bio-samtools 0.2.5 → 0.3.0

data/lib/bio/db/sam.rb

@@ -15,6 +15,15 @@ module Bio
     class Sam
       attr_reader :sam_file
 
+      # 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. 
+      #
       def initialize(optsa={})
         opts =  { :fasta => nil,  :bam => nil,:tam => nil, :compressed => true, :write => false }.merge!(optsa)
 
@@ -43,7 +52,9 @@ module Bio
 
         ObjectSpace.define_finalizer(self,  self.class.method(:finalize).to_proc)
       end
-
+      
+      #Function that actually opens the sam file
+      #Throws a SAMException if the file can't be open.
       def open()
 
         raise SAMException.new(), "Writing not supported yet" if @write
@@ -72,10 +83,13 @@ module Bio
 
       end
 
+      #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
       def to_s()
         (@binary ? "Binary" : "Text") + " file: " + @sam + " with fasta: " + @fasta_path
       end
 
+      #Closes the sam file and destroys the C pointers using the functions provided by libbam
       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?
@@ -84,11 +98,17 @@ module Bio
         @fasta_index = nil
       end
 
+      # Destructor method that closes the file before letting the object be garbage collected.
       def Sam.finalize(id)
         id.close()
         puts "Finalizing #{id}  at #{Time.new}"       
       end
 
+      #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.
       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)
@@ -100,6 +120,9 @@ module Bio
         end
       end
 
+      #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.
       def load_reference()
         raise SAMException.new(), "No path for the refernce fasta file. " if @fasta_path.nil?
 
@@ -114,6 +137,7 @@ module Bio
 
       end
 
+      #Returns the average coverage of a region in a bam file.
       def average_coverage(chromosome, qstart, len)
 
         #reference = fetch_reference(chromosome, qstart,len)
@@ -128,6 +152,9 @@ module Bio
         avg_cov
       end
 
+      #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.
       def chromosome_coverage(chromosome, qstart, len)
         #  reference = fetch_reference(chromosome, qstart,len)
         #  len = reference.length if len > reference.length
@@ -160,6 +187,7 @@ module Bio
         coverages
       end
 
+      #Returns the sequence for a given region.
       def fetch_reference(chromosome, qstart,qend)
         load_reference if @fasta_index.nil? || @fasta_index.null? 
         query = query_string(chromosome, qstart,qend)
@@ -170,11 +198,14 @@ module Bio
         reference
       end
 
+      #Generates a query sting to be used by the region parser in samtools. 
+      #In principle, you shouldn't need to use this function.
       def query_string(chromosome, qstart,qend)
         query = chromosome + ":" + qstart.to_s + "-" + qend.to_s 
         query
       end
 
+      #Returns an array of Alignments on a given region.
       def fetch(chromosome, qstart, qend)
         als = Array.new
         fetchAlignment = Proc.new do |alignment|
@@ -185,6 +216,11 @@ module Bio
         als
       end  
 
+      #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?)
       def fetch_with_function(chromosome, qstart, qend, function)
         load_index if @sam_index.nil? || @sam_index.null?
         chr = FFI::MemoryPointer.new :int
@@ -211,6 +247,132 @@ module Bio
         #LibC.free qpointer
         count
       end
+      
+      #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. 
+      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
+
+      #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 
+      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 
+        opts.each_pair do |k,v|
+          if long_opts[k]
+            opts[long_opts[k]] = v 
+            opts.delete(k)
+          end
+        end
+        
+        ##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) }
+        
+        strptrs = []
+        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
+          strptrs << FFI::MemoryPointer.from_string(k)
+          strptrs << FFI::MemoryPointer.from_string(v.to_s) unless ["-R", "-B", "-E", "-6", "-A"].include?(k) #these are just flags so don't pass a value...
+        end
+        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
+      
+      # 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
+      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
 
