Class: Bio::DB::Sam

Inherits:
Object
  • Object
show all
Defined in:
lib/bio/db/sam.rb

Instance Attribute Summary collapse

Class Method Summary collapse

Instance Method Summary collapse

Constructor Details

#initialize(optsa = {}) ⇒ Sam

To make a new sam object. Initialize expects a hash optsa with the following elemets:

fasta

The fasta file with the reference. (nil)

bam

path to a binary SAM file (nil)

tam

path to a text SAM file (nil)

compressed

If the binary file is compressed (true)

write

If the file is to be writen (false). Not supported yet.

NOTE: you can’t use binary and text formats simultaneusly. To make queries, the file has to be a sorted binary. This function doesn’t actually open the file, it just prepares the object to be opened in a later stage.



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# File 'lib/bio/db/sam.rb', line 29

def initialize(optsa={})
  opts =  { :fasta => nil,  :bam => nil,:tam => nil, :compressed => true, :write => false }.merge!(optsa)



  @fasta_path = opts[:fasta]
  @compressed = opts[:compressed]
  @write = opts[:write]
  bam = opts[:bam]
  tam = opts[:tam]

  if bam == nil && tam == nil && @fasta_path == nil then
    raise SAMException.new(), "No alignment or reference file"
  elsif bam != nil && tam != nil then
    raise SAMException.new(), "Alignment has to be in either text or binary format, not both"
  elsif bam != nil then
    @binary = true
    @sam = bam     
  elsif tam != nil then
    @sam = tam     
    @binary = false

  end
  @fasta_file = nil
  @sam_file   = nil

  ObjectSpace.define_finalizer(self,  self.class.method(:finalize).to_proc)
end

Instance Attribute Details

#sam_file ⇒ Object (readonly)

Returns the value of attribute sam_file.



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# File 'lib/bio/db/sam.rb', line 18

def sam_file
  @sam_file
end

Class Method Details

.finalize(id) ⇒ Object

Destructor method that closes the file before letting the object be garbage collected.



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# File 'lib/bio/db/sam.rb', line 104

def Sam.finalize(id)
  id.close()
  puts "Finalizing #{id}  at #{Time.new}"       
end

.merge(files, merged_file, headers, add_RG, by_qname) ⇒ Object

Merges n BAM files. This doesn’t require to create a SAM object

files

An array with the paths to the files.

merged_file

The path to the merged file

headers

The BAM file containing the header

add_RG

If true, the RG tag is added (infered from the filenames)

by_qname

If true, the bamfiles should by ordered by query name, if false, by coordinates.



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# File 'lib/bio/db/sam.rb', line 259

def self.merge(files, merged_file, headers, add_RG, by_qname)
  strptrs = []
  strptrs << FFI::MemoryPointer.from_string("merge")
  files.each do |file|
    strptrs << FFI::MemoryPointer.from_string(file)
  end
  strptrs << nil

  # Now load all the pointers into a native memory block
  argv = FFI::MemoryPointer.new(:pointer, strptrs.length)
  strptrs.each_with_index do |p, i|
     argv[i].put_pointer(0,  p)
  end
  #void bam_merge_core(int by_qname, const char *out, const char *headers, int n, char * const *fn, int add_RG)
  Bio::DB::SAM::Tools.bam_merge_core(by_qname, merged_file, headers, strptrs.length, argv, add_RG)
end

Instance Method Details

#average_coverage(chromosome, qstart, len) ⇒ Object

Returns the average coverage of a region in a bam file.



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# File 'lib/bio/db/sam.rb', line 143

def average_coverage(chromosome, qstart, len)

  #reference = fetch_reference(chromosome, qstart,len)
  # len = reference.length if len > reference.length


  coverages = chromosome_coverage(chromosome, qstart, len)
  total = 0
  len.times{ |i| total= total + coverages[i] }
  avg_cov = total.to_f / len
  #LibC.free reference
  avg_cov
end

#chromosome_coverage(chromosome, qstart, len) ⇒ Object

Returns an array with the coverage at each possition in the queried region This is a simple average coverage just calculated with the first and last possition of the alignment, ignoring the gaps.



