false);
}
if(raaln->score >= adapter_thresh){
num_pretty_print++;
pretty_print_alignment_stdaln(ppaw,sqp,raaln,false,true,false);
}
}
//do stuff to it
//assume full length adapter and squish it down to the read with no gaps
int rpos,fpos;
rpos = fpos = (- MAX_SEQ_LEN);
if(faaln->score >= adapter_thresh){
fpos = max(faaln->start1 - faaln->start2,0);
}
if(raaln->score >= adapter_thresh){
rpos = max(raaln->start1 - raaln->start2,0);
}
//make rlen the minimum of the two adapter search methods
if(rpos >= 0){
sqp->rlen = min(sqp->rlen,rpos);
}
//make flen the minimum of the two adapter search methods
if(fpos >= 0){
sqp->flen = min(sqp->flen,fpos);
}
if(sqp->flen < min_read_len || sqp->rlen < min_read_len){
num_discarded++;
if(write_discard){
write_fastq(dffqw, sqp->fid, untrim_fseq, untrim_fqual);
write_fastq(drfqw, sqp->rid, untrim_rseq, untrim_rqual);
}
goto CLEAN_ADAPTERS;
}else{ //trim the adapters
sqp->fseq[sqp->flen] = '\0';
sqp->fqual[sqp->flen] = '\0';
sqp->rseq[sqp->rlen] = '\0';
sqp->rqual[sqp->rlen] = '\0';
strncpy(sqp->rc_rseq,sqp->rseq,sqp->rlen+1); //move regular reads now trimmed into RC read's place
strncpy(sqp->rc_rqual,sqp->rqual,sqp->rlen+1);
rev_qual(sqp->rc_rqual, sqp->rlen); //amd re-reverse the RC reads
revcom_seq(sqp->rc_rseq, sqp->rlen);
}
//do a nice global alignment between two reads, and print consensus
fraln = aln_stdaln_aux(sqp->fseq, sqp->rc_rseq, &aln_param_rd2rd,
ALN_TYPE_GLOBAL, 1, sqp->flen, sqp->rlen);
//calculate the minimum score we are willing to accept to merge the reads
//basically this is saying that 7/8 of the read must overlap perfectly
read_thresh = (((int)sqp->flen) + ((int)sqp->rlen)) -
(((int)sqp->flen) * read_frac_thresh * aln_param_rd2rd.gap_ext) -
(((int)sqp->rlen) * read_frac_thresh * aln_param_rd2rd.gap_ext) -
(aln_param_rd2rd.gap_open*2) - (aln_param_rd2rd.gap_end*2);
//now lets put something useful in the alignment suboptimal score thing since right now it
//is just left blank:
//fprintf(stderr, "rt:%d\tfl:%d\trl:%d\trft:%f\tgx:%d\tgo:%d\tge%d\n", read_thresh,((int)sqp->flen),((int)sqp->rlen),read_frac_thresh,aln_param_rd2rd.gap_ext,aln_param_rd2rd.gap_open,aln_param_rd2rd.gap_end);
fraln->subo = read_thresh;
if(do_read_merging && fraln->score > read_thresh){
//if we want read merging,
//and the alignment score is better than the threshold just calculated...
//write the merged sequence
fill_merged_sequence(sqp, fraln, true);
if(pretty_print && num_pretty_print < max_pretty_print){
num_pretty_print++;
pretty_print_alignment_stdaln(ppaw,sqp,fraln,false,false,true);
}
if(strlen(sqp->merged_seq) >= min_read_len && strlen(sqp->merged_qual) >= min_read_len){
num_merged++;
write_fastq(mfqw,sqp->fid,sqp->merged_seq,sqp->merged_qual);
}
else{
num_discarded++;
if(write_discard){
write_fastq(dffqw, sqp->fid, untrim_fseq, untrim_fqual);
write_fastq(drfqw, sqp->rid, untrim_rseq, untrim_rqual);
}
}
}else if(fraln->score > read_thresh){
// we know that the adapters are present, trimmed, and the resulting
// read lengths are both long enough to print.
