From 6775c66a97fd06fed1aa9c4f651dd1b6edee708a Mon Sep 17 00:00:00 2001 From: Tim Reddy Tim Date: Mon, 8 Dec 2008 14:17:52 +0000 Subject: [PATCH] Added framework for methylseq analysis. --- .../reference_data/hg18_methylseq_regions | 90612 ++++++++++++++++ htswanalysis/scripts/ConfigureTasks.pm | 37 +- htswanalysis/scripts/SummarizeProject2.pm | 27 + htswanalysis/scripts/Summarize_Methylseq.pm | 66 + htswanalysis/src/Makefile | 7 +- .../src/Methylseq/Methylseq_Analysis.cpp | 285 + 6 files changed, 91031 insertions(+), 3 deletions(-) create mode 100644 htswanalysis/reference_data/hg18_methylseq_regions create mode 100755 htswanalysis/scripts/Summarize_Methylseq.pm create mode 100755 htswanalysis/src/Methylseq/Methylseq_Analysis.cpp diff --git a/htswanalysis/reference_data/hg18_methylseq_regions b/htswanalysis/reference_data/hg18_methylseq_regions new file mode 100644 index 0000000..9310093 --- /dev/null +++ b/htswanalysis/reference_data/hg18_methylseq_regions @@ -0,0 +1,90612 @@ +1 chr1 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-36,6 +36,7 @@ my $WINGPEAKSGENOMEDIR = "$root_dir/reference_data"; my $PROFILEDIR = "$root_dir/bin"; my $GENOMEDIR = "/Volumes/Genomes"; my $QPCRDIR = "$root_dir/bin"; +my $METHYLSEQDIR = "$root_dir/bin"; my $QPCRTESTDIR = "$root_dir/reference_data/qPCR_Tests"; my $QPCRBACKGROUND = "$root_dir/reference_data/GenericBackground"; @@ -56,7 +57,7 @@ my $projects_file = getProjectsXML($parm); my $xmldoc = XML::Simple->new(); my $xmlfile = $projects_file; -my $xml = $xmldoc->XMLin($xmlfile, ForceArray => ['Project','ComparePeakCalls','CompareLibraries','PeakCalling','ProfileReads','qPCR','MotifFinding'], KeepRoot=>1); +my $xml = $xmldoc->XMLin($xmlfile, ForceArray => ['Project','MethylSeq', 'ComparePeakCalls','CompareLibraries','PeakCalling','ProfileReads','qPCR','MotifFinding'], KeepRoot=>1); my $projects = ""; @@ -109,6 +110,7 @@ sub writeProject { my $projectdir = $projectid; my $tasks = ""; + $tasks .= WriteMethylseqTasks($project); $tasks .= WriteQPCRTasks($project); $tasks .= WriteProfileTasks($project); $tasks .= WriteCompareLibTasks($project); @@ -164,6 +166,39 @@ sub writeTask { registerTask($taskid); } +sub WriteMethylseqTasks { + my $project = shift; + my $root = "Methylseq"; + my $tasks = ""; + if(exists($project->{$root})) { + for my $mseq (@{$project->{$root}}) { + my $task = $mseq->{TaskId}; + my $genome = $mseq->{Genome}; + my $name = $mseq->{Name}; $name =~ tr/ \(\)\./____/; + + my $msp1 = $mseq->{Msp1}->{Library}; + my $hpa2 = $mseq->{Hpa2}->{Library}; + + my $outfile = "methylseq_summary"; + + my $seqcheck = "if [ ! -e $data_dir/Libraries/$msp1.txt ]; then $root_dir/scripts/analys_track_main.py updsts $task \"Waiting for sequencing.\"; fi;"; + $seqcheck .= "if [ ! -e $data_dir/Libraries/$hpa2.txt ]; then $root_dir/scripts/analys_track_main.py updsts $task \"Waiting for sequencing.\"; fi;"; + $seqcheck .= "if [ ! -e $root_dir/reference_data/".$genome."_methylseq_regions ]; then $root_dir/scripts/analys_track_main.py updsts $task \"No methylseq regions available for $genome.\"; fi;"; + + my $cmd = "$outfile: $data_dir/Libraries/$msp1.txt $data_dir/Libraries/$hpa2.txt\n"; + $cmd .= "\t$METHYLSEQDIR/Methylseq_Analysis $data_dir/Libraries/$msp1.txt $root_dir/reference_data/".$genome."_methylseq_regions $data_dir/Libraries/$hpa2.txt > \$@\n"; + writeTask($mseq, $root, $outfile, $cmd, $seqcheck); + + $tasks .