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# File 'lib/bio/db/sam.rb', line 160

def chromosome_coverage(chromosome, qstart, len)
  #  reference = fetch_reference(chromosome, qstart,len)
  #  len = reference.length if len > reference.length
  #p qend.to_s + "-" + qstart.to_s + "framesize " + (qend - qstart).to_s
  coverages = Array.new(len, 0)

  chr_cov_proc = Proc.new do |alignment|
    #last = qstart + len
    #first = qstart
    #last = alignment.calend if last > alignment.calend
    #first = alignment.pos if first < alignment.pos
    # p first
    last = alignment.calend - qstart
    first = alignment.pos - qstart
    if last < first
      tmp = last
      last = first
      first = last
    end

    #  STDERR.puts "#{first} #{last}\n"
    first.upto(last-1) { |i|

      coverages[i-1] = 1 + coverages[i-1]  if i-1 < len && i > 0
    }
  end

  fetch_with_function(chromosome, qstart, qstart+len,  chr_cov_proc)
  #p coverages
  coverages
end

#close ⇒ Object

Closes the sam file and destroys the C pointers using the functions provided by libbam



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# File 'lib/bio/db/sam.rb', line 95

def close()
  Bio::DB::SAM::Tools.fai_destroy(@fasta_index) unless @fasta_index.nil? || @fasta_index.null?
  Bio::DB::SAM::Tools.bam_index_destroy(@sam_index) unless @sam_index.nil? || @sam_index.null?
  Bio::DB::SAM::Tools.samclose(@sam_file) unless @sam_file.nil? 
  @sam_file = nil
  @fasta_index = nil
end

#deprecated_pileup(cmd) ⇒ Object

utility method that does not use the samtools API, it calls samtools directly as if on the command line and catches the output, to use this method you must have a version of samtools that supports the pileup command (< 0.1.17) otherwise the command will fail. mpileup is the preferred method for getting pileups. With this method the sam object should be created as usual, but you need to pass this method a string of options for samtools you don’t need to provide the call to samtools pileup itself or -f <fasta file> or the bam file itself, these are taken from the sam object

Raises:



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# File 'lib/bio/db/sam.rb', line 473

def deprecated_pileup( cmd )
  
  system('samtools pileup > /dev/null 2>&1')
  ##assumes samtools is in the path...
  if $?.exitstatus > 1
    raise RuntimeError, "samtools is required on the path. A version of samtools with the pileup function is required"
  end
  
  raise SAMException.new(), "No BAMFile provided" unless @sam and @binary
  raise SAMException.new(), "No FastA provided" unless @fasta_path
  
  command = 'samtools pileup ' + cmd + " -f #{@fasta_path}" + " #{@sam}" 
  
  pipe = IO.popen(command)
  while line = pipe.gets
    yield Pileup.new(line)
  end
  pipe.close
end

#each_reference ⇒ Object

yields each reference name and its length



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# File 'lib/bio/db/sam.rb', line 535

def each_reference
  refs = index_stats
  refs.each_pair do |k, v|
    yield k, v[:length]
  end
end

#fetch(chromosome, qstart, qend) ⇒ Object

Returns an array of Alignments on a given region.



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# File 'lib/bio/db/sam.rb', line 211

def fetch(chromosome, qstart, qend)
  als = Array.new
  fetchAlignment = Proc.new do |alignment|
    als.push(alignment.clone)   
    0  
  end
  fetch_with_function(chromosome, qstart, qend, fetchAlignment)
  als
end

#fetch_reference(chromosome, qstart, qend) ⇒ Object

Returns the sequence for a given region.