// We also know that we aren't doing merging.
// Now we just need to print.
if(pretty_print && num_pretty_print < max_pretty_print){
num_pretty_print++;
pretty_print_alignment_stdaln(ppaw,sqp,fraln,false,false,true);
}
//do end polishing to take care of examples like the following:
// Read Alignment Score:59, Suboptimal Score:-85
// ID:HWI-ST593:1:1101:14566:7002#ACA/1
// READ1: ------------ATACAACTCGCTGACTTTGTCCTGGCATTTGACATATGCCTCGTAGTCTGCAAAGACTTTAAACCGGTCATGGTGGAACAGCATGTTGA
// ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
// READ2: CTCTTCCGATCTATACAACTCGCTGACTTTGTCCTGGCATTTGACATATGCCTCGTAGTCTGCAAAGACTTTAAACCGGTCATGGTGGAACAGCATGTTG-
make_blunt_ends(sqp,fraln);
if(strlen(sqp->fseq) >= min_read_len &&
strlen(sqp->fqual) >= min_read_len &&
strlen(sqp->rseq) >= min_read_len &&
strlen(sqp->rqual) >= min_read_len){
write_fastq(ffqw, sqp->fid, sqp->fseq, sqp->fqual);
write_fastq(rfqw, sqp->rid, sqp->rseq, sqp->rqual);
}else{
num_discarded++;
if(write_discard){
write_fastq(dffqw, sqp->fid, untrim_fseq, untrim_fqual);
write_fastq(drfqw, sqp->rid, untrim_rseq, untrim_rqual);
}
}
}else{ //there was a bad looking read-read alignment, so lets not risk it and junk it
num_discarded++;
if(write_discard){
//write_fastq(dffqw, sqp->fid, sqp->fseq, sqp->fqual);
//write_fastq(drfqw, sqp->rid, sqp->rseq, sqp->rqual);
write_fastq(dffqw, sqp->fid, untrim_fseq, untrim_fqual);
write_fastq(drfqw, sqp->rid, untrim_rseq, untrim_rqual);
}
}
}else{
//no adapters present
//check for strong read overlap to assist trimming ends of adapters from end of read
if(do_read_merging){
if(read_merge(sqp, min_ol_reads, min_match_reads, max_mismatch_reads, qcut)){
//print merged output
if(strlen(sqp->merged_seq) >= min_read_len &&
strlen(sqp->merged_qual) >= min_read_len){
num_merged++;
write_fastq(mfqw,sqp->fid,sqp->merged_seq,sqp->merged_qual);
if(pretty_print && num_pretty_print < max_pretty_print){
num_pretty_print++;
pretty_print_alignment(ppaw,sqp,qcut,false); //false b/c merged input in fixed order
}
}else{
num_discarded++;
if(write_discard){
write_fastq(dffqw, sqp->fid, untrim_fseq, untrim_fqual);
write_fastq(drfqw, sqp->rid, untrim_rseq, untrim_rqual);
}
}
}else{
//no significant overlap so just write them
if(strlen(sqp->fseq) >= min_read_len &&
strlen(sqp->fqual) >= min_read_len &&
strlen(sqp->rseq) >= min_read_len &&
strlen(sqp->rqual) >= min_read_len){
write_fastq(ffqw, sqp->fid, sqp->fseq, sqp->fqual);
write_fastq(rfqw, sqp->rid, sqp->rseq, sqp->rqual);
}else{
num_discarded++;
if(write_discard){
write_fastq(dffqw, sqp->fid, untrim_fseq, untrim_fqual);
write_fastq(drfqw, sqp->rid, untrim_rseq, untrim_rqual);
}
}
}
//done
goto CLEAN_ADAPTERS;
}else{ //just write reads to output fastqs
if(strlen(sqp->fseq) >= min_read_len &&
strlen(sqp->fqual) >= min_read_len &&
strlen(sqp->rseq) >= min_read_len &&
strlen(sqp->rqual) >= min_read_len){
write_fastq(ffqw, sqp->fid, sqp->fseq, sqp->fqual);
write_fastq(rfqw, sqp->rid, sqp->rseq, sqp->rqual);
}else{
num_discarded++;
if(write_discard){
write_fastq(dffqw, sqp->fid, untrim_fseq, untrim_fqual);
write_fastq(drfqw, sqp->rid, untrim_rseq, untrim_rqual);
}
}
goto CLEAN_ADAPTERS;
}
}
/**
* Section for heirarchial cleanup
*
* In every case we will at least have to free up the alignment between the adapter and two reads.