     end
 

data/lib/bio/db/sam/pileup.rb

@@ -0,0 +1,98 @@
+# :title:Pileup
+# = Pileup 
+# A class representing information in SAMTools pileup format
+# Author:: Dan MacLean (dan.maclean@tsl.ac.uk)
+# Pileup is described at http://sourceforge.net/apps/mediawiki/samtools/index.php?title=SAM_FAQ#I_do_not_understand_the_columns_in_the_pileup_output.
+# Briefly (when you invoke pileup with the -c option):
+# * 1 reference sequence name
+# * 2 reference coordinate
+# * (3) reference base, or `*' for an indel line
+# * (4) genotype where heterozygotes are encoded in the IUB code: M=A/C, R=A/G, W=A/T, S=C/G, Y=C/T and K=G/T; indels are indicated by, for example, */+A, -A/* or +CC/-C. There is no difference between */+A or +A/*.
+# * (5) Phred-scaled likelihood that the genotype is wrong, which is also called `consensus quality'.
+# * (6) Phred-scaled likelihood that the genotype is identical to the reference, which is also called `SNP quality'. Suppose the reference base is A and in alignment we see 17 G and 3 A. We will get a low consensus quality because it is difficult to distinguish an A/G heterozygote from a G/G homozygote. We will get a high SNP quality, though, because the evidence of a SNP is very strong.
+# * (7) root mean square (RMS) mapping quality
+# * 8 # reads covering the position
+# * 9 read bases at a SNP line (check the manual page for more information); the 1st indel allele otherwise
+# * 10 base quality at a SNP line; the 2nd indel allele otherwise
+# * (11) indel line only: # reads directly supporting the 1st indel allele
+# * (12) indel line only: # reads directly supporting the 2nd indel allele
+# * (13) indel line only: # reads supporting a third indel allele
+# If pileup is invoked without `-c', indel lines and columns between 3 and 7 inclusive will not be outputted.
+# 
+# NB mpileup uses the 6 column output format eg
+# "seq2\t151\tG\tG\t36\t0\t99\t12\t...........A\t:9<;;7=<<<<<"
+# Pileup provides accessors for all columns (6 or 10 column format) and a few other useful methods
+# 
+# 
+
+class Pileup
+  attr_accessor :ref_name, :pos, :ref_base, :coverage, :read_bases, :read_quals, :consensus, :consensus_quality, :snp_quality, :rms_mapq, :ar1, :ar2, :ar3
+  
+  #creates the Pileup object
+  #    pile_up_line = "seq2\t151\tG\tG\t36\t0\t99\t12\t...........A\t:9<;;7=<<<<<"
+  #    pile = Pileup.new(pile_up_line)
+  def initialize(pile_up_line)
+    cols = pile_up_line.split(/\t/)
+    if cols.length == 6 ##should only be able to get 6 lines from mpileup
+      @ref_name, @pos, @ref_base, @coverage, @read_bases, @read_quals = cols
+    elsif (10..13).include?(cols.length) ##incase anyone tries to use deprecated pileup with -c flag we get upto 13 cols...
+      @ref_name, @pos, @ref_base, @consensus, @consensus_quality, @snp_quality, @rms_mapq, @coverage, @read_bases, @read_quals, @ar1, @ar2, @ar3 = cols
+      @consensus_quality = @consensus_quality.to_f
+      @snp_quality = @snp_quality.to_f
+      @rms_mapq = @rms_mapq.to_f
+    else
+      #raise RuntimeError, "parsing line '#{pile_up_line.chomp}' failed"
+    end
+      
+    @pos = @pos.to_i
+    @coverage = @coverage.to_f
+    @ref_count = nil
+    @non_ref_count_hash = nil 
+    @non_ref_count = nil
+  end
+  
+  # Calculate the total count of each non-reference nucleotide and return a hash of all 4 nt counts, returns a hash
+  #    pile.non_refs #{:A => 1, :C => 0, :T => 0, :G => 0}
+  def non_refs
+    if @non_ref_count_hash.nil?
+       @non_ref_count_hash = {:A => self.read_bases.count("Aa"), :C => self.read_bases.count("Cc"), :G => self.read_bases.count("Gg"), :T => self.read_bases.count("Tt")}
+    end
+      @non_ref_count_hash
+  end
+  
+  # returns the total non-reference bases in the reads at this position
+  def non_ref_count
+    if @non_ref_count.nil?
+      @non_ref_count = @read_bases.count("ATGCatgc").to_f
+    end
+    @non_ref_count
+  end
+  
+  # returns the count of reference-bases in the reads at this position
+  def ref_count
+    if @ref_count.nil?
+      @ref_count = self.read_bases.count(".,")
+    end
+    @ref_count
+  end
+  
+  # returns the consensus (most frequent) base from the pileup, if there are equally represented bases returns a string containing all equally represented bases in alphabetical order   
+  def consensus
+      if @consensus.nil?
+        max = self.non_refs.values.max
+        if (self.ref_count / self.coverage) > 0.5
+          @consensus = self.ref_base 
+        elsif self.ref_count > max
+          @consensus = self.ref_base
+        else
+          arr = self.non_refs.select {|k,v| v == max }
+          bases = arr.collect {|b| b[0].to_s }
+          bases << self.ref_base if self.ref_count == max
+          @consensus = bases.sort.join
+        end
+      end
+      @consensus
+  end
+  
+end
+