Raises:



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# File 'lib/bio/db/sam.rb', line 193

def fetch_reference(chromosome, qstart,qend)
  load_reference if @fasta_index.nil? || @fasta_index.null? 
  query = query_string(chromosome, qstart,qend)
  len = FFI::MemoryPointer.new :int
  reference = Bio::DB::SAM::Tools.fai_fetch(@fasta_index, query, len)
  raise SAMException.new(), "Unable to get sequence for reference: "+query if reference.nil?

  reference
end

#fetch_with_function(chromosome, qstart, qend, function) ⇒ Object

Executes a function on each Alignment inside the queried region of the chromosome. The chromosome can be either the textual name or a FixNum with the internal index. However, you need to get the internal index with the provided API, otherwise the pointer is outside the scope of the C library. Returns the count of alignments in the region. WARNING: Accepts an index already parsed by the library. It fails when you use your own FixNum (FFI-bug?)



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# File 'lib/bio/db/sam.rb', line 226

def fetch_with_function(chromosome, qstart, qend, function)
  load_index if @sam_index.nil? || @sam_index.null?
  chr = FFI::MemoryPointer.new :int
  beg = FFI::MemoryPointer.new :int
  last = FFI::MemoryPointer.new :int
  query = query_string(chromosome, qstart,qend)
  qpointer = FFI::MemoryPointer.from_string(query)
  header = @sam_file[:header]
  Bio::DB::SAM::Tools.bam_parse_region(header,qpointer, chr, beg, last) 
  #raise SAMException.new(), "invalid query: " + query  if(chr.read_int < 0)
  count = 0;

  fetchAlignment = Proc.new do |bam_alignment, data|
    alignment =  Alignment.new
    alignment.set(bam_alignment, header)
    function.call(alignment)
    count = count + 1
    0  
  end
  Bio::DB::SAM::Tools.bam_fetch(@sam_file[:x][:bam], @sam_index,chr.read_int,beg.read_int, last.read_int, nil, fetchAlignment)
  #LibC.free chr
  #LibC.free beg
  #LibC.free last
  #LibC.free qpointer
  count
end

#index_stats ⇒ Object

Raises:



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# File 'lib/bio/db/sam.rb', line 494

def index_stats 
  raise SAMException.new(), "No BAMFile provided" unless @sam and @binary
  raise SAMException.new(), "No FastA provided" unless @fasta_path
  strptrs = []
  strptrs << FFI::MemoryPointer.from_string("idxstats")
  strptrs << FFI::MemoryPointer.from_string(@sam)
  strptrs << nil

  # Now load all the pointers into a native memory block
  argv = FFI::MemoryPointer.new(:pointer, strptrs.length)
  strptrs.each_with_index do |p, i|
     argv[i].put_pointer(0,  p)
  end
  
  index_stats = {}
  
  old_stdout = STDOUT.clone
  read_pipe, write_pipe = IO.pipe()
  STDOUT.reopen(write_pipe)
  
  #int bam_idxstats(int argc, char *argv[])
  Bio::DB::SAM::Tools.bam_idxstats(strptrs.length - 1,argv)
  if fork
    write_pipe.close
    STDOUT.reopen(old_stdout) #beware .. stdout from other processes eg tests calling this method can get mixed in...
    begin
      
      while line = read_pipe.readline #TAB delimited with each line consisting of reference sequence name, sequence length, # mapped reads and # unmapped reads.
          info = line.split(/\t/)
          next unless info.length == 4
          index_stats[ info[0] ] = {:length => info[1].to_i, :mapped_reads => info[2].to_i, :unmapped_reads => info[3].to_i } 
      end
      rescue EOFError
        read_pipe.close
        Process.wait
      end
  end #fork
  index_stats
end

#load_index ⇒ Object

Loads the bam index to be used for fetching. If the index doesn’t exists the index is built provided that the user has writing access to the folder where the BAM file is located. If the creation of the file fails a SAMException is thrown. If the index doesn’t exist, loading it will take more time. It is suggested to generate the index separatedly if the bam file sits on a server where the executing user may not have writing permissions in the server.