* however in some cases there will be an additional alignment between the two reads. We can do
* good cleanup in this case with gotos
*/
aln_free_AlnAln(fraln);
CLEAN_ADAPTERS:
aln_free_AlnAln(faaln);
aln_free_AlnAln(raaln);
//End the loop over reads
}
end = clock();
double cpu_time_used = ((double) (end - start)) / CLOCKS_PER_SEC;
fprintf(stderr,"\nPairs Processed:\t%lld\n",num_pairs);
fprintf(stderr,"Pairs Merged:\t%lld\n",num_merged);
fprintf(stderr,"Pairs With Adapters:\t%lld\n",num_adapter);
fprintf(stderr,"Pairs Discarded:\t%lld\n",num_discarded);
fprintf(stderr,"CPU Time Used (Minutes):\t%lf\n",cpu_time_used/60.0);
SQP_destroy(sqp);
gzclose(ffq);
gzclose(ffqw);
gzclose(rfq);
gzclose(rfqw);
if(mfqw != NULL)
gzclose(mfqw);
if(ppaw != NULL)
gzclose(ppaw);
if(dffqw != NULL)
gzclose(dffqw);
if(drfqw != NULL)
gzclose(drfqw);
return 0;
}
// extract differences between the pair of strings
std::vector<Variant> extract_variants(const std::string& reference,
const std::string& haplotype)
{
AlnParam par = aln_param_nt2nt;
par.band_width = std::max(20, abs(reference.size() - haplotype.size()) * 2);
AlnAln* aln = aln_stdaln(reference.c_str(), haplotype.c_str(), &par, 1, 1);
// Make aligned strings where gaps are padded with '-'
std::string pad_ref(aln->out1);
std::string pad_hap(aln->out2);
assert(pad_ref.size() == pad_hap.size());
//std::cout << "PR: " << pad_ref << "\n";
//std::cout << "PH: " << pad_hap << "\n";
// parse variants from the alignment
std::vector<Variant> variants;
// generate a map from padded bases to positions in the original reference sequence
std::vector<size_t> ref_positions(pad_ref.size(), 0);
size_t pos = 0;
for(size_t i = 0; i < pad_ref.size(); ++i) {
ref_positions[i] = pad_ref[i] != '-' ? pos : std::string::npos;
pos += pad_ref[i] != '-';
}
// diff_start iterates over the places where these sequences are different
size_t diff_start = 0;
while(1) {
// find the start point of the next difference between the strings
while(diff_start < pad_ref.size() && pad_ref[diff_start] == pad_hap[diff_start]) {
diff_start++;
}
// check for end of alignment
if(diff_start == pad_ref.size())
break;
// find the end point of the difference
bool is_indel = false;
size_t diff_end = diff_start;
while(diff_end < pad_ref.size() && pad_ref[diff_end] != pad_hap[diff_end]) {
is_indel = is_indel || pad_ref[diff_end] == '-' || pad_hap[diff_end] == '-';
diff_end++;
}
// If the difference is an indel, we include the previous matching reference base
diff_start -= is_indel;
Variant v;
v.ref_name = "noctg";
assert(ref_positions[diff_start] != std::string::npos);
v.ref_position = ref_positions[diff_start];
v.ref_seq = remove_gaps(pad_ref.substr(diff_start, diff_end - diff_start).c_str());
v.alt_seq = remove_gaps(pad_hap.substr(diff_start, diff_end - diff_start).c_str());
variants.push_back(v);
diff_start = diff_end;
}
aln_free_AlnAln(aln);
return variants;
}