data/test/basictest.rb

@@ -276,6 +276,20 @@ class TestBioDbSam < Test::Unit::TestCase
     assert(true, "Average coverage ran")
     #assert(3 == cov, "The coverage is 3")
   end
+  
+  #test whether the call to mpileup works and returns 10 objects of class pileup
+  def test_pileup
+    sam = Bio::DB::Sam.new(:fasta=>@testReference, :bam=>@testBAMFile )
+    pileup_list = []
+    sam.mpileup(:region => "chr_1:100-110") do |pile|
+      next unless pile.ref_name == 'chr_1' ##required because in the test environment stdout gets mixed in with the captured stdout in the function and non pileup lines are passed...
+      pileup_list << pile
+    end
+    assert_equal(10,pileup_list.length)
+    pileup_list.each  do |p|
+      assert_kind_of(Pileup, p)
+    end
+  end
 
 end
 

data/test/pileup.rb

@@ -0,0 +1,68 @@
+$: << File.expand_path(File.dirname(__FILE__) + '/../lib')
+$: << File.expand_path('.')
+
+require "bio/db/sam/pileup"
+require "test/unit"
+
+class TestPileup < Test::Unit::TestCase
+  
+  def setup
+    @six_col = Pileup.new("seq1\t272\tT\t24\t,.$.....,,.,.,...,,,.,..^+.\t<<<+;<<<<<<<<<<<=<;<;7<&")
+    @ten_col = Pileup.new("seq2\t151\tG\tG\t36\t0\t99\t12\t...........A\t:9<;;7=<<<<<")
+    @snp = Pileup.new("seq1\t272\tT\t24\t,.$.....,,.gGgGgGgGgGgGg^+.\t<<<+;<<<<<<<<<<<=<;<;7<&") 
+    @snp_2 = Pileup.new("seq1\t272\tT\t24\t......aaaaaaggggggcccccc$^+\t<<<+;<<<<<<<<<<<=<;<;7<&")
+    @snp_3 = Pileup.new("seq1\t272\tT\t24\t......aaaaaaaagggggccccc$^+\t<<<+;<<<<<<<<<<<=<;<;7<&")
+  end
+  
+  def test_new_from_6_column
+    assert_equal("seq1", @six_col.ref_name)
+    assert_equal(272, @six_col.pos)
+    assert_equal("T", @six_col.ref_base)
+    assert_equal(24, @six_col.coverage)
+    assert_equal(",.$.....,,.,.,...,,,.,..^+.", @six_col.read_bases)
+    assert_equal("<<<+;<<<<<<<<<<<=<;<;7<&", @six_col.read_quals)
+  end
+  
+  def test_new_from_10_column
+    assert_equal("seq2", @ten_col.ref_name)
+    assert_equal(151, @ten_col.pos)
+    assert_equal("G", @ten_col.ref_base)
+    assert_equal("G", @ten_col.consensus)
+    assert_equal(36, @ten_col.consensus_quality)
+    assert_equal(0, @ten_col.snp_quality)
+    assert_equal(99, @ten_col.rms_mapq)
+    assert_equal(12, @ten_col.coverage)
+    assert_equal("...........A", @ten_col.read_bases)
+    assert_equal(":9<;;7=<<<<<", @ten_col.read_quals)
+  end
+  
+  def test_non_refs
+    assert_equal({:A => 1, :C => 0, :T => 0, :G => 0}, @ten_col.non_refs)
+    assert_equal({:A => 0, :C => 0, :T => 0, :G => 0}, @six_col.non_refs)
+  end
+  
+  def test_consensus
+    assert_equal("G", @snp.consensus)
+    assert_equal("ACGT", @snp_2.consensus)
+    assert_equal("A", @snp_3.consensus)
+  end
+  
+  def test_non_ref_count
+    assert_equal(13,@snp.non_ref_count)
+    assert_equal(18,@snp_2.non_ref_count)
+    assert_equal(18,@snp_3.non_ref_count)
+  end
+  
+  def test_ref_count
+    assert_equal(11,@snp.ref_count)
+    assert_equal(6,@snp_2.ref_count)
+    assert_equal(6,@snp_3.ref_count)
+  end
+  
+  def test_ref_plus_non_ref_equal_to_coverage
+    assert_equal(@snp.coverage,@snp.ref_count + @snp.non_ref_count)
+   assert_equal(@snp_2.coverage,@snp_2.ref_count + @snp_2.non_ref_count)
+   assert_equal(@snp_3.coverage,@snp_3.ref_count + @snp_3.non_ref_count)
+  end
+ 
+end