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# File 'lib/bio/db/sam.rb', line 114

def load_index()
  raise SAMException.new(), "Indexes are only supported by BAM files, please use samtools to convert your SAM file" unless @binary
  @sam_index = Bio::DB::SAM::Tools.bam_index_load(@sam)
  if @sam_index.null? then
    p "Generating index for: " + @sam
    Bio::DB::SAM::Tools.bam_index_build(@sam)
    @sam_index = Bio::DB::SAM::Tools.bam_index_load(@sam)
    raise SAMException.new(), "Unable to generate bam index for: " + @sam if @sam_index.nil? || @sam_index.null?
  end
end

#load_reference ⇒ Object

Loads the reference file to be able to query regions of it. This requires the fai index to exist in the same folder than the reference. If it doesn’t exisits, this functions attempts to generate it. If user doesn’t have writing permissions on the folder, or the creation of the fai fails for any reason, a SAMException is thrown.

Raises:



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# File 'lib/bio/db/sam.rb', line 128

def load_reference()
  raise SAMException.new(), "No path for the refernce fasta file. " if @fasta_path.nil?

  @fasta_index = Bio::DB::SAM::Tools.fai_load(@fasta_path)

  if @fasta_index.null? then
    p "Generating index for: " + @fasta_path
    Bio::DB::SAM::Tools.fai_build(@fasta_path)
    @fasta_index =  Bio::DB::SAM::Tools.fai_load(@fasta_path)
    raise SAMException.new(), "Unable to generate fasta index for: " + @fasta_path if @fasta_index.nil? ||  @fasta_index.null?
  end

end

#mpileup(opts = {}) ⇒ Object

calls the mpileup function, opts is a hash of options identical to the command line options for mpileup. is an iterator that yields a Pileup object for each postion the command line options that generate/affect BCF/VCF are ignored ie (g,u,e,h,I,L,o,p) call the option as a symbol of the flag, eg -r for region is called :r => “some SAM compatible region” eg bam.mpileup(:r => “chr1:1000-2000”, :q => 50) gets the bases with quality > 50 on chr1 between 1000-5000

Raises:



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# File 'lib/bio/db/sam.rb', line 281

def mpileup( opts={})

  raise SAMException.new(), "No BAMFile provided" unless @sam and @binary
  raise SAMException.new(), "No FastA provided" unless @fasta_path
  #long option form to short samtools form..
  long_opts = {
  :region => :r,
  :illumina_quals => :six,
  :count_anomalous => :A,
  :no_baq => :B,
  :adjust_mapq => :C,
  :max_per_bam_depth => :d,
  :extended_baq => :E,
  :exclude_reads_file => :G,
  :list_of_positions => :l,
  :mapping_quality_cap => :M,
  :ignore_rg => :R,
  :min_mapping_quality => :q,
  :min_base_quality => :Q
  }
  ##convert any long_opts to short opts 
  temp_opts = opts.dup
  opts.each_pair do |k,v|
    if long_opts[k]
      temp_opts[long_opts[k]] = v 
      temp_opts.delete(k)
    end
  end
  opts = temp_opts
  ##remove any calls to -g or -u for mpileup, bcf output is not yet supported
  ##and also associated output options
  [:g, :u, :e, :h, :I, :L, :o, :p].each {|x| opts.delete(x) }