= $task." "; + } + } + return $tasks; +} + +sub WriteProfileTasks { + +} + sub WriteQPCRTasks { my $project = shift; my $tasks = ""; diff --git a/htswanalysis/scripts/SummarizeProject2.pm b/htswanalysis/scripts/SummarizeProject2.pm index b509ac3..25ffa02 100755 --- a/htswanalysis/scripts/SummarizeProject2.pm +++ b/htswanalysis/scripts/SummarizeProject2.pm @@ -74,6 +74,32 @@ for my $i (0..scalar(@{$xml->{qPCR}})-1) { $qPCR_Summary .= "\n"; } +my $methylseq_summary = ""; +if(exists($xml->{Methylseq})) { + $methylseq_sumary "\n"; + $methylseq_summary .= "\n"; + $methylseq_summary .= "\n"; + $methylseq_summary .= "\n"; + $methylseq_summary .= "\n"; + $methylseq_summary .= "\n"; + $methylseq_summary .= "\n"; + $methylseq_summary .= "\n"; + + for my $i (0..scalar(@{$xml->{Methylseq}})-1) { + my $task = $xml->{Methylseq}->[$i]->{TaskId}; + my $genome = $xml->{Methylseq}->[$i]->{Genome}; + my $name = $xml->{Methylseq}->[$i]->{Name}; + + my $msp1 = $xml->{Methylseq}->[$i]->{Msp1}->{Library}; + my $hpa2 = $xml->{Methylseq}->[$i]->{Hpa2}->{Library}; + + $methylseq_summary .= `$root_dir/scripts/SummarizeMethylseq.pm $name $task $msp1 $hpa2 $genome`; + + } + $methylseq_summary .= "
msp1hpa2AssayedMethylatedErrorsData
\n"; +} + + my $profile_summary = ""; if(exists($xml->{ProfileReads})) { $profile_summary = "

Read Profiles (Genome Browser)

\n"; @@ -180,6 +206,7 @@ print "$project_name Analysis Summary\n" print "

$project_name Analysis Summary

\n"; print SummarizeLibrary($library_info); print $profile_summary; +print $methylseq_summary; print $library_comparisons; print $peak_call_comparisons; print $motif_finding; diff --git a/htswanalysis/scripts/Summarize_Methylseq.pm b/htswanalysis/scripts/Summarize_Methylseq.pm new file mode 100755 index 0000000..044f6c9 --- /dev/null +++ b/htswanalysis/scripts/Summarize_Methylseq.pm @@ -0,0 +1,66 @@ +#!/usr/bin/perl -w + +use strict; +use warnings; + +my $name = shift; +if(!defined($name)) { print STDERR "Error: No name provided to summarize methylseq\n"; exit(1); } +my $task = shift; +if(!defined($task)) { print STDERR "Error: No task provided to summarize methylseq\n"; exit(1); } +my $msp1_lib = shift; +if(!defined($msp1_lib)) { print STDERR "Error: No msp1 library provided to summarize methylseq\n"; exit(1); } +my $hpa2_lib = shift; +if(!defined($hpa2_lib)) { print STDERR "Error: No hpa2 library provided to summarize methylseq\n"; exit(1); } +my $genome = shift; + +my $assayed_bed = "Assayed.bed"; +my $methylated_bed = "Methylated.bed"; +my $methylseq_calls = "MethylseqCalls.tab"; +my $summary = "methylseq_summary"; +if(!defined($summary)) { return; } + +open(FILE,$summary); + +my $tag_count = ; chomp $tag_count; +$tag_count =~ /^(\d+) MSP1 tags, (\d+) HPA2 tags$/; +my($msp_count,$hpa_count) = ($1,$2); + +my $assayed_count = ; chomp $assayed_count; + $assayed_count =~ /^(\d+) of (\d+) regions assayed/; + my($n_assayed,$n_regions) = ($1,$2); + +my $methylated_count = ; chomp $methylated_count; + $methylated_count =~ /^(\d+) of \d+ regions methylated/; + my $n_methylated = $1; + +my $error_count = ; chomp $error_count; + $error_count =~ /^(\d+) of \d+ regions with error/; + my $n_error = $1; + +my $pcnt_assayed = $n_assayed / $n_regions; +my $pcnt_methylated = $n_methylated / $n_assayed; +my $pcnt_error = $n_error / $n_regions; + +my $assayed_color; +if($pcnt_assayed < 0.7) { $assayed_color = "FF6600"; } +elsif($pcnt_assayed < 0.9) { $assayed_color = "4444FF"; } +else {$assayed_color= "66FF66"; } + +my $methylated_color; +if($pcnt_methylated < 0.3) { $methylated_color = "FF6600"; } +elsif($pcnt_methylated < 0.6) { $methylated_color = "4444FF"; } +else {$methylated_color= "66FF66"; } + +my $error_color; +if($pcnt_error > 0.05) { $error_color = "FF6600"; } +elsif($pcnt_error > 0.01) { $error_color = "4444FF"; } +else {$error_color= "66FF66"; } + +print ""; +printf "$msp1_lib$hpa2_lib$n_assayed (%.4f)$n_methylated (%0.4f)$n_error (%0.4f)",$pcnt_assayed,$pcnt_methylated,$pcnt_error; +print "Calls
\n"; +print "Assayed Regions"; +print "(Genome Browser)
\n"; +print "Methylated Regions "; +print "(Genome Browser)\n"; +print ""; diff --git a/htswanalysis/src/Makefile b/htswanalysis/src/Makefile index 02ce979..4b22aa9 100644 --- a/htswanalysis/src/Makefile +++ b/htswanalysis/src/Makefile @@ -1,12 +1,15 @@ CPPFLAGS=-g -Wall -O3 -I/opt/local/include LDFLAGS=-lgsl -lgslcblas -lm -L/opt/local/lib -TARGETS=complexity_count qPCR profile_reads_against_features profile_reads_wig count_reads_in_peaks ValidationDesign/refine_peaks +TARGETS=complexity_count qPCR profile_reads_against_features profile_reads_wig count_reads_in_peaks ValidationDesign/refine_peaks Methylseq/Methylseq_Analysis all: $(TARGETS) ValidationDesign/refine_peaks: ValidationDesign/refine_peaks.cpp - cd ValidationDesign && make + cd ValidationDesign && $(MAKE) + +Methhylseq/Methylseq_Analysis: Methylseq/Methylseq_Analysis.cpp + cd Methylseq && $(MAKE) install: $(TARGETS) cp $^ ../bin/ diff --git a/htswanalysis/src/Methylseq/Methylseq_Analysis.cpp b/htswanalysis/src/Methylseq/Methylseq_Analysis.cpp new file mode 100755 index 0000000..14a1248 --- /dev/null +++ b/htswanalysis/src/Methylseq/Methylseq_Analysis.cpp @@ -0,0 +1,285 @@ +#include +#include +#include +#include +#include +#include +#include +#include +#include +#include + +#include + +#define WINDOW 25 + +using namespace std; + +void split (const string& text, const string& separators, vector& words); + +class Read { + public: + string chr; + int pos; + bool strand; + + Read(string chr, int pos, bool strand) { this->chr = chr; this->pos = pos; this->strand = strand; } + Read(const Read& r) { this->chr = r.chr; this->pos = r.pos; this->strand = r.strand; } + Read& operator=(const Read& r) {this->chr = r.chr; this->pos = r.pos; this->strand = r.strand; return *this;} +}; + +typedef enum ReadType {MSP1,HPA2}; + +class Region { + public: + + int start; + int end; + bool strand; + + string chr; + string name; + + unsigned int msp1_count; + unsigned int hpa2_count; + + + Region(string name, string chr, int start, int end, bool strand) { + this->name = name; + this->chr = chr; + this->start = start; + this->end = end; + this->strand = strand; + this->msp1_count = 0; + this->hpa2_count = 0; + } + + Region(const Region& h) { + this->name = h.name; + this->chr = h.chr; + this->start = h.start; + this->end = h.end; + this->strand = h.strand; + this->msp1_count = h.msp1_count; + this->hpa2_count = h.hpa2_count; + } + + Region& operator=(const Region& h) { + this->name = h.name; + this->chr = h.chr; + this->start = h.start; + this->end = h.end; + this->strand = h.strand; + this->msp1_count = h.msp1_count; + this->hpa2_count = h.hpa2_count; + return *this; + } +}; + +bool Region_lt_Read(const Region& feat, const Read& read) { + //First compare based on chromosomes + if(read.chr == feat.chr) { return feat.end < read.pos; } + else { return feat.chr < read.chr; } +} + +ostream &operator<<( ostream &out, const Region &h ) { + out << h.name.c_str() << "\tchr" << h.chr.c_str() << "\t" << h.start << "\t" << h.end << "\t" << h.strand << "\t" << h.msp1_count << "\t" << h.hpa2_count; + return out; +} + +typedef vector Reads; +typedef vector Regions; + +bool compare_regions(const Region &a, const Region &b) { if(a.chr == b.chr) { return a.end < b.start; } else { return a.chr < b.chr; } } +bool compare_reads(const Read &a, const Read &b) { if(a.chr == b.chr) { return a.pos < b.pos; } else { return a.chr < b.chr; } } + +void read_features(const char* filename, Regions& features) { + string delim("\t"); + + ifstream feat(filename); + size_t N = 0; + while(feat.peek() != EOF) { + char line[1024]; + feat.getline(line,1024,'\n'); + N++; + string line_str(line); + vector fields; + split(line_str, delim, fields); + if(fields.size() < 5) { cerr << "Error (" << filename << "): wrong number of fields in feature list (line " << N << " has " << fields.size() << " fields)\n"; } + + string name = fields[0]; + string chr = fields[1]; + if(chr == "NA") { continue; } + int start = atoi(fields[2].c_str()); + int stop = atoi(fields[3].c_str()); + bool strand = 0; + + Region feat(name,chr,start,stop,strand); + features.push_back(feat); + } + + //sort the features so we can run through it once + std::stable_sort(features.begin(),features.end(),compare_regions); + feat.close(); +} + +void read_align_file(char* filename, Reads& features) { + string delim(" \n"); + string location_delim(":"); + char strand_str[2]; strand_str[1] = '\0'; + ifstream seqs(filename); + string name(""); + while(seqs.peek() != EOF) { + char line[2048]; + seqs.getline(line,2048,'\n'); + + string line_str(line); + vector fields; + split(line_str, delim, fields); + if(fields.size() != 7) { continue; } + + vector location; split(fields[3], location_delim, location); + string chr = location[0]; + if(chr == "NA") { continue; } + int pos = atoi(location[1].c_str()); + bool strand = ((fields[4].c_str())[0] == 'F')?0:1; + + if(strand == 0) { + Read read(chr,pos,false); + features.push_back(read); + } else { + Read read(chr,pos+25,true); + features.push_back(read); + } + } + seqs.close(); + + //sort the data so we can run through it once + std::sort(features.begin(),features.end(),compare_reads); + cerr << "Reads sorted\n"; +} + +unsigned int count_regions_assayed(Regions& r, unsigned int threshold = 1) { + unsigned int n_assayed = 0; + for(Regions::iterator i = r.begin(); i != r.end(); ++i) { + if(i->msp1_count >= threshold) { n_assayed++; } + } + return n_assayed; +} + +unsigned int count_regions_methylated(Regions& r, unsigned int msp_threshold = 1, unsigned int hpa_threshold = 1) { + unsigned int n_methylated = 0; + for(Regions::iterator i = r.begin(); i != r.end(); ++i) { + if(i->msp1_count >= msp_threshold && i->hpa2_count >= hpa_threshold) { n_methylated++; } + } + return n_methylated; +} + +unsigned int count_errors(Regions& r, unsigned int msp_threshold = 1, unsigned int hpa_threshold = 1) { + unsigned int n_error = 0; + for(Regions::iterator i = r.begin(); i != r.end(); ++i) { + if(i->msp1_count < msp_threshold && i->hpa2_count >= hpa_threshold) { n_error++; } + } + return n_error; +} + +pair count_region_tags(Regions& r) { + pair counts; counts.first = 0; counts.second = 0; + for(Regions::iterator i = r.begin(); i != r.end(); ++i) { + counts.first += i->msp1_count; + counts.second += i->hpa2_count; + } + return counts; +} + +void count_read_in_features(Regions& features, Reads& data, ReadType type) { + Regions::iterator