module ViralSeq # Core functions for `tcs` pipeline class TcsCore class << self # methods to calculate TCS consensus cut-off based on the maximum numbers of PIDs and platform error rate. def calculate_cut_off(m, error_rate = 0.02) n = 0 case error_rate when 0.005...0.015 if m <= 10 n = 2 else n = 1.09*10**-26*m**6 + 7.82*10**-22*m**5 - 1.93*10**-16*m**4 + 1.01*10**-11*m**3 - 2.31*10**-7*m**2 + 0.00645*m + 2.872 end when 0...0.005 if m <= 10 n = 2 else n = -9.59*10**-27*m**6 + 3.27*10**-21*m**5 - 3.05*10**-16*m**4 + 1.2*10**-11*m**3 - 2.19*10**-7*m**2 + 0.004044*m + 2.273 end else if m <= 10 n = 2 elsif m <= 8500 n = -1.24*10**-21*m**6 + 3.53*10**-17*m**5 - 3.90*10**-13*m**4 + 2.12*10**-9*m**3 - 6.06*10**-6*m**2 + 1.80*10**-2*m + 3.15 else n = 0.0079 * m + 9.4869 end end n = n.round n = 2 if n < 3 return n end # identify which file in the directory is R1 file, and which is R2 file based on file names # input as directory (Dir object or a string of path) # by default, .gz files will be unzipped. # return as an hash of {r1_file: file1, r1_file: file2} def r1r2(directory, unzip = true) files = [] Dir.chdir(directory) { files = Dir.glob "*" } r1_file = "" r2_file = "" files.each do |f| tag = parser_file_name(f)[:tag] if tag.include? "R1" unzip ? r1_file = unzip_r(directory, f) : r1_file = File.join(directory, f) elsif tag.include? "R2" unzip ? r2_file = unzip_r(directory, f) : r2_file = File.join(directory, f) end end return { r1_file: r1_file, r2_file: r2_file } end # end of ViralSeq:TcsCore.r1r2 # sort directories containing mulitple r1 and r2 files. # use the library name (first string before "_") to seperate libraries # out_dir is the Dir object or string of the output directory, by default named as directory + "_sorted" # return a hash as { with_both_r1_r2: [lib1, lib2, ...], missing_r1: [lib1, lib2, ...], missing_r2: [lib1, lib2, ...], error: [lib1, lib2, ...]} def sort_by_lib(directory, out_dir = directory + "_sorted") Dir.mkdir(out_dir) unless File.directory?(out_dir) files = [] Dir.chdir(directory) {files = Dir.glob("*")} files.each do |file| path = File.join(directory,file) index = file.split("_")[0] index_dir = File.join(out_dir, index) Dir.mkdir(index_dir) unless File.directory?(index_dir) File.rename(path, File.join(index_dir, file)) end return_obj = { with_both_r1_r2: [], missing_r1: [], missing_r2: [], error: [] } libs = [] Dir.chdir(out_dir) { libs = Dir.glob('*') } libs.each do |lib| file_check = ViralSeq::TcsCore.r1r2(File.join(out_dir, lib)) if !file_check[:r1_file].empty? and !file_check[:r2_file].empty? return_obj[:with_both_r1_r2] << lib elsif file_check[:r1_file].empty? and !file_check[:r2_file].empty? return_obj[:missing_r1] << lib elsif file_check[:r2_file].empty? and !file_check[:r1_file].empty? return_obj[:missing_r2] << lib else return_obj[:error] << lib end end return return_obj end # sort array of file names to determine if there is potential errors # @param name_array [Array] array of file names # @return [hash] name check results def validate_file_name(name_array) errors = { file_type_error: [] , missing_r1_file: [] , missing_r2_file: [] , extra_r1_r2_file: [], no_region_tag: [] , multiple_region_tag: [] } passed_libs = {} name_with_r1_r2 = [] name_array.each do |name| tag = parser_file_name(name)[:tag] if name !~ /\.fastq\Z|\.fastq\.gz\Z/ errors[:file_type_error] << name elsif tag.count("R1") == 0 and tag.count("R2") == 0 errors[:no_region_tag] << name elsif tag.count("R1") > 0 and tag.count("R2") > 0 errors[:multiple_region_tag] << name elsif tag.count("R1") > 1 or tag.count("R2") > 1 errors[:multiple_region_tag] << name else name_with_r1_r2 << name end end libs = {} name_with_r1_r2.map do |name| libname = parser_file_name(name)[:libname] libs[libname] ||= [] libs[libname] << name end libs.each do |libname, files| count_r1_file = 0 count_r2_file = 0 files.each do |name| tag = parser_file_name(name)[:tag] if tag.include? "R1" count_r1_file += 1 elsif tag.include? "R2" count_r2_file += 1 end end if count_r1_file > 1 or count_r2_file > 1 errors[:extra_r1_r2_file] += files elsif count_r1_file.zero? errors[:missing_r1_file] += files elsif count_r2_file.zero? errors[:missing_r2_file] += files else passed_libs[libname] = files end end file_name_with_lib_name = {} passed_libs.each do |lib_name, files| files.each do |f| file_name_with_lib_name[f] = lib_name end end passed_names = [] passed_libs.values.each { |names| passed_names += names} if passed_names.size < name_array.size pass = false else pass = true end file_name_with_error_type = {} errors.each do |type, files| files.each do |f| file_name_with_error_type[f] ||= [] file_name_with_error_type[f] << type.to_s.tr("_", "\s") end end file_check = [] name_array.each do |name| file_check_hash = {} file_check_hash[:fileName] = name file_check_hash[:errors] = file_name_with_error_type[name] file_check_hash[:libName] = file_name_with_lib_name[name] file_check << file_check_hash end return { allPass: pass, files: file_check } end # filter r1 raw sequences for non-specific primers. # input r1_sh, SeqHash obj. # return filtered Hash of sequence name and seq pair, in the object { r1_filtered_seq: r1_filtered_seq_pair } def filter_r1(r1_sh, forward_primer) if forward_primer.match(/(N+)(\w+)$/) forward_n = $1.size forward_bio_primer = $2 else forward_n = 0 forward_bio_primer = forward_primer end forward_bio_primer_size = forward_bio_primer.size forward_starting_number = forward_n + forward_bio_primer_size forward_primer_ref = forward_bio_primer.nt_parser r1_passed_seq = {} r1_raw = r1_sh.dna_hash proc_filter = proc do |name| seq = r1_raw[name] next unless general_filter seq primer_region_seq = seq[forward_n, forward_bio_primer_size] if primer_region_seq =~ forward_primer_ref new_name = remove_tag name r1_passed_seq[new_name] = seq end end r1_raw.keys.map do |name| proc_filter.call name end return { r1_passed_seq: r1_passed_seq, forward_starting_number: forward_starting_number } end # end of filter_r1 # filter r2 raw sequences for non-specific primers. # input r2_sh, SeqHash obj. # return filtered Hash of sequence name and seq pair, as well as the length of PID. def filter_r2(r2_sh, cdna_primer) r2_raw = r2_sh.dna_hash cdna_primer.match(/(N+)(\w+)$/) pid_length = $1.size cdna_bio_primer = $2 cdna_bio_primer_size = cdna_bio_primer.size reverse_starting_number = pid_length + cdna_bio_primer_size cdna_primer_ref = cdna_bio_primer.nt_parser r2_passed_seq = {} proc_filter = proc do |name| seq = r2_raw[name] next unless general_filter seq primer_region_seq = seq[pid_length, cdna_bio_primer_size] if primer_region_seq =~ cdna_primer_ref new_name = remove_tag name r2_passed_seq[new_name] = seq end end r2_raw.keys.map do |name| proc_filter.call name end return { r2_passed_seq: r2_passed_seq, pid_length: pid_length, reverse_starting_number: reverse_starting_number } end # end of filter_r2 # puts error message in the log file handler, and abort with the same infor def log_and_abort(log, infor) log.puts Time.now.to_s + "\t" + infor log.close abort infor.red.bold end # lower detection sensitivity for minority mutations given the number of TCS, calculated based on binomial distribution. # R required. # @param tcs_number [Integer] number of TCS # @return [Float] lower detection limit # @example calculate lower detection limit # ViralSeq::TcsCore.detection_limit(100) # => 0.0362 def detection_limit(tcs_number) dl = `Rscript -e "library(dplyr); ifelse(#{tcs_number} > 2, (binom.test(0,#{tcs_number})['conf.int'] %>% unlist %>% unname)[2] %>% round(4) %>% cat, 0)" 2>/dev/null` dl.to_f end private def unzip_r(indir, f) r_file = File.join(indir, f) if f =~ /.gz/ `gzip -d #{r_file}` new_f = f.sub ".gz", "" r_file = File.join(indir, new_f) end return r_file end def parser_file_name(file_name) t = file_name.split(".")[0].split("_") if t.size == 1 libname = "lib" tag = [ t[0].upcase ] else libname = t[0] tag = t[1..-1].map(&:upcase) end return {libname: libname, tag: tag} end def general_filter(seq) return false unless seq if seq.size < ($platform_sequencing_length - 10) return false elsif seq[1..-2] =~ /N/ # sequences with ambiguities except the 1st and last position removed return false elsif seq =~ /A{11}/ # a string of poly-A indicates adaptor sequence return false elsif seq =~ /T{11}/ # a string of poly-T indicates adaptor sequence return false else return true end end # remove region info tags from the raw MiSeq sequences. def remove_tag(seq_name) if seq_name =~ /\s/ new_tag = $` else new_tag = seq_name[0..-3] end end end # end of class << self end # end of TcsCore module end # end of main module