  sam_opts = []
  #strptrs << FFI::MemoryPointer.from_string("mpileup")
  opts.each do |k,v|
    next unless opts[k] ##dont bother unless the values provided are true.. 
    k = '6' if k == :six
    k = '-' + k.to_s
    sam_opts << k #strptrs << FFI::MemoryPointer.from_string(k)
    sam_opts << v.to_s  unless ["-R", "-B", "-E", "-6", "-A"].include?(k) #these are just flags so don't pass a value... strptrs << FFI::MemoryPointer.from_string(v.to_s)
  end
  sam_opts = sam_opts + ['-f', @fasta_path, @sam]
  sam_command = "#{File.join(File.expand_path(File.dirname(__FILE__)),'sam','external','samtools')} mpileup #{sam_opts.join(' ')} 2> /dev/null"

  sam_pipe = IO.popen(sam_command)
  while line = sam_pipe.gets
    yield Bio::DB::Pileup.new(line)
  end
  sam_pipe.close
  #strptrs << FFI::MemoryPointer.from_string('-f')
  #strptrs << FFI::MemoryPointer.from_string(@fasta_path)
  #strptrs << FFI::MemoryPointer.from_string(@sam)
  #strptrs << nil

  # Now load all the pointers into a native memory block
  #argv = FFI::MemoryPointer.new(:pointer, strptrs.length)
  #strptrs.each_with_index do |p, i|
  #   argv[i].put_pointer(0,  p)
  #end

  #old_stdout = STDOUT.clone
  #read_pipe, write_pipe = IO.pipe()
  #STDOUT.reopen(write_pipe)
    #int bam_mpileup(int argc, char *argv[])
   # Bio::DB::SAM::Tools.bam_mpileup(strptrs.length - 1,argv)
    #if fork
    #  write_pipe.close
    #  STDOUT.reopen(old_stdout) #beware .. stdout from other processes eg tests calling this method can get mixed in...
    #  begin
    #    while line = read_pipe.readline
    #      yield Pileup.new(line)
    #    end
    #  rescue EOFError
    #    read_pipe.close
    #    Process.wait
    #  end
    #end
end

#mpileup_plus(opts) ⇒ Object

experimental method that spawns a samtools mpileup | bcftools view process and supports returning of pileup vcf otherwise works like mpileup

Raises:



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# File 'lib/bio/db/sam.rb', line 363

def mpileup_plus( opts )

  raise SAMException.new(), "No BAMFile provided" unless @sam and @binary
  raise SAMException.new(), "No FastA provided" unless @fasta_path
  #long option form to short samtools form..
  long_opts = {
  :region => :r,
  :illumina_quals => :six,
  :count_anomalous => :A,
  :no_baq => :B,
  :adjust_mapq => :C,
  :max_per_bam_depth => :d,
  :extended_baq => :E,
  :exclude_reads_file => :G,
  :list_of_positions => :l,
  :mapping_quality_cap => :M,
  :ignore_rg => :R,
  :min_mapping_quality => :q,
  :min_base_quality => :Q,
  ###following options are for the -g -u option
  :genotype_calling => :g,
  :uncompressed_bcf => :u,
  :extension_sequencing_probability => :e,
  :homopolymer_error_coefficient => :h,
  :no_indels => :I,
  :skip_indel_over_average_depth => :L,
  :gap_open_sequencing_error_probability => :o,
  :platforms => :P 
  }

  ##convert any long_opts to short opts 
  temp_opts = opts.dup
  opts.each_pair do |k,v|
    if long_opts[k]
      temp_opts[long_opts[k]] = v 
      temp_opts.delete(k)
    end
  end
  opts = temp_opts
  ##remove any calls to -g or -u for mpileup, bcf output is not yet supported
  ##and also associated output options
  #[:g, :u, :e, :h, :I, :L, :o, :p].each {|x| opts.delete(x) }
  opts[:u] = true if opts[:g] #so that we always get uncompressed output
  opts.delete(:g)
  
  sam_opts = []
  #strptrs << FFI::MemoryPointer.from_string("mpileup")
  opts.each do |k,v|
    next unless opts[k] ##dont bother unless the values provided are true.. 
    k = '6' if k == :six
    k = '-' + k.to_s
    sam_opts << k #strptrs << FFI::MemoryPointer.from_string(k)
    sam_opts << v.to_s  unless ["-R", "-B", "-E", "-6", "-A", "-g", "-u", "-I"].include?(k) #these are just flags so don't pass a value... strptrs << FFI::MemoryPointer.from_string(v.to_s)
  end
  sam_opts = sam_opts + ['-f', @fasta_path, @sam]
  
  command = "#{File.join(File.expand_path(File.dirname(__FILE__)),'sam','external','samtools')} mpileup #{sam_opts.join(' ')} 2> /dev/null"
  if opts[:u]
    command = command + " | #{File.join(File.expand_path(File.dirname(__FILE__)),'sam','external','bcftools')} view -cg -"
  end
  pipe = IO.popen(command)
  $stderr.puts command
  if opts[:u]
    while line = pipe.gets
      next if line[0,1] == '#' #skip any header or meta-lines, we dont do anything with those 
      yield Bio::DB::Vcf.new(line) 
    end
  else
    while line = pipe.gets
      yield Bio::DB::Pileup.new(line)
    end
  end
  pipe.close
  #strptrs << FFI::MemoryPointer.from_string('-f')
  #strptrs << FFI::MemoryPointer.from_string(@fasta_path)
  #strptrs << FFI::MemoryPointer.from_string(@sam)
  #strptrs << nil

  # Now load all the pointers into a native memory block
  #argv = FFI::MemoryPointer.new(:pointer, strptrs.length)
  #strptrs.each_with_index do |p, i|
  #   argv[i].put_pointer(0,  p)
  #end

  #old_stdout = STDOUT.clone
  #read_pipe, write_pipe = IO.pipe()
  #STDOUT.reopen(write_pipe)
    #int bam_mpileup(int argc, char *argv[])
   # Bio::DB::SAM::Tools.bam_mpileup(strptrs.length - 1,argv)
    #if fork
    #  write_pipe.close
    #  STDOUT.reopen(old_stdout) #beware .. stdout from other processes eg tests calling this method can get mixed in...
    #  begin
    #    while line = read_pipe.readline
    #      yield Pileup.new(line)
    #    end
    #  rescue EOFError
    #    read_pipe.close
    #    Process.wait
    #  end
    #end
end

#open ⇒ Object

Function that actually opens the sam file Throws a SAMException if the file can’t be open.

Raises:



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# File 'lib/bio/db/sam.rb', line 60

def open()

  raise SAMException.new(), "Writing not supported yet" if @write
  raise SAMException.new(), "No SAM file specified" unless @sam 

  opts = @write ? "w" : "r"
  if @binary then  
    opts += "b" 
    if @write then
      unless @compressed then 
        opts += "u"
      end
    end
  end
  valid = ["r", "w", "wh", "rb", "wb" , "wbu"]
  unless valid.include?(opts) then
    raise SAMException.new(), "Invalid options for samopen: " + opts 
  end

  samFile = Bio::DB::SAM::Tools.samopen(@sam, opts, nil)
  if samFile.null? then
    @sam_file = nil
    raise SAMException.new(), "File not opened:  " + @sam
  end
  @sam_file = Bio::DB::SAM::Tools::SamfileT.new(samFile)

end

#query_string(chromosome, qstart, qend) ⇒ Object

Generates a query sting to be used by the region parser in samtools. In principle, you shouldn’t need to use this function.



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# File 'lib/bio/db/sam.rb', line 205

def query_string(chromosome, qstart,qend)
  query = chromosome + ":" + qstart.to_s + "-" + qend.to_s 
  query
end

#to_s ⇒ Object

Prints a description of the sam file in a text format containg if it is binary or text, the path and the fasta file of the reference



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# File 'lib/bio/db/sam.rb', line 90

def to_s()
  (@binary ? "Binary" : "Text") + " file: " + @sam + " with fasta: " + @fasta_path
end