metadata

@@ -1,7 +1,7 @@
 --- !ruby/object:Gem::Specification
 name: bio-samtools
 version: !ruby/object:Gem::Version
-  version: 0.2.5
+  version: 0.3.0
   prerelease: 
 platform: ruby
 authors:
@@ -15,7 +15,7 @@ date: 2011-10-26 00:00:00.000000000Z
 dependencies:
 - !ruby/object:Gem::Dependency
   name: ffi
-  requirement: &2152414440 !ruby/object:Gem::Requirement
+  requirement: &2170620360 !ruby/object:Gem::Requirement
     none: false
     requirements:
     - - ! '>='
@@ -23,10 +23,10 @@ dependencies:
         version: '0'
   type: :runtime
   prerelease: false
-  version_requirements: *2152414440
+  version_requirements: *2170620360
 - !ruby/object:Gem::Dependency
   name: bio
-  requirement: &2152413500 !ruby/object:Gem::Requirement
+  requirement: &2170612920 !ruby/object:Gem::Requirement
     none: false
     requirements:
     - - ! '>='
@@ -34,10 +34,10 @@ dependencies:
         version: 1.4.2
   type: :runtime
   prerelease: false
-  version_requirements: *2152413500
+  version_requirements: *2170612920
 - !ruby/object:Gem::Dependency
   name: shoulda
-  requirement: &2152412540 !ruby/object:Gem::Requirement
+  requirement: &2170610380 !ruby/object:Gem::Requirement
     none: false
     requirements:
     - - ! '>='
@@ -45,10 +45,10 @@ dependencies:
         version: '0'
   type: :development
   prerelease: false
-  version_requirements: *2152412540
+  version_requirements: *2170610380
 - !ruby/object:Gem::Dependency
   name: bundler
-  requirement: &2152411700 !ruby/object:Gem::Requirement
+  requirement: &2170607620 !ruby/object:Gem::Requirement
     none: false
     requirements:
     - - ~>
@@ -56,10 +56,10 @@ dependencies:
         version: 1.0.0
   type: :development
   prerelease: false
-  version_requirements: *2152411700
+  version_requirements: *2170607620
 - !ruby/object:Gem::Dependency
   name: jeweler
-  requirement: &2152410900 !ruby/object:Gem::Requirement
+  requirement: &2170606580 !ruby/object:Gem::Requirement
     none: false
     requirements:
     - - ! '>='
@@ -67,10 +67,10 @@ dependencies:
         version: '0'
   type: :development
   prerelease: false
-  version_requirements: *2152410900
+  version_requirements: *2170606580
 - !ruby/object:Gem::Dependency
   name: rcov
-  requirement: &2152410220 !ruby/object:Gem::Requirement
+  requirement: &2170597180 !ruby/object:Gem::Requirement
     none: false
     requirements:
     - - ! '>='
@@ -78,10 +78,10 @@ dependencies:
         version: '0'
   type: :development
   prerelease: false
-  version_requirements: *2152410220
+  version_requirements: *2170597180
 - !ruby/object:Gem::Dependency
   name: bio
-  requirement: &2152409600 !ruby/object:Gem::Requirement
+  requirement: &2170593180 !ruby/object:Gem::Requirement
     none: false
     requirements:
     - - ! '>='
@@ -89,10 +89,10 @@ dependencies:
         version: 1.4.2
   type: :development
   prerelease: false
-  version_requirements: *2152409600
+  version_requirements: *2170593180
 - !ruby/object:Gem::Dependency
   name: ffi
-  requirement: &2152408900 !ruby/object:Gem::Requirement
+  requirement: &2170591660 !ruby/object:Gem::Requirement
     none: false
     requirements:
     - - ! '>='
@@ -100,7 +100,7 @@ dependencies:
         version: '0'
   type: :development
   prerelease: false
-  version_requirements: *2152408900
+  version_requirements: *2170591660
 description: ! "Binder of samtools for ruby, on the top of FFI. \n\n  This project