feat_it = features.begin(); + + for(Reads::iterator i = data.begin(); i != data.end(); ++i) { + //skip to first feature after read + string start_chr = feat_it->chr; + while(feat_it != features.end() && Region_lt_Read(*feat_it, *i)) { + feat_it++; + } + + //stop if we have run out of features. + if(feat_it == features.end()) { break; } + + if(i->pos >= feat_it->start && i->pos <= feat_it->end) { + if(type == MSP1) { feat_it->msp1_count++; } + if(type == HPA2) { feat_it->hpa2_count++; } + } + } +} + +int compare_double(const void* a, const void* b) { + return *(double*)a - *(double*)b; +} + +pair count_region_tags(Regions& r); +unsigned int count_regions_assayed(Regions& r, unsigned int threshold); +unsigned int count_regions_methylated(Regions& r, unsigned int msp_threshold, unsigned int hpa_threshold); +unsigned int count_errors(Regions& r, unsigned int msp_threshold, unsigned int hpa_threshold); + +int main(int argc, char** argv) { + if(argc < 4) { cerr << "Usage: " << argv[0] << " msp1_reads hpa2_reads regions\n"; exit(1); } + + char msp1_filename[1024]; strcpy(msp1_filename,argv[1]); + char hpa2_filename[1024]; strcpy(hpa2_filename,argv[2]); + char region_filename[1024]; strcpy(region_filename,argv[3]); + + Reads msp1; read_align_file(msp1_filename, msp1); + Reads hpa2; read_align_file(hpa2_filename, hpa2); + + Regions regions; read_features(region_filename, regions); + + count_read_in_features(regions, msp1, MSP1); + count_read_in_features(regions, hpa2, HPA2); + + pair total = count_region_tags(regions); + cout << total.first << " MSP1 tags, " << total.second << " HPA2 tags" << endl; + + unsigned int n_assayed = count_regions_assayed(regions); + cout << n_assayed << " of " << regions.size() << " regions assayed (" << (double)n_assayed/(double)regions.size() << ")\n"; + + unsigned int n_methylated = count_regions_methylated(regions); + cout << n_methylated << " of " << n_assayed << " regions methylated (" << (double)n_methylated/(double)n_assayed << ")\n"; + + unsigned int n_error = count_errors(regions); + cout << n_error << " of " << regions.size() << " regions with error (" << (double)n_error/(double)regions.size() << ")\n"; + + + ofstream assayed("Assayed.bed"); + ofstream methylated("Methylated.bed"); + ofstream methylcalls("MethylseqCalls.tab"); + + unsigned int msp_threshold = 1; + unsigned int hpa_threshold = 1; + + for(unsigned int idx = 0; idx < regions.size(); idx++) { + bool is_assayed = 0; + bool is_methylated = 0; + if(regions[idx].msp1_count >= msp_threshold) { + assayed << regions[idx].chr << "\t" << regions[idx].start << "\t" << regions[idx].end << endl; + is_assayed = 1; + if(regions[idx].hpa2_count >= hpa_threshold) { + methylated << regions[idx].chr << "\t" << regions[idx].start << "\t" << regions[idx].end << endl; + is_methylated = 1; + } + } + methylcalls << regions[idx].name << "\t" << regions[idx].chr << "\t" << regions[idx].start << "\t" << regions[idx].end << "\t" << regions[idx].msp1_count << "\t" << regions[idx].hpa2_count << "\t" << is_assayed << "\t" << is_methylated << endl; + } +} + +void split (const string& text, const string& separators, vector& words) { + + size_t n = text.length (); + size_t start = text.find_first_not_of (separators); + + while (start < n) { + size_t stop = text.find_first_of (separators, start); + if (stop > n) stop = n; + words.push_back (text.substr (start, stop-start)); + start = text.find_first_not_of (separators, stop+1); + } +} -- 2.30.2