   was born from the need to add support of BAM files to \n  the gee_fu genome browser
   (http://github.com/danmaclean/gee_fu)."
@@ -120,6 +120,70 @@ files:
 - Rakefile
 - VERSION
 - bio-samtools.gemspec
+- doc/basic_styles.css
+- doc/classes/Bio.html
+- doc/classes/Bio/DB.html
+- doc/classes/Bio/DB/Alignment.html
+- doc/classes/Bio/DB/Alignment.src/M000012.html
+- doc/classes/Bio/DB/Alignment.src/M000013.html
+- doc/classes/Bio/DB/Alignment.src/M000014.html
+- doc/classes/Bio/DB/Alignment.src/M000015.html
+- doc/classes/Bio/DB/SAM.html
+- doc/classes/Bio/DB/SAM/Library.html
+- doc/classes/Bio/DB/SAM/Library.src/M000006.html
+- doc/classes/Bio/DB/SAM/Tools.html
+- doc/classes/Bio/DB/SAM/Tools.src/M000007.html
+- doc/classes/Bio/DB/SAM/Tools/Bam1CoreT.html
+- doc/classes/Bio/DB/SAM/Tools/Bam1T.html
+- doc/classes/Bio/DB/SAM/Tools/Bam1T.src/M000010.html
+- doc/classes/Bio/DB/SAM/Tools/BamHeaderT.html
+- doc/classes/Bio/DB/SAM/Tools/BamHeaderT.src/M000008.html
+- doc/classes/Bio/DB/SAM/Tools/BamHeaderT.src/M000009.html
+- doc/classes/Bio/DB/SAM/Tools/BamPileup1T.html
+- doc/classes/Bio/DB/SAM/Tools/SamfileT.html
+- doc/classes/Bio/DB/SAM/Tools/SamfileTX.html
+- doc/classes/Bio/DB/SAMException.html
+- doc/classes/Bio/DB/SAMException.src/M000016.html
+- doc/classes/Bio/DB/Sam.src/M000017.html
+- doc/classes/Bio/DB/Sam.src/M000018.html
+- doc/classes/Bio/DB/Sam.src/M000019.html
+- doc/classes/Bio/DB/Sam.src/M000020.html
+- doc/classes/Bio/DB/Sam.src/M000021.html
+- doc/classes/Bio/DB/Sam.src/M000022.html
+- doc/classes/Bio/DB/Sam.src/M000023.html
+- doc/classes/Bio/DB/Sam.src/M000024.html
+- doc/classes/Bio/DB/Sam.src/M000025.html
+- doc/classes/Bio/DB/Sam.src/M000026.html
+- doc/classes/Bio/DB/Sam.src/M000027.html
+- doc/classes/Bio/DB/Sam.src/M000028.html
+- doc/classes/Bio/DB/Sam.src/M000029.html
+- doc/classes/Bio/DB/Sam.src/M000030.html
+- doc/classes/Bio/DB/Sam.src/M000031.html
+- doc/classes/Bio/DB/Sam.src/M000032.html
+- doc/classes/Bio/DB/Tag.html
+- doc/classes/Bio/DB/Tag.src/M000011.html
+- doc/classes/LibC.html
+- doc/classes/Pileup.html
+- doc/classes/Pileup.src/M000001.html
+- doc/classes/Pileup.src/M000002.html
+- doc/classes/Pileup.src/M000003.html
+- doc/classes/Pileup.src/M000004.html
+- doc/classes/Pileup.src/M000005.html
+- doc/created.rid
+- doc/files/lib/bio-samtools_rb.html
+- doc/files/lib/bio/db/sam/bam_rb.html
+- doc/files/lib/bio/db/sam/faidx_rb.html
+- doc/files/lib/bio/db/sam/library_rb.html
+- doc/files/lib/bio/db/sam/pileup_rb.html
+- doc/files/lib/bio/db/sam/sam_rb.html
+- doc/files/lib/bio/db/sam_rb.html
+- doc/fr_class_index.html
+- doc/fr_file_index.html
+- doc/fr_method_index.html
+- doc/index.html
+- doc/rdoc-style.css
+- doc/tutorial.html
+- doc/tutorial.pdf
 - ext/mkrf_conf.rb
 - lib/bio-samtools.rb
 - lib/bio/.DS_Store
@@ -129,12 +193,14 @@ files:
 - lib/bio/db/sam/external/VERSION
 - lib/bio/db/sam/faidx.rb
 - lib/bio/db/sam/library.rb
+- lib/bio/db/sam/pileup.rb
 - lib/bio/db/sam/sam.rb
 - test/basictest.rb
 - test/coverage.rb
 - test/coverage_plot.rb
 - test/feature.rb
 - test/helper.rb
+- test/pileup.rb
 - test/samples/small/ids2.txt
 - test/samples/small/sorted.bam
 - test/samples/small/test
@@ -171,7 +237,7 @@ required_ruby_version: !ruby/object:Gem::Requirement
       version: '0'
       segments:
       - 0
-      hash: -1844225119409437910
+      hash: 1941204301952225721
 required_rubygems_version: !ruby/object:Gem::Requirement
   none: false
   requirements: