bio-gngm 0.1.0
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- data/.document +5 -0
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- data/Gemfile.lock +33 -0
- data/LICENSE.txt +20 -0
- data/README.rdoc +33 -0
- data/Rakefile +53 -0
- data/VERSION +1 -0
- data/bio-gngm.gemspec +173 -0
- data/doc/Bio.html +129 -0
- data/doc/Bio/DB.html +128 -0
- data/doc/Bio/DB/Pileup.html +316 -0
- data/doc/Bio/DB/Vcf.html +683 -0
- data/doc/Bio/Util.html +135 -0
- data/doc/Bio/Util/Gngm.html +1655 -0
- data/doc/LICENSE_txt.html +111 -0
- data/doc/_index.html +169 -0
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- data/doc/images/threads.png +0 -0
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- data/doc/images/zoom.png +0 -0
- data/doc/index.html +88 -0
- data/doc/js/app.js +205 -0
- data/doc/js/darkfish.js +153 -0
- data/doc/js/full_list.js +167 -0
- data/doc/js/jquery.js +18 -0
- data/doc/js/navigation.js +142 -0
- data/doc/js/search.js +94 -0
- data/doc/js/search_index.js +1 -0
- data/doc/js/searcher.js +228 -0
- data/doc/lib/bio-gngm_rb.html +103 -0
- data/doc/lib/bio/util/bio-gngm_rb.html +96 -0
- data/doc/method_list.html +382 -0
- data/doc/rdoc.css +543 -0
- data/doc/table_of_contents.html +161 -0
- data/examples/.DS_Store +0 -0
- data/examples/make_histograms.rb +40 -0
- data/examples/make_threads.rb +42 -0
- data/examples/make_threads_isize.rb +41 -0
- data/examples/use_indels.rb +36 -0
- data/lib/bio-gngm.rb +12 -0
- data/lib/bio/util/bio-gngm.rb +1029 -0
- data/scripts/get_subseq.rb +16 -0
- data/scripts/make_histograms_laerfyve.rb +83 -0
- data/scripts/make_histograms_laerfyve_stitched.rb +59 -0
- data/scripts/make_threads_isize_laerfyfe.rb +52 -0
- data/scripts/make_threads_unmapped_laerfyfe.rb +72 -0
- data/scripts/make_threads_unmapped_laerfyfe_pseudo.rb +56 -0
- data/scripts/make_threads_unmapped_simulation.rb +54 -0
- data/scripts/make_threads_unmapped_simulation_immediate_region.rb +59 -0
- data/scripts/optimise_freq_window_size.rb +82 -0
- data/stitched_contigs.zip +0 -0
- data/test/data/ids2.txt +1 -0
- data/test/data/sorted.bam +0 -0
- data/test/data/test +0 -0
- data/test/data/test.bam +0 -0
- data/test/data/test.fa +20 -0
- data/test/data/test.fai +0 -0
- data/test/data/test.sai +0 -0
- data/test/data/test.tam +10 -0
- data/test/data/test_chr.fasta +1000 -0
- data/test/data/test_chr.fasta.amb +2 -0
- data/test/data/test_chr.fasta.ann +3 -0
- data/test/data/test_chr.fasta.bwt +0 -0
- data/test/data/test_chr.fasta.fai +1 -0
- data/test/data/test_chr.fasta.pac +0 -0
- data/test/data/test_chr.fasta.rbwt +0 -0
- data/test/data/test_chr.fasta.rpac +0 -0
- data/test/data/test_chr.fasta.rsa +0 -0
- data/test/data/test_chr.fasta.sa +0 -0
- data/test/data/testu.bam +0 -0
- data/test/data/testu.bam.bai +0 -0
- data/test/helper.rb +18 -0
- data/test/test_bio-gngm.rb +126 -0
- metadata +276 -0
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<body class="indexpage">
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<h1>Table of Contents</h1>
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<a href="Bio/Util/Gngm.html">Bio::Util::Gngm</a>
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<li><a href="Bio/Util/Gngm.html#label-Background">Background</a>
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<li><a href="Bio/Util/Gngm.html#label-Example">Example</a>
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<li><a href="Bio/Util/Gngm.html#label-Polymorphisms+and+statistics">Polymorphisms and statistics</a>
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<li><a href="Bio/Util/Gngm.html#label-Acknowledgements">Acknowledgements</a>
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<li><a href="Bio/Util/Gngm.html#label-Using+bio-gngm">Using bio-gngm</a>
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<li class="method"><a href="Bio/Util/Gngm.html#method-c-new">::new — Bio::Util::Gngm</a>
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<p><a href="http://validator.w3.org/check/referer">[Validate]</a>
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<p>Generated by <a href="https://github.com/rdoc/rdoc">RDoc</a> 3.11.
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<p>Generated with the <a href="http://deveiate.org/projects/Darkfish-Rdoc/">Darkfish Rdoc Generator</a> 3.
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data/examples/.DS_Store
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#!/usr/bin/env ruby
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#
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# make_histograms
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#
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# Created by Dan MacLean (TSL) on 2012-01-17.
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# Copyright (c) . All rights reserved.
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###################################################
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### An example script to get SNP positions and make histograms
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### of the frequncy of discordant SNPs. Generates plots for each.
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$LOAD_PATH.unshift(File.join(File.dirname(__FILE__), '..', 'lib'))
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$LOAD_PATH.unshift(File.dirname(__FILE__))
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require 'bio-gngm'
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require 'bio-samtools'
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#open the BAM file and specify the region of interest
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g = Bio::Util::Gngm.new(:file => "aln.bam",
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:format => :bam,
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:fasta => "reference.fasta",
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:samtools => {:r => "Chr1:1-6000000",
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:q => 20,
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:Q => 50
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}
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)
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#retrieve the SNPs from the BAM file
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g.snp_positions
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#plot a frequency histogram for different bin sizes
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[100000, 250000, 500000].each do |bin_width|
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file_name = "#{bin_width}.png"
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g.frequency_histogram("#{file_name}",bin_width)
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end
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#close the BAM file
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g.close
|
|
40
|
+
|
|
@@ -0,0 +1,42 @@
|
|
|
1
|
+
#!/usr/bin/env ruby
|
|
2
|
+
#
|
|
3
|
+
# make_threads
|
|
4
|
+
#
|
|
5
|
+
# Created by Dan MacLean (TSL) on 2012-01-17.
|
|
6
|
+
# Copyright (c) . All rights reserved.
|
|
7
|
+
###################################################
|
|
8
|
+
|
|
9
|
+
### An example script to specify a region, get SNP positions, make density threads for different kernels,
|
|
10
|
+
### cluster for different values of k and then draw the threads, bands and signal. Generates plots for each new set of parameters
|
|
11
|
+
|
|
12
|
+
|
|
13
|
+
$LOAD_PATH.unshift(File.join(File.dirname(__FILE__), '..', 'lib'))
|
|
14
|
+
$LOAD_PATH.unshift(File.dirname(__FILE__))
|
|
15
|
+
require 'bio-gngm'
|
|
16
|
+
|
|
17
|
+
|
|
18
|
+
g = Bio::Util::Gngm.new(:file => "/Users/macleand/Desktop/ngm/fli.sorted.bam",
|
|
19
|
+
:format => :bam,
|
|
20
|
+
:fasta => "/Users/macleand/Desktop/ngm/TAIR9_chr_all.fas",
|
|
21
|
+
:samtools => {:r => "Chr1:1000000-2000000",
|
|
22
|
+
:q => 20,
|
|
23
|
+
:Q => 50
|
|
24
|
+
},
|
|
25
|
+
)
|
|
26
|
+
g.snp_positions( :min_depth => 10, :mapping_quality => 40.0, :min_non_ref_count => 5)
|
|
27
|
+
g.collect_threads(:start => 0.3, :stop => 0.8, :slide => 0.01, :size => 0.2 )
|
|
28
|
+
[0.25, 0.5, 1.0].each do |kernel_adjust|
|
|
29
|
+
[4, 9, 11].each do | k |
|
|
30
|
+
filename = "#{name}_#{k}_#{kernel_adjust}_all_threads.png"
|
|
31
|
+
g.calculate_clusters(:k => k, :adjust => kernel_adjust, :control_chd => 1.0, :expected_chd => 0.5)
|
|
32
|
+
g.draw_threads(filename)
|
|
33
|
+
filename = "#{name}_#{k}_#{kernel_adjust}_clustered_bands.png"
|
|
34
|
+
g.draw_bands(filename)
|
|
35
|
+
filename = "#{name}_#{k}_#{kernel_adjust}_signal.png"
|
|
36
|
+
g.draw_signal(filename)
|
|
37
|
+
end
|
|
38
|
+
end
|
|
39
|
+
g.close
|
|
40
|
+
end
|
|
41
|
+
|
|
42
|
+
sam.close
|
|
@@ -0,0 +1,41 @@
|
|
|
1
|
+
#!/usr/bin/env ruby
|
|
2
|
+
#
|
|
3
|
+
# make_threads_isize
|
|
4
|
+
#
|
|
5
|
+
# Created by Dan MacLean (TSL) on 2012-01-17.
|
|
6
|
+
# Copyright (c) . All rights reserved.
|
|
7
|
+
###################################################
|
|
8
|
+
|
|
9
|
+
### An example script to luse th insert size between paired end reads to infer large deletions. The script, make density threads based on the proportion of reads with insert size above 0.5 for different kernels,
|
|
10
|
+
### cluster for different values of k and then draw the threads, bands and signal. Generates plots for each new set of parameters. Contains a rescue clause in case something goes wrong, these sorts of calculations
|
|
11
|
+
### can be very data-sparse and density curves can be hard to make and cluster when lots of window are empty
|
|
12
|
+
|
|
13
|
+
require 'bio-gngm'
|
|
14
|
+
|
|
15
|
+
|
|
16
|
+
g = Bio::Util::Gngm.new(:file => "aln.sort.bam",
|
|
17
|
+
:format => :bam,
|
|
18
|
+
:fasta => "reference.fasta",
|
|
19
|
+
:samtools => {:r => "1:1000000-10000000",
|
|
20
|
+
:q => 20,
|
|
21
|
+
:Q => 50
|
|
22
|
+
}
|
|
23
|
+
)
|
|
24
|
+
g.get_insert_size_frequency(:ref_window_size => 152, :ref_window_slide => 152, :isize => 150)
|
|
25
|
+
g.collect_threads
|
|
26
|
+
[0.25, 0.5, 1.0].each do |kernel_adjust|
|
|
27
|
+
[4, 9, 11].each do | k |
|
|
28
|
+
begin
|
|
29
|
+
g.calculate_clusters(:k => k, :adjust => kernel_adjust, :control_chd => 0.5, :expected_chd => 0.9)
|
|
30
|
+
filename = "isize_#{k}_#{kernel_adjust}_all_threads.png"
|
|
31
|
+
g.draw_threads(filename, :draw_legend => "isize_#{k}_#{kernel_adjust}_legend.png")
|
|
32
|
+
filename = "isize_#{k}_#{kernel_adjust}_bands.png"
|
|
33
|
+
g.draw_bands(filename)
|
|
34
|
+
filename = "isize_#{k}_#{kernel_adjust}_signal.png"
|
|
35
|
+
g.draw_signal(filename)
|
|
36
|
+
rescue Exception => e
|
|
37
|
+
puts "failed on #{k} #{kernel_adjust}"
|
|
38
|
+
puts e.message, e.backtrace
|
|
39
|
+
end
|
|
40
|
+
end
|
|
41
|
+
end
|
|
@@ -0,0 +1,36 @@
|
|
|
1
|
+
#!/usr/bin/env ruby
|
|
2
|
+
#
|
|
3
|
+
# use_indels
|
|
4
|
+
#
|
|
5
|
+
# Created by Dan MacLean (TSL) on 2012-01-17.
|
|
6
|
+
# Copyright (c) . All rights reserved.
|
|
7
|
+
###################################################
|
|
8
|
+
|
|
9
|
+
### An example script to specify a region, get small deletion positions, make density threads for different kernels,
|
|
10
|
+
### cluster for different values of k and then draw the threads, bands and signal. Generates plots for each new set of parameters
|
|
11
|
+
|
|
12
|
+
require 'bio-gngm'
|
|
13
|
+
|
|
14
|
+
|
|
15
|
+
|
|
16
|
+
g = Bio::Util::Gngm.new(:file => "aln.sorted.bam",
|
|
17
|
+
:format => :bam,
|
|
18
|
+
:fasta => "reference.fasta",
|
|
19
|
+
:samtools => {:r => "Chr1:1-3000000",
|
|
20
|
+
:q => 20,
|
|
21
|
+
:Q => 50
|
|
22
|
+
}
|
|
23
|
+
)
|
|
24
|
+
g.snp_positions(:deletions_only => true)
|
|
25
|
+
g.collect_threads
|
|
26
|
+
g.calculate_clusters(:k => 9, :adjust => 0.5, :control_chd => 0.5, :expected_chd => 1.0)
|
|
27
|
+
filename = "indels_all_threads.png"
|
|
28
|
+
g.draw_threads(filename)
|
|
29
|
+
filename = "indels_clustered_bands.png"
|
|
30
|
+
g.draw_bands(filename)
|
|
31
|
+
filename = "indels_signal.png"
|
|
32
|
+
g.draw_signal(filename)
|
|
33
|
+
filename = "indels_peaks.png"
|
|
34
|
+
g.draw_peaks(filename)
|
|
35
|
+
g.close
|
|
36
|
+
|
data/lib/bio-gngm.rb
ADDED
|
@@ -0,0 +1,12 @@
|
|
|
1
|
+
# Please require your code below, respecting the bioruby directory tree.
|
|
2
|
+
# For instance, perhaps the only uncommented line in this file might
|
|
3
|
+
# be something like this:
|
|
4
|
+
#
|
|
5
|
+
# require 'bio/sequence/awesome_sequence_plugin_thingy'
|
|
6
|
+
#
|
|
7
|
+
# and then create the ruby file 'lib/bio/sequence/awesome_sequence_thingy.rb'
|
|
8
|
+
# and put your plugin's code there. It is bad practice to write other code
|
|
9
|
+
# directly into this file, because doing so causes confusion if this biogem
|
|
10
|
+
# was ever to get merged into the main bioruby tree.
|
|
11
|
+
|
|
12
|
+
require 'bio/util/bio-gngm'
|
|
@@ -0,0 +1,1029 @@
|
|
|
1
|
+
#!/usr/bin/env ruby
|
|
2
|
+
#
|
|
3
|
+
# bio-gngm
|
|
4
|
+
#
|
|
5
|
+
# Created by Dan MacLean (TSL) on 2011-12-07.
|
|
6
|
+
# Copyright (c) . All rights reserved.
|
|
7
|
+
###################################################
|
|
8
|
+
|
|
9
|
+
require 'rinruby'
|
|
10
|
+
require 'bio-samtools'
|
|
11
|
+
require 'bio/db/pileup'
|
|
12
|
+
require 'bio/db/vcf'
|
|
13
|
+
require 'pp'
|
|
14
|
+
|
|
15
|
+
=begin
|
|
16
|
+
Extends the methods of the Bio::DB::Pileup class in bio-samtools. A pileup object represents the SAMtools pileup format at
|
|
17
|
+
http://samtools.sourceforge.net/pileup.shtml. These extension methods are used by the Bio::Util::Gngm object internally and
|
|
18
|
+
are not exposed to the user of the Bio::Util::Gngm object through that.
|
|
19
|
+
=end
|
|
20
|
+
class Bio::DB::Pileup
|
|
21
|
+
|
|
22
|
+
#attributes set by call to Bio::DB::Pileup#discordant_chastity
|
|
23
|
+
attr_accessor :top_non_ref_count, :second_non_ref_count, :third_non_ref_count
|
|
24
|
+
|
|
25
|
+
#calculates the discordant chastity statistic as defined in Austin et al (2011) http://bar.utoronto.ca/ngm/description.html and http://onlinelibrary.wiley.com/doi/10.1111/j.1365-313X.2011.04619.x/abstract;jsessionid=F73E2DA628523B26205297CEE95526DA.d02t04
|
|
26
|
+
#Austin _et_ _al_ (2011) *Next-generation* *mapping* *of* *Arabidopsis* *genes* _Plant_ _Journal_ *67*(4):7125-725
|
|
27
|
+
#
|
|
28
|
+
#Briefly,
|
|
29
|
+
# The statistic measures the degree of difference between the SNP and the expected reference base.
|
|
30
|
+
# Using the mapping information comprising a SNP, the most frequent base that is not the reference base
|
|
31
|
+
# is compared to the next most common base after it.
|
|
32
|
+
# (from http://bar.utoronto.ca/ngm/description.html )
|
|
33
|
+
def discordant_chastity
|
|
34
|
+
arr = self.non_refs.to_a.sort {|a,b| b.last <=> a.last }
|
|
35
|
+
@top_non_ref_count, @second_non_ref_count, @third_non_ref_count = arr.collect {|c| c.last}
|
|
36
|
+
case
|
|
37
|
+
when self.non_ref_count == 0 then 0.0
|
|
38
|
+
when @top_non_ref_count == @coverage then 1.0
|
|
39
|
+
when @second_non_ref_count > 0 then @top_non_ref_count.to_f / (@top_non_ref_count + @second_non_ref_count).to_f
|
|
40
|
+
else @top_non_ref_count.to_f / @coverage.to_f
|
|
41
|
+
end
|
|
42
|
+
end
|
|
43
|
+
|
|
44
|
+
#returns true if self is a SNP with minimum coverage depth of +:min_depth+ and minimum non-reference bases of +:min_non_ref_count+
|
|
45
|
+
#returns false for every position where the reference base is N or n if +:ignore_reference_n+ is set to true
|
|
46
|
+
#
|
|
47
|
+
#Options and Defaults:
|
|
48
|
+
#- :min_depth => 2
|
|
49
|
+
#- :min_non_ref_count => 2
|
|
50
|
+
#- :ignore_reference_n => false
|
|
51
|
+
#
|
|
52
|
+
#Example
|
|
53
|
+
# pileup.is_snp?(:min_depth => 5, :min_non_ref_count => 2)
|
|
54
|
+
# pileup.is_snp?(:min_depth => 5, :min_non_ref_count => 1, :ignore_reference_n => true)
|
|
55
|
+
def is_snp?(opts)
|
|
56
|
+
if opts[:ignore_reference_n] and self.ref_base == "N" or self.ref_base == "n"
|
|
57
|
+
return false
|
|
58
|
+
elsif self.coverage >= opts[:min_depth] and self.non_ref_count >= opts[:min_non_ref_count]
|
|
59
|
+
return true
|
|
60
|
+
end
|
|
61
|
+
false
|
|
62
|
+
end
|
|
63
|
+
end
|
|
64
|
+
|
|
65
|
+
|
|
66
|
+
|
|
67
|
+
#Extends the methods of the Bio::DB::Vcf class in bio-samtools. A Vcf object represents the VCF format described at
|
|
68
|
+
#http://www.1000genomes.org/node/101 . The Bio::DB::Vcf object returns all information in the VCF line, but the implementation here acts as if
|
|
69
|
+
#there is only possibly one variant at each position and ignores positions at which there may be multiple variants. Vcf format is only used when
|
|
70
|
+
#the Bio::Util::Gngm object requests information about indels using SAMtools mpileup method.
|
|
71
|
+
class Bio::DB::Vcf
|
|
72
|
+
#returns true if the +alt+ column of the Vcf is not *.*
|
|
73
|
+
#
|
|
74
|
+
#Examples
|
|
75
|
+
#
|
|
76
|
+
#vcf record = 20 14370 rs6054257 G A 29 PASS ...
|
|
77
|
+
# vcf.variant? #=> true
|
|
78
|
+
#vcf record = 20 1230237 . T . 47 PASS ...
|
|
79
|
+
# vcf.variant? #=> false
|
|
80
|
+
def variant?
|
|
81
|
+
not self.alt == "." rescue false
|
|
82
|
+
end
|
|
83
|
+
|
|
84
|
+
#Return a short string representing chromosome, position, reference sequence, alt sequence(s) and the info string of the Vcf object.
|
|
85
|
+
def to_s
|
|
86
|
+
"#{self.chrom} #{self.pos} #{self.ref} #{self.alt} #{self.info}"
|
|
87
|
+
end
|
|
88
|
+
|
|
89
|
+
#The depth of reads actually used in the genotype call by Vcftools. The sum of the DP4 attribute. Returns 0.0 if no value is calculated.
|
|
90
|
+
def used_depth
|
|
91
|
+
self.info["DP4"].split(",").inject {|sum,n| sum.to_f + n.to_f} rescue 0.0
|
|
92
|
+
end
|
|
93
|
+
|
|
94
|
+
#List of alternate alleles at this locus, obtained by splitting the vcf.alt attribute string on commas
|
|
95
|
+
#
|
|
96
|
+
#Example
|
|
97
|
+
#vcf.alt = "ACT,TCA"
|
|
98
|
+
# vcf.alternatives = ["ACT", "TCA"]
|
|
99
|
+
#vcf.alt = "T"
|
|
100
|
+
# vcf.alternatives = ["T"]
|
|
101
|
+
def alternatives
|
|
102
|
+
self.alt.split(",") rescue []
|
|
103
|
+
end
|
|
104
|
+
|
|
105
|
+
##Returns the depth of reads containing the non reference allele. IE the sum of the last two figures in the DP4 attribute.
|
|
106
|
+
def non_ref_allele_count
|
|
107
|
+
self.info["DP4"].split(",")[2..3].inject {|sum,n| sum.to_f + n.to_f } rescue 0.0
|
|
108
|
+
end
|
|
109
|
+
|
|
110
|
+
#Returns the non-reference allele frequency based on depth of reads used for the genotype call,
|
|
111
|
+
#
|
|
112
|
+
#IE
|
|
113
|
+
# vcf.non_ref_allele_count / vcf.used_depth
|
|
114
|
+
def non_ref_allele_freq
|
|
115
|
+
self.non_ref_allele_count / self.used_depth
|
|
116
|
+
end
|
|
117
|
+
|
|
118
|
+
#Returns the mean Mapping Quality from the reads over this position as defined by the Vcf MQ attribute.
|
|
119
|
+
def mq
|
|
120
|
+
self.info["MQ"].to_f rescue 0.0
|
|
121
|
+
end
|
|
122
|
+
|
|
123
|
+
##Returns the genotype quality score from the sample data (as defined by the Vcf GQ attribute) for the first sample in the Vcf only.
|
|
124
|
+
def gq
|
|
125
|
+
self.samples["1"]["GQ"].to_f rescue 0.0
|
|
126
|
+
end
|
|
127
|
+
|
|
128
|
+
#Returns the phred scaled likelihood of the first non-reference allele (as defined by the Vcf PL attribute) for the first sample in the Vcf only.
|
|
129
|
+
def pl
|
|
130
|
+
self.samples["1"]["PL"].split(",")[1].to_f rescue 0.0
|
|
131
|
+
end
|
|
132
|
+
|
|
133
|
+
#Returns true if only one variant allele is recorded. Loci with more than one allele are too complicated for now, so are discarded...
|
|
134
|
+
def has_just_one_variant?
|
|
135
|
+
self.alternatives.length == 1 and self.variant?
|
|
136
|
+
end
|
|
137
|
+
|
|
138
|
+
#Returns true if the position passes criteria
|
|
139
|
+
#
|
|
140
|
+
#Options and Defaults:
|
|
141
|
+
#- :min_depth => 2
|
|
142
|
+
#- :min_non_ref_count => 2
|
|
143
|
+
#- :mapping_quality => 10
|
|
144
|
+
#
|
|
145
|
+
#Example
|
|
146
|
+
# vcf.pass_quality?(:min_depth => 5, :min_non_ref_count => 2, :mapping_quality => 25)
|
|
147
|
+
def pass_quality?(options)
|
|
148
|
+
(self.used_depth >= options[:min_depth] and self.mq >= options[:mapping_quality] and self.non_ref_allele_count >= options[:min_non_ref_count])
|
|
149
|
+
end
|
|
150
|
+
|
|
151
|
+
#Returns true if the length of the alt column is less than that of the ref column in the Vcf and if Vcf.pass_quality? is true.
|
|
152
|
+
#Looks only at the positions that are predicted simple deletions, any positions where the alt alleles includes more than one deletion or a SNP or an insertion also is ignored.
|
|
153
|
+
def is_deletion?(options)
|
|
154
|
+
case
|
|
155
|
+
when (not self.has_just_one_variant?) then false
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when ( self.alt.length < self.ref.length and self.pass_quality?(options) ) then true
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else false
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end
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rescue ## if something goes wrong, skip the postion,
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false
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+
end
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+
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#Returns true if the length of the alt column is greater than that of the ref column in the Vcf and if Vcf.pass_quality? is true.
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#Looks only at the positions that are predicted simple deletions, any positions where the alt alleles includes more than one deletion or a SNP or an insertion also is ignored.
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def is_insertion?(options)
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case
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when (not self.has_just_one_variant?) then false
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when ( self.alt.length > self.ref.length and self.pass_quality?(options) ) then true
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else false
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+
end
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rescue ## if something goes wrong, skip the postion,
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false
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|
+
end
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+
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#Returns true if either Vcf.is_insertion? or Vcf.is_deletion? is true
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def is_indel?(opts)
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self.is_insertion?(opts) || self.is_deletion?(opts)
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+
end
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+
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+
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end
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module Bio
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class Util
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=begin
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A Bio::Util::Gngm object represents a single region on a reference genome that is to be examined using the NGM technique described in Austin et al (2011) http://bar.utoronto.ca/ngm/description.html and http://onlinelibrary.wiley.com/doi/10.1111/j.1365-313X.2011.04619.x/abstract;jsessionid=F73E2DA628523B26205297CEE95526DA.d02t04
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Austin _et_ _al_ (2011) *Next-generation* *mapping* *of* *Arabidopsis* *genes* _Plant_ _Journal_ *67*(4):7125-725 .
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+
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Bio::Util::Gngm provides methods for finding SNPs, small INDELS and larger INDELS, creating histograms of polymorphism frequency,
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creating and clustering density curves, creating signal plots and finding peaks. The ratio of reference-agreeing and reference-differing reads can be specified.
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+
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== Background
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The basic concept of the technique is that density curves of polymorphism frequency across the region of interest are plotted and analysed. Each curve is called a thread, as it represents a polymorphism that
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+
was called with a statistic within a certain user-specified range, eg if a SNP was called with 50% non-reference bases from sequence reads (say all A), and 50% reference reads (all T) then a discordant
|
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+
chastity statistic (ChD) of 0.5 would be calculated and assigned to that SNP. Depending on the width and slide of the windows the user had specified, the frequency of SNPs with ChD in the specified range
|
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+
would be drawn in the same density curve. In the figure below each different coloured curve represents the frequency of SNPs with similar ChD.
|
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+
|
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link:images/threads.png
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+
|
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Each of these density curves is called a thread. Threads are clustered into groups called bands and the bands containing the expected and control polymorphisms extracted. In the figure below, the control band is 0.5, the expected mutation in 1.0.
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|
+
Typically and in the Austin et al (2011) description of NGM the control band is the heterophasic band that represents natural variation, the thing taken to be the baseline. For a simple SNP, numerically the discordant chastity is expected to be 0.5.
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|
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|
+
Conversely the expected band is the homophasic band that represents the selected for SNP region. Normally the discordant chastity is expected to be 1.0.
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|
+
|
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|
+
link:images/bands.png
|
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|
+
|
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207
|
+
The points where the signal from the control and expected band converge most is a likely candidate region for the causative mutation, so here at about the 1.6 millionth nucleotide.
|
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|
+
|
|
209
|
+
link:images/signal.png
|
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+
|
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211
|
+
== Example
|
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|
+
require 'bio-gngm'
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|
+
|
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214
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+
|
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215
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+
|
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216
|
+
|
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217
|
+
g = Bio::Util::Gngm.new(:file => "aln.sorted.bam",
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|
+
:format => :bam,
|
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|
+
:fasta => "reference.fasta",
|
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|
+
:samtools => {:r => "chr1:1-100000",
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|
+
:q => 20,
|
|
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|
+
:Q => 50
|
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|
+
},
|
|
224
|
+
:min_non_ref_freq => 0.5,
|
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225
|
+
:min_non_ref => 3
|
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|
+
)
|
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|
+
g.snp_positions
|
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|
+
g.collect_threads(:start => 0.2, :stop => 1.0, :slide => 0.01, :size => 0.1 )
|
|
229
|
+
[0.25, 0.5, 1.0].each do |kernel_adjust| # loop through different kernel values
|
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|
+
[4, 9, 11].each do | k | # loop through different cluster numbers
|
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|
+
|
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232
|
+
#cluster
|
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233
|
+
g.calculate_clusters(:k => k, :adjust => kernel_adjust, :control_chd => 0.7, :expected_chd => 0.5)
|
|
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|
+
#draw thread and bands
|
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235
|
+
filename = "#{name}_#{k}_#{kernel_adjust}_all_threads.png"
|
|
236
|
+
g.draw_threads(filename)
|
|
237
|
+
|
|
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|
+
filename = "#{name}_#{k}_#{kernel_adjust}_clustered_bands.png"
|
|
239
|
+
g.draw_bands(filename, :add_lines => [100,30000,675432])
|
|
240
|
+
|
|
241
|
+
#draw signal
|
|
242
|
+
filename = "#{name}_#{k}_#{kernel_adjust}_signal.png"
|
|
243
|
+
g.draw_signal(filename)
|
|
244
|
+
|
|
245
|
+
#auto-guess peaks
|
|
246
|
+
filename = "#{name}_#{k}_#{kernel_adjust}_peaks.png"
|
|
247
|
+
g.draw_peaks(filename)
|
|
248
|
+
end
|
|
249
|
+
end
|
|
250
|
+
g.close #close BAM file
|
|
251
|
+
|
|
252
|
+
== Polymorphisms and statistics
|
|
253
|
+
Bio::Util::Gngm will allow you to look for polymorphisms that are SNPs, INDELS (as insertions uniquely, deletions uniquely or both) and longer insertions or deletions based on the insert size on paired-end read alignments.
|
|
254
|
+
Each has a different statistic attached to it.
|
|
255
|
+
|
|
256
|
+
=== SNPs
|
|
257
|
+
Simple Single Nucleotide Polymorphisms are called and its ChD statistic calculated as described in Austin et al (2011).
|
|
258
|
+
|
|
259
|
+
=== Short INDELS
|
|
260
|
+
These are called via SAMtools/BCFtools so are limited to the INDELs that can be called that way. The implementation at the moment only considers positions with one INDEL, sites with
|
|
261
|
+
more than one potential INDEL (ie multiple alleles) are disregarded as a position at all. See the Bio::DB::Vcf extensions in this package for a description of what constitutes an INDEL.
|
|
262
|
+
The Vcf attribute Bio::DB::Vcf#non_ref_allele_freq is used as the statistic in this case.
|
|
263
|
+
|
|
264
|
+
=== Insertion Size
|
|
265
|
+
Paired-end alignments have an expected distance between the paired reads (called insert size, or isize). Groups of reads in one position with larger or smaller than expected isize can indicate large deletions or insertions.
|
|
266
|
+
Due to the details of read preparation the actual isize varies around a mean value with an expected proportion of 50% of reads having isize above the mean, and 50% below. To create density curves of insertion size frequency a moves along the
|
|
267
|
+
window of user-specified size is moved along the reference genome in user-specified steps and all alignments in that window are examined. The Bio::DB::Sam#isize attribute is inspected for all alignments passing
|
|
268
|
+
user-specified quality and the proportion of reads in that window that have an insert size > the expected insert size is used as the statistic in this case. Proportions approaching 1 indicate that the sequenced organism has a deletion in that section relative to the reference.
|
|
269
|
+
Proportions approaching 0 indicate an insertion in that section relative to the reference. Proportions around 0.5 indicate random variation of insert size, IE no INDEL. Seems to be a good idea to keep the window size similar to the read + isize. Useful in conjunction with assessing unmapped mates.
|
|
270
|
+
|
|
271
|
+
=== Unmapped Mate Pairs / Paired Ends.
|
|
272
|
+
Paired-end alignments where one mate finds a mapping but the other doesnt, can indicate an insertion/deletion larger than the insert size of the reads used (IE one read disappeared into the deleted section). This method uses a statistic based on proportion of
|
|
273
|
+
mapped/unmapped reads in a window. Proportions of reads that are mapped but the mate is unmapped should be about 0.5 in a window over an insertion/deletion (since the reads can go in either direction..). With no insertion deletion, the
|
|
274
|
+
proportion should be closer to 0.
|
|
275
|
+
|
|
276
|
+
== Input types
|
|
277
|
+
A sorted BAM file is used as the source of alignments. Pileup is not used nor likely to be as it is a deprecated function within SAMtools. With the BAM file you will need the reference FASTA and the BAM index (.bai).
|
|
278
|
+
|
|
279
|
+
== Workflow
|
|
280
|
+
1. Create Bio::Util::Gngm object for a specific region in the reference genome
|
|
281
|
+
2. Polymorphisms are found
|
|
282
|
+
3. Density curves (threads) are calculated
|
|
283
|
+
4. Clustering density threads into bands is done
|
|
284
|
+
5. Signal is compared between band of interest and control
|
|
285
|
+
6. Figures are printed
|
|
286
|
+
|
|
287
|
+
== Prerequisites
|
|
288
|
+
- Ruby 1.9.3 or greater (if you have an earlier version, try RVM for installing different versions of Ruby alongside your system install and switching nicely between them)
|
|
289
|
+
- R 2.11.1 or greater
|
|
290
|
+
|
|
291
|
+
The following ruby-gems are required
|
|
292
|
+
- rinruby >= 2.0.2
|
|
293
|
+
- bio-samtools >= 0.5.0
|
|
294
|
+
|
|
295
|
+
The following R packages are required
|
|
296
|
+
- ggplot2
|
|
297
|
+
- peaks
|
|
298
|
+
|
|
299
|
+
== Acknowledgements
|
|
300
|
+
Thanks very much indeed to Ryan Austin, who invented NGM in the first place and was very forthcoming with R code, around which this implementation is based.
|
|
301
|
+
|
|
302
|
+
== Using bio-gngm
|
|
303
|
+
The package is not yet released, a gem will be prepared soon. Until then scripts run fine when saved in the package scripts from within the package directory with the below pre-amble at the top of the script. Run scripts from the root of the package directory.
|
|
304
|
+
$LOAD_PATH.unshift(File.join(File.dirname(__FILE__), '..', 'lib'))
|
|
305
|
+
$LOAD_PATH.unshift(File.dirname(__FILE__))
|
|
306
|
+
require 'bio-samtools'
|
|
307
|
+
require 'bio-gngm'
|
|
308
|
+
|
|
309
|
+
== API
|
|
310
|
+
=end
|
|
311
|
+
class Gngm
|
|
312
|
+
|
|
313
|
+
attr_accessor :file
|
|
314
|
+
|
|
315
|
+
#Ruby 1.9.3 has a rounding error in the Range#step function such that some decimal places are rounded off to 0.00000000000000...1 above their place. So this constant is used to identify
|
|
316
|
+
#windows within a short distance and prevent any rounding errors. Hopefully I should be able to remove this in later versions.
|
|
317
|
+
ERROR_MARGIN = 0.000001
|
|
318
|
+
|
|
319
|
+
public
|
|
320
|
+
#Returns a new Bio::Util::Gngm object.
|
|
321
|
+
#
|
|
322
|
+
# g = Bio::Util::Gngm.new(:file => "aln.sort.bam",
|
|
323
|
+
# :format => :bam,
|
|
324
|
+
# :samtools => {:q => 20, :Q => 50, :r => "Chr1:1-100000"},
|
|
325
|
+
# :fasta => "reference.fa"
|
|
326
|
+
#
|
|
327
|
+
# )
|
|
328
|
+
#
|
|
329
|
+
#Required parameters and defaults:
|
|
330
|
+
#- <tt>:file => nil</tt> -the path to the bam file containing the alignments, a .bai index must be present
|
|
331
|
+
#- <tt>:format => :bam</tt> -always bam
|
|
332
|
+
#- <tt>:fasta => nil</tt> -the path to the FASTA formatted reference sequence
|
|
333
|
+
#- <tt>:samtools => {:q => 20, :Q => 50, :r => "Chr1:100-1100"}</tt> -options for samtools, see bio-samtools documentation for further details. The :r option is required to specify the region of interest
|
|
334
|
+
#Optional parameters and defaults:
|
|
335
|
+
#Most of these are parameters for specific methods and can be over-ridden when particular methods are called
|
|
336
|
+
#- <tt>:variant_call => {:indels => false, :deletions_only => false, :insertions_only => false, :min_depth => 2, :max_depth => 10000000, :mapping_quality => 10.0, :min_non_ref_count => 2, :ignore_reference_n => true}</tt> -for SNP/Indel calling only one of <tt>:indels, :deletions_only, :insertions_only</tt> should be used.
|
|
337
|
+
#- <tt>:threads => {:start => 0.2, :stop => 1.0, :slide => 0.01, :size => 0.1 }</tt> -options for thread windows
|
|
338
|
+
#- <tt>:insert_size_opts => {:ref_window_size => 200, :ref_window_slide => 50, :isize => 150}</tt> -options for insert size calculations
|
|
339
|
+
#- <tt>:histo_bin_width => 250000</tt> -bin width for histograms of SNP frequency
|
|
340
|
+
#- <tt>:graphics => {:width => 1000, :height => 500, :draw_legend => false, :add_boxes => nil}</tt> -graphics output options, +:draw_legend+ draws a legend plot for band figures only
|
|
341
|
+
#- <tt>:peaks => {:sigma => 3.0, :threshold => 10.0, :background => false, :iterations => 13, :markov => false, :window => 3, :range => 10000}</tt> -parameters for automated peak calling, parameters relate to R package Peaks. +:range+ is the width of the box to draw on the peak plot
|
|
342
|
+
def initialize(options)
|
|
343
|
+
@file = nil
|
|
344
|
+
@snp_positions = nil
|
|
345
|
+
@threads = nil
|
|
346
|
+
@densities = nil
|
|
347
|
+
@clusters = nil
|
|
348
|
+
@control_band = nil
|
|
349
|
+
@expected_band = nil
|
|
350
|
+
@signal = nil
|
|
351
|
+
@peak_indices = nil
|
|
352
|
+
@peak_y_values = nil
|
|
353
|
+
@density_max_y = nil #the maximum y value needed to plot the entire set density plots of threads and maintain a consistent scale for plots
|
|
354
|
+
@colours = %w[#A6CEE3 #1F78B4 #B2DF8A #33A02C #FB9A99 #E31A1C #FDBF6F #FF7F00 #CAB2D6 #6A3D9A #FFFF99 #B15928]
|
|
355
|
+
@thread_colours = {}
|
|
356
|
+
@opts = {
|
|
357
|
+
:file => nil,
|
|
358
|
+
:format => :bam,
|
|
359
|
+
:fasta => nil,
|
|
360
|
+
:samtools => {:q => 20, :Q => 50},
|
|
361
|
+
##indels = call any and only indels.. :deletions_only :insertions_only = only one tyoe
|
|
362
|
+
## some options are designed to be equivalent to vcfutils.pl from bvftools options when using vcf
|
|
363
|
+
##:min_depth (-d)
|
|
364
|
+
##:max_depth (-D)
|
|
365
|
+
##:mapping_quality (-Q) minimum RMS mappinq quality for SNPs (mq in info fields)
|
|
366
|
+
##:min_non_ref_count (-a) minimum num of alt bases ... the sum of the last two numbers in DP4 in info fields
|
|
367
|
+
##doesnt do anything with window filtering or pv values...
|
|
368
|
+
:insert_size_opts => {:ref_window_size => 200, :ref_window_slide => 50, :isize => 150},
|
|
369
|
+
:variant_call => {:indels => false, :deletions_only => false, :insertions_only => false, :min_depth => 2, :max_depth => 10000000, :mapping_quality => 10.0, :min_non_ref_count => 2, :ignore_reference_n => true},
|
|
370
|
+
:histo_bin_width => 250000,
|
|
371
|
+
:graphics => {:width => 1000, :height => 500, :draw_legend => false, :add_boxes => nil},
|
|
372
|
+
:adjust => 1,
|
|
373
|
+
:control_chd => 0.5,
|
|
374
|
+
:expected_chd => 1.0,
|
|
375
|
+
:threads => {:start => 0.2, :stop => 1.0, :slide => 0.01, :size => 0.1 },
|
|
376
|
+
:peaks => {:sigma => 3.0, :threshold => 10.0, :background => false, :iterations => 13, :markov => false, :window => 3, :range => 10000} ##range is the width of the box to draw on the peak plot
|
|
377
|
+
}
|
|
378
|
+
@opts.merge!(options)
|
|
379
|
+
open_file
|
|
380
|
+
end
|
|
381
|
+
|
|
382
|
+
private
|
|
383
|
+
#opens the file
|
|
384
|
+
def open_file
|
|
385
|
+
case @opts[:format]
|
|
386
|
+
when :bam then open_bam
|
|
387
|
+
end
|
|
388
|
+
end
|
|
389
|
+
|
|
390
|
+
private
|
|
391
|
+
#calls Bio::DB::Sam.open
|
|
392
|
+
def open_bam
|
|
393
|
+
@file = Bio::DB::Sam.new(:bam => @opts[:file], :fasta => @opts[:fasta] )
|
|
394
|
+
@file.open
|
|
395
|
+
end
|
|
396
|
+
|
|
397
|
+
public
|
|
398
|
+
#for BAM files calls Bio::DB::Sam#close to close the connections to input files safely
|
|
399
|
+
def close
|
|
400
|
+
case @opts[:format]
|
|
401
|
+
when :bam then @file.close
|
|
402
|
+
end
|
|
403
|
+
end
|
|
404
|
+
|
|
405
|
+
public
|
|
406
|
+
#Returns array of arrays <tt>[[position, statistic]]</tt> for polymorphisms passing filters in +optsa+
|
|
407
|
+
#Default options are those in the +:variant_call+ global options hash which can be over ridden in the method call
|
|
408
|
+
#
|
|
409
|
+
#Options and defaults:
|
|
410
|
+
#- <tt>:indels => false</tt> -call small insertions AND deletions instead of simple SNPs
|
|
411
|
+
#- <tt>:deletions_only => false</tt> -call just deletions instead of simple SNPs
|
|
412
|
+
#- <tt>:insertions_only => false</tt> -call small insertions instead of simple SNPs
|
|
413
|
+
#- <tt>:min_depth => 2</tt> -minimum quality passing depth of coverage at a position for a SNP call
|
|
414
|
+
#- <tt>:max_depth => 10000000</tt> -maximum quality passing depth of coverage at a position for a SNP call
|
|
415
|
+
#- <tt>:mapping_quality => 10.0</tt> -minimum mapping quality required for a read to be used in depth calculation
|
|
416
|
+
#- <tt>:min_non_ref_count => 2</tt> -minimum number of reads not matching the reference for SNP to be called
|
|
417
|
+
#- <tt>:ignore_reference_n => true</tt> -ignore positions where the reference is N or n
|
|
418
|
+
#
|
|
419
|
+
#When INDEL calling only one of <tt>:indels, :deletions_only, :insertions_only</tt> should be used. If all are +false+, SNPs are called.
|
|
420
|
+
#
|
|
421
|
+
#Sets the instance variable @snp_positions. Only gets positions the first time it is called, in subsequent calls pre-computed positions and statistics are returned, so changing parameters has no effect.
|
|
422
|
+
def snp_positions(optsa={})
|
|
423
|
+
opts = @opts[:variant_call].merge(optsa)
|
|
424
|
+
return @snp_positions if @snp_positions
|
|
425
|
+
case
|
|
426
|
+
when @file.instance_of?(Bio::DB::Sam) then get_snp_positions_from_bam(opts)
|
|
427
|
+
end
|
|
428
|
+
end
|
|
429
|
+
|
|
430
|
+
private
|
|
431
|
+
#Calls SNP/short INDEL positions from a BAM file and the appropriate statistic according to quality criteria passed by Bio::Util::Gngm#snp_positions.
|
|
432
|
+
#Sets @snp_positions
|
|
433
|
+
def get_snp_positions_from_bam(options={})
|
|
434
|
+
opts = @opts[:variant_call].merge(options)
|
|
435
|
+
if opts[:indels] and (opts[:deletions_only] or opts[:insertions_only])
|
|
436
|
+
raise "Cant have indels and deletions only or insertions only, need to specify ':indels => true' to get both"
|
|
437
|
+
end
|
|
438
|
+
arr = []
|
|
439
|
+
##when we are calling mpileup_plus we need to add :g to the samtools options
|
|
440
|
+
if opts[:indels] or opts[:deletions_only] or opts[:insertions_only]
|
|
441
|
+
@opts[:samtools][:g] = true
|
|
442
|
+
end
|
|
443
|
+
|
|
444
|
+
if not @opts[:samtools][:g]
|
|
445
|
+
@file.mpileup(@opts[:samtools]) do |pileup|
|
|
446
|
+
arr << [pileup.pos, pileup.discordant_chastity] if pileup.is_snp?(opts)
|
|
447
|
+
end
|
|
448
|
+
else
|
|
449
|
+
@file.mpileup_plus(@opts[:samtools]) do |vcf|
|
|
450
|
+
next if not vcf.variant? ##we dont care about the calls for reference agreeing positions
|
|
451
|
+
next if (opts[:ignore_reference_n] and vcf.ref =~ /N/i)
|
|
452
|
+
##indel use returns the vcf allele_frequency, not the ChDs (because calculating it is a mess... )
|
|
453
|
+
if opts[:indels]
|
|
454
|
+
arr << [vcf.pos, vcf.non_ref_allele_freq] if vcf.is_indel?(opts)
|
|
455
|
+
elsif opts[:deletions_only]
|
|
456
|
+
arr << [vcf.pos, vcf.non_ref_allele_freq] if vcf.is_deletion?(opts)
|
|
457
|
+
elsif opts[:insertions_only]
|
|
458
|
+
arr << [vcf.pos, vcf.non_ref_allele_freq] if vcf.is_insertion?(opts)
|
|
459
|
+
end
|
|
460
|
+
end
|
|
461
|
+
end
|
|
462
|
+
|
|
463
|
+
@snp_positions = arr
|
|
464
|
+
arr
|
|
465
|
+
end
|
|
466
|
+
|
|
467
|
+
private
|
|
468
|
+
#Gets the insert size for each alignment in the BAM positions from a BAM file according to quality criteria passed by Bio::Util::Gngm#get_insert_size_frequency.
|
|
469
|
+
def get_insert_size_frequency_from_bam(opts={})
|
|
470
|
+
reference_window_size,reference_window_slide = opts[:ref_window_size], opts[:ref_window_slide]
|
|
471
|
+
arr = []
|
|
472
|
+
@opts[:samtools][:r] =~ /(.*):(.*)-(.*)/
|
|
473
|
+
chr,rstart,rstop = $1.to_s,$2.to_i,$3.to_i
|
|
474
|
+
(rstart..rstop).step(reference_window_slide) do |win_start|
|
|
475
|
+
win_tot = 0.0
|
|
476
|
+
win_over_isize = 0.0
|
|
477
|
+
@file.fetch(chr, win_start, win_start + reference_window_size).each do |alignment|
|
|
478
|
+
next if not alignment_passes(alignment)
|
|
479
|
+
win_tot = win_tot + 1
|
|
480
|
+
win_over_isize = win_over_isize + 1 if alignment.isize.abs > opts[:isize]
|
|
481
|
+
end
|
|
482
|
+
prop = win_over_isize / win_tot
|
|
483
|
+
arr << [win_start, prop]
|
|
484
|
+
end
|
|
485
|
+
@snp_positions = arr
|
|
486
|
+
end
|
|
487
|
+
#Gets the proportion of reads with unmapped mates in a window
|
|
488
|
+
def get_unmapped_mate_frequency_from_bam(opts={})
|
|
489
|
+
reference_window_size,reference_window_slide = opts[:ref_window_size], opts[:ref_window_slide]
|
|
490
|
+
arr = []
|
|
491
|
+
@opts[:samtools][:r] =~ /(.*):(.*)-(.*)/
|
|
492
|
+
chr,rstart,rstop = $1.to_s,$2.to_i,$3.to_i
|
|
493
|
+
(rstart..rstop).step(reference_window_slide) do |win_start|
|
|
494
|
+
#puts "__________________#{win_start}____________________"
|
|
495
|
+
win_tot = 0.0
|
|
496
|
+
win_mates_unmapped = 0.0
|
|
497
|
+
@file.fetch(chr, win_start, win_start + reference_window_size).each do |alignment|
|
|
498
|
+
next if (alignment.failed_quality) # or @opts[:samtools][:q] <= alignment.mapq or not alignment.is_paired)
|
|
499
|
+
win_tot = win_tot + 1
|
|
500
|
+
win_mates_unmapped = win_mates_unmapped + 1 if alignment.mate_unmapped
|
|
501
|
+
end
|
|
502
|
+
|
|
503
|
+
#puts "win tot #{win_tot}"
|
|
504
|
+
#puts "win mates #{win_mates_unmapped}"
|
|
505
|
+
prop = win_mates_unmapped / win_tot
|
|
506
|
+
#puts "prop #{prop}"
|
|
507
|
+
arr << [win_start, prop]
|
|
508
|
+
end
|
|
509
|
+
@snp_positions = arr
|
|
510
|
+
end
|
|
511
|
+
|
|
512
|
+
private
|
|
513
|
+
#Returns true if the passed Bio::DB::Sam passes the quality criteria
|
|
514
|
+
def alignment_passes(aln)
|
|
515
|
+
not aln.failed_quality && @opts[:samtools][:q] <= aln.mapq && aln.is_paired and not aln.mate_unmapped
|
|
516
|
+
end
|
|
517
|
+
|
|
518
|
+
public
|
|
519
|
+
#Returns array of arrays <tt>[[window start position, proportion of alignments > insert size]]</tt>.
|
|
520
|
+
#Does this by taking successive windows across reference and collects the proportion of the reads in that window
|
|
521
|
+
#that have an insert size > the expected insert size. Proportions approaching 1 indicate that the sequenced organism has a deletion in that section, proportions approaching 0 indicate an insertion in that section, proportions around 0.5 indicate random variation of insert size, IE no indel.
|
|
522
|
+
#
|
|
523
|
+
#Each section should be approximately the size of the insertion you expect to find and should increment in as small steps as possible.
|
|
524
|
+
#
|
|
525
|
+
#Options and defaults:
|
|
526
|
+
#- <tt>:ref_window_size => 200</tt> width of window in which to calculate proportions
|
|
527
|
+
#- <tt>:ref_window_slide => 50</tt> number of bases to move window in each step
|
|
528
|
+
#- <tt>:isize => 150</tt> expected insert size
|
|
529
|
+
#
|
|
530
|
+
#Sets the instance variable @snp_positions. Only gets positions the first time it is called, in subsequent calls pre-computed positions and statistics are returned, so changing parameters has no effect
|
|
531
|
+
def get_insert_size_frequency(options={})
|
|
532
|
+
opts = @opts[:insert_size_opts].merge(options)
|
|
533
|
+
return @snp_positions if @snp_positions
|
|
534
|
+
case
|
|
535
|
+
when @file.instance_of?(Bio::DB::Sam) then get_insert_size_frequency_from_bam(opts)
|
|
536
|
+
end
|
|
537
|
+
end
|
|
538
|
+
|
|
539
|
+
#Returns array of arrays <tt>[[window start position, proportion of reads with unmapped mates]]</tt>.
|
|
540
|
+
#Does this by taking successive windows across reference and counting the reads with unmapped mates
|
|
541
|
+
#Proportions approaching 0.5 indicate that the sequenced organism has an insertion in that section, proportions approaching 0 indicate nothing different in that section.
|
|
542
|
+
#
|
|
543
|
+
#Each section should be approximately the size of the insertion you expect to find and should increment in as small steps as possible.
|
|
544
|
+
#
|
|
545
|
+
#Options and defaults:
|
|
546
|
+
#- <tt>:ref_window_size => 200</tt> width of window in which to calculate proportions
|
|
547
|
+
#- <tt>:ref_window_slide => 50</tt> number of bases to move window in each step
|
|
548
|
+
#
|
|
549
|
+
#Sets the instance variable @snp_positions. Only gets positions the first time it is called, in subsequent calls pre-computed positions and statistics are returned, so changing parameters has no effect
|
|
550
|
+
def get_unmapped_mate_frequency(options={})
|
|
551
|
+
opts = @opts[:insert_size_opts].merge(options)
|
|
552
|
+
return @snp_positions if @snp_positions
|
|
553
|
+
case
|
|
554
|
+
when @file.instance_of?(Bio::DB::Sam) then get_unmapped_mate_frequency_from_bam(opts)
|
|
555
|
+
end
|
|
556
|
+
end
|
|
557
|
+
|
|
558
|
+
|
|
559
|
+
|
|
560
|
+
public
|
|
561
|
+
#Draws a histogram of polymorphism frequencies across the reference genome section defined in Bio::Util::Gngm#initialize with bin width +bin_width+ and writes it to a PNG file +file+
|
|
562
|
+
def frequency_histogram(file="myfile.png", bin_width=@opts[:histo_bin_width], opts=@opts[:graphics])
|
|
563
|
+
posns = self.snp_positions.collect {|a| a.first}
|
|
564
|
+
r = new_r
|
|
565
|
+
r.eval "suppressMessages ( library(ggplot2) )" #setup R environment...
|
|
566
|
+
r.posns = posns
|
|
567
|
+
r.eval "data = data.frame(position=posns)"
|
|
568
|
+
r.eval "png('#{file}', width=#{opts[:width]}, height=#{opts[:height]})"
|
|
569
|
+
graph_cmd = "qplot(position,data=data, geom='histogram', binwidth = #{bin_width}, alpha=I(1/3), main='#{file}', color='red')"
|
|
570
|
+
r.eval(graph_cmd)
|
|
571
|
+
r.eval "dev.off()"
|
|
572
|
+
r.quit
|
|
573
|
+
end
|
|
574
|
+
|
|
575
|
+
#Returns contents of @threads, an array of arrays <tt>[[window 1, snp position 1, snp position 2 ... snp position n],[window 2, snp position 1, snp position 2 ... snp position n] ]</tt>.
|
|
576
|
+
#If @threads is nil (because snps have not yet been gathered into threads) the Bio::Util::Gngm#collect_threads method is called and @threads is set before returning
|
|
577
|
+
#
|
|
578
|
+
#Options and defaults:
|
|
579
|
+
#- <tt>:start => 0.2</tt> -first window
|
|
580
|
+
#- <tt>:stop => 1.0</tt> -last window
|
|
581
|
+
#- <tt>:slide => 0.01</tt> -distance between windows
|
|
582
|
+
#- <tt>:size => 0.1</tt> -window width
|
|
583
|
+
public
|
|
584
|
+
def threads(opts=@opts[:threads])
|
|
585
|
+
@threads ||= collect_threads(opts)
|
|
586
|
+
end
|
|
587
|
+
|
|
588
|
+
public
|
|
589
|
+
#Resets contents of instance variable @threads and returns an array of arrays <tt>[[window 1, snp position 1, snp position 2 ... snp position n],[window 2, snp position 1, snp position 2 ... snp position n] ]</tt>.
|
|
590
|
+
#Always sets @threads regardless of whether it contains anything or not so is useful for trying out different window sizes etc
|
|
591
|
+
#
|
|
592
|
+
#Options and defaults:
|
|
593
|
+
#- <tt>:start => 0.2</tt> -first window
|
|
594
|
+
#- <tt>:stop => 1.0</tt> -last window
|
|
595
|
+
#- <tt>:slide => 0.01</tt> -distance between windows
|
|
596
|
+
#- <tt>:size => 0.1</tt> -window width
|
|
597
|
+
def collect_threads(options={})
|
|
598
|
+
opts = @opts[:threads].merge(options)
|
|
599
|
+
opts[:slide] = 0.000001 if opts[:slide] < 0.000001 ##to allow for the rounding error in the step function...
|
|
600
|
+
raise RuntimeError, "snp positions have not been calculated yet" if not @snp_positions
|
|
601
|
+
start,stop,slide,size = opts[:start].to_f, opts[:stop].to_f, opts[:slide].to_f, opts[:size].to_f
|
|
602
|
+
arr = []
|
|
603
|
+
(start..stop).step(slide) do |win|
|
|
604
|
+
arr << [win, @snp_positions.select {|x| x.last >= win and x.last < win + size }.collect {|y| y.first} ]
|
|
605
|
+
end
|
|
606
|
+
@threads = arr
|
|
607
|
+
end
|
|
608
|
+
|
|
609
|
+
private
|
|
610
|
+
#Returns the value of @density_max_y or if nil, calls Bio::Util::Gngm#get_density_max_y to work out the maximum y axis value for plots
|
|
611
|
+
#Might not work properly as seems to call non-existent method...
|
|
612
|
+
def density_max_y
|
|
613
|
+
@density_max_y ||= get_density_max_y
|
|
614
|
+
end
|
|
615
|
+
|
|
616
|
+
private
|
|
617
|
+
def calculate_density_max_y
|
|
618
|
+
mx = 0.0
|
|
619
|
+
self.densities.each do |x|
|
|
620
|
+
m = x[2].max
|
|
621
|
+
mx = m if m > mx
|
|
622
|
+
end
|
|
623
|
+
@density_max_y = mx
|
|
624
|
+
end
|
|
625
|
+
|
|
626
|
+
public
|
|
627
|
+
#Draws the threads in a single PNG file +file+
|
|
628
|
+
#
|
|
629
|
+
#Options and defaults
|
|
630
|
+
#- <tt>:draw_legend => nil</tt> -if a filename is provided a legend will be drawn in a second plot
|
|
631
|
+
#- <tt>:width => 1000</tt> -width of the PNG in pixels
|
|
632
|
+
#- <tt>:height => 500</tt> -height of the PNG in pixels
|
|
633
|
+
def draw_threads(file="myfile.png", options={})
|
|
634
|
+
opts = @opts[:graphics].merge(options)
|
|
635
|
+
#uses R's standard plot functions.. needed because ggplot can die unexpectedly...
|
|
636
|
+
raise RuntimeError, "Can't draw threads until clustering is done" unless @clusters
|
|
637
|
+
r = new_r
|
|
638
|
+
r.eval "png('#{file}', width=#{opts[:width]}, height=#{opts[:height]})"
|
|
639
|
+
plot_open = false
|
|
640
|
+
self.densities.each do |t|
|
|
641
|
+
r.curr_win = t.last
|
|
642
|
+
r.dx = t[1]
|
|
643
|
+
r.dy = t[2]
|
|
644
|
+
if plot_open
|
|
645
|
+
r.eval "lines(dx,dy, col=\"#{@thread_colours[t.first]}\", xlab='position', ylab='density')"
|
|
646
|
+
else
|
|
647
|
+
r.eval "plot(dx,dy, type=\"l\", col=\"#{@thread_colours[t.first]}\",ylim=c(0,#{density_max_y}), main='#{file}',xlab='position', ylab='density')"
|
|
648
|
+
plot_open = true
|
|
649
|
+
end
|
|
650
|
+
end
|
|
651
|
+
r.eval "dev.off()"
|
|
652
|
+
if opts[:draw_legend]
|
|
653
|
+
r.eval "png('#{opts[:draw_legend]}', width=#{opts[:width]}, height=#{opts[:height]})"
|
|
654
|
+
colours = @thread_colours.each.sort.collect {|x| x.last}.join("','")
|
|
655
|
+
names = @thread_colours.each.sort.collect {|x| x.first}.join("','")
|
|
656
|
+
r.eval "plot(1,xlab="",ylab="",axes=FALSE)"
|
|
657
|
+
r.eval "legend('top', c('#{names}'), lty=c(1),lwd=c(1),col=c('#{colours}'), ncol=4)"
|
|
658
|
+
r.eval "dev.off()"
|
|
659
|
+
end
|
|
660
|
+
r.quit
|
|
661
|
+
end
|
|
662
|
+
|
|
663
|
+
|
|
664
|
+
public
|
|
665
|
+
#Returns the instance variable @densities array of arrays <tt>[window, [density curve x values], [density curve y values] ]</tt>. The R function +density()+ is used to calculate the values. If @densities is nil when called this method will run the Bio::Util::Gngm#calculate_densities method and set @densities
|
|
666
|
+
#With this method you cannot recalculate the densities after they have been done once.
|
|
667
|
+
#
|
|
668
|
+
#Options and defaults
|
|
669
|
+
#- <tt>adjust = 1</tt>, -the kernel adjustment parameter for the R +density+ function
|
|
670
|
+
def densities(adjust=1)
|
|
671
|
+
@densities ||= calculate_densities(adjust)
|
|
672
|
+
end
|
|
673
|
+
|
|
674
|
+
public
|
|
675
|
+
#Sets and returns the array of arrays <tt>[window, [density curve x values], [density curve y values] ]</tt> Calculates the density curve using the R function +density()+ Always sets @densities regardless of whether it contains anything or not so is useful for trying out adjustment values.
|
|
676
|
+
#Ignores threads with fewer than 2 polymorphisms since density can't be computed with so few polymorphisms.
|
|
677
|
+
#
|
|
678
|
+
#Options and defaults
|
|
679
|
+
#- <tt>adjust = 1</tt>, -the kernel adjustment parameter for the R +density+ function
|
|
680
|
+
def calculate_densities(adjust=1)
|
|
681
|
+
r = new_r
|
|
682
|
+
densities = []
|
|
683
|
+
self.threads.each do |t|
|
|
684
|
+
next if t.last.length < 2 ##length of density array is smaller or == threads, since too small windows are ignored...
|
|
685
|
+
r.curr_win = t.last
|
|
686
|
+
r.eval "d = density(curr_win,n=240,kernel=\"gaussian\", from=#{@snp_positions.first[0]}, to=#{@snp_positions.last[0]}, adjust=#{adjust})"
|
|
687
|
+
densities << [t.first, r.pull("d$x"), r.pull("d$y")]
|
|
688
|
+
end
|
|
689
|
+
r.quit
|
|
690
|
+
@densities = densities
|
|
691
|
+
calculate_density_max_y ##need to re-do every time we get new densities
|
|
692
|
+
densities
|
|
693
|
+
end
|
|
694
|
+
|
|
695
|
+
|
|
696
|
+
public
|
|
697
|
+
#Draws the clustered bands that correspond to the expected and control window in a single PNG file +file+
|
|
698
|
+
#
|
|
699
|
+
#Options and defaults
|
|
700
|
+
#- <tt>:add_lines => nil</tt> -if an array of positions is provided eg +[100,345] , vertical lines will be drawn at these positions. Useful for indicating feature positions on the plot
|
|
701
|
+
#- <tt>:width => 1000</tt> -width of the PNG in pixels
|
|
702
|
+
#- <tt>:height => 500</tt> -height of the PNG in pixels
|
|
703
|
+
def draw_bands(file="myfile.png", optsa={})
|
|
704
|
+
opts = @opts[:graphics].merge(optsa)
|
|
705
|
+
pp optsa
|
|
706
|
+
raise RuntimeError, "Can't draw threads until clustering is done" unless @clusters
|
|
707
|
+
#uses R's standard plot functions.
|
|
708
|
+
##same as draw_threads, but skips threads that aren't on the bands lists
|
|
709
|
+
##
|
|
710
|
+
r = new_r
|
|
711
|
+
r.eval "png('#{file}', width=#{opts[:width]}, height=#{opts[:height]})"
|
|
712
|
+
plot_open = false
|
|
713
|
+
self.densities.each do |t|
|
|
714
|
+
if @control_band.include?(t[0]) or @expected_band.include?(t[0])
|
|
715
|
+
r.dx = t[1]
|
|
716
|
+
r.dy = t[2]
|
|
717
|
+
r.curr_win = t.last
|
|
718
|
+
#r.eval "d = density(curr_win,n=240,kernel=\"gaussian\", from=#{@snp_positions.first[0]}, to=#{@snp_positions.last[0]})"
|
|
719
|
+
if plot_open
|
|
720
|
+
r.eval "lines(dx, dy, col=\"#{@thread_colours[t.first]}\")"
|
|
721
|
+
else
|
|
722
|
+
r.eval "plot(dx, dy, type=\"l\", col=\"#{@thread_colours[t.first]}\",ylim=c(0,#{density_max_y}), main='#{file}',xlab='position', ylab='density')"
|
|
723
|
+
plot_open = true
|
|
724
|
+
end
|
|
725
|
+
end
|
|
726
|
+
end
|
|
727
|
+
label1 = "Control band: " + @control_band.min.to_s + " < ChD < " + @control_band.max.to_s
|
|
728
|
+
label2 = "Expected band: " + @expected_band.min.to_s + " < ChD < " + @expected_band.max.to_s
|
|
729
|
+
r.eval "legend('top', c('#{label1}','#{label2}'), lty=c(1,1),lwd=c(2.5,2.5),col=c('#{@thread_colours[@control_band.first]}','#{@thread_colours[@expected_band.first]}'))"
|
|
730
|
+
if opts[:add_lines] and opts[:add_lines].instance_of?(Array)
|
|
731
|
+
opts[:add_lines].each do |pos|
|
|
732
|
+
r.eval "abline(v=#{pos})"
|
|
733
|
+
end
|
|
734
|
+
end
|
|
735
|
+
r.eval "dev.off()"
|
|
736
|
+
r.quit
|
|
737
|
+
end
|
|
738
|
+
|
|
739
|
+
public
|
|
740
|
+
#Returns the array instance variable @clusters. The R function +kmeans()+ is used to calculate the clusters based on a correlation matrix of the density curves. If @clusters is nil when called this method will run the Bio::Util::Gngm#calculate_clusters method and set @clusters
|
|
741
|
+
#With this method you cannot recalculate the clusters after they have been done once.
|
|
742
|
+
#
|
|
743
|
+
#Options and defaults
|
|
744
|
+
#- <tt>:k => 9</tt>, -the number of clusters for the R +kmeans+ function
|
|
745
|
+
#- <tt>:seed => false</tt> -set this to a number to make the randomized clustering reproducible
|
|
746
|
+
#- <tt>:control_chd => 0.5</tt> -the value of the control thread/window
|
|
747
|
+
#- <tt>:expected_chd => 1.0</tt> -the value of the expected thread/window
|
|
748
|
+
#- <tt>:adjust => 1.0</tt> -the kernel adjustment parameter for the R +density+ function
|
|
749
|
+
def clusters(opts={})
|
|
750
|
+
@clusters ||= calculate_clusters(opts={})
|
|
751
|
+
end
|
|
752
|
+
|
|
753
|
+
public
|
|
754
|
+
#Calculates the k-means clusters of density curves (groups threads into bands), [density curve y values] ]</tt> Calculates the clusters using the R function +kmeans()+ Recalculates @densities as it does with Bio::Util::Gngm#calculate_densities, so clustering can be done without having to explicitly call Bio::Util::Gngm#calculate_densities.
|
|
755
|
+
#Clusters are recalulated every time regardless of whether its been done before contains anything or not so is useful for trying out different values for the parameters. When clusters are calculated the expected and control
|
|
756
|
+
#bands are compared with the Bio::Util::Gngm#calculate_signal method and the @signal array populated. Resets the instance variables @control_band, @expected_band, @signal, @peak_indices, @peak_y_values and @clusters
|
|
757
|
+
#
|
|
758
|
+
#Options and defaults
|
|
759
|
+
#- <tt>:k => 9</tt>, -the number of clusters for the R +kmeans+ function
|
|
760
|
+
#- <tt>:seed => false</tt> -set this to a number to make the randomized clustering reproducible
|
|
761
|
+
#- <tt>:control_chd => 0.5</tt> -the value of the control thread/window
|
|
762
|
+
#- <tt>:expected_chd => 1.0</tt> -the value of the expected thread/window
|
|
763
|
+
#- <tt>:adjust => 1.0</tt> -the kernel adjustment parameter for the R +density+ function
|
|
764
|
+
#- <tt>:pseudo => false</tt> - force the densities into a single thread cluster when the number of distinct threads with SNPs is < the value of k. This is only useful in a situation where the spread of the statistic is very limited. EG for using mapped/unmapped mate pairs then almost all windows will have proportion 1.0 but a tiny number will be close to 0.5 with few other values considered.
|
|
765
|
+
def calculate_clusters( opts={} )
|
|
766
|
+
options = {:k => 9, :seed => false, :adjust => 1, :control_chd => 0.5, :expected_chd => 1.0, :pseudo => false}
|
|
767
|
+
options = options.merge(opts)
|
|
768
|
+
if options[:pseudo]
|
|
769
|
+
put_threads_into_individual_clusters(options)
|
|
770
|
+
return
|
|
771
|
+
end
|
|
772
|
+
r = new_r
|
|
773
|
+
names = []
|
|
774
|
+
name = "a"
|
|
775
|
+
@control_band = nil #needs resetting as we are working with new clusters
|
|
776
|
+
@expected_band = nil #needs resetting as we are working with new clusters
|
|
777
|
+
@signal = nil #needs resetting as we are working with new clusters
|
|
778
|
+
@peak_indices = nil #needs resetting as we are working with new cluster
|
|
779
|
+
@peak_y_values = nil #needs resetting as we are working with new cluster
|
|
780
|
+
self.calculate_densities(options[:adjust]).each do |d|
|
|
781
|
+
density_array = d.last
|
|
782
|
+
r.assign name, density_array ##although windows go in in numeric order, r wont allow numbers as names in data frames so we need a proxy
|
|
783
|
+
names << "#{name}=#{name}"
|
|
784
|
+
name = name.next
|
|
785
|
+
end
|
|
786
|
+
data_frame_command = "data = data.frame(" + names.join(",") + ")"
|
|
787
|
+
r.eval data_frame_command
|
|
788
|
+
r.eval "set.seed(#{options[:seed]})" if options[:seed]
|
|
789
|
+
r.eval "k = kmeans(cor(data),#{options[:k]},nstart=1000)"
|
|
790
|
+
@clusters = r.pull "k$cluster" ##clusters are returned in the order in densities
|
|
791
|
+
r.quit
|
|
792
|
+
##now set the cluster colours..
|
|
793
|
+
colours = %w[#A6CEE3 #1F78B4 #B2DF8A #33A02C #FB9A99 #E31A1C #FDBF6F #FF7F00 #CAB2D6 #6A3D9A #FFFF99 #B15928]
|
|
794
|
+
ci = 0
|
|
795
|
+
col_nums = {} ##hash of cluster numbers and colours
|
|
796
|
+
@clusters.each_index do |i|
|
|
797
|
+
if not col_nums[@clusters[i]]
|
|
798
|
+
col_nums[@clusters[i]] = colours[ci]
|
|
799
|
+
ci += 1
|
|
800
|
+
ci = 0 if ci > 11
|
|
801
|
+
end
|
|
802
|
+
@thread_colours[self.densities[i].first] = col_nums[@clusters[i]]
|
|
803
|
+
end
|
|
804
|
+
@control_band = get_band(options[:control_chd])
|
|
805
|
+
@expected_band = get_band(options[:expected_chd])
|
|
806
|
+
calculate_signal
|
|
807
|
+
end
|
|
808
|
+
|
|
809
|
+
private
|
|
810
|
+
##gives each window/thread a seperate and arbitrary cluster, used when you suspect the statistic will not spread across all possible windows very well. Wont specifiy @control_band or @expected_band and therefore wont directly calculate the signal
|
|
811
|
+
def put_threads_into_individual_clusters(options)
|
|
812
|
+
@control_band = nil #needs resetting as we are working with new clusters
|
|
813
|
+
@expected_band = nil #needs resetting as we are working with new clusters
|
|
814
|
+
@signal = nil #needs resetting as we are working with new clusters
|
|
815
|
+
@peak_indices = nil #needs resetting as we are working with new cluster
|
|
816
|
+
@peak_y_values = nil #needs resetting as we are working with new cluster
|
|
817
|
+
self.calculate_densities(options[:adjust])
|
|
818
|
+
@clusters = Array.new (@densities.length) {|x| 1 + x}
|
|
819
|
+
##now set the cluster colours..
|
|
820
|
+
colours = %w[#A6CEE3 #1F78B4 #B2DF8A #33A02C #FB9A99 #E31A1C #FDBF6F #FF7F00 #CAB2D6 #6A3D9A #FFFF99 #B15928]
|
|
821
|
+
ci = 0
|
|
822
|
+
col_nums = {} ##hash of cluster numbers and colours
|
|
823
|
+
@clusters.each_index do |i|
|
|
824
|
+
if not col_nums[@clusters[i]]
|
|
825
|
+
col_nums[@clusters[i]] = colours[ci]
|
|
826
|
+
ci += 1
|
|
827
|
+
ci = 0 if ci > 11
|
|
828
|
+
end
|
|
829
|
+
@thread_colours[self.densities[i].first] = col_nums[@clusters[i]]
|
|
830
|
+
end
|
|
831
|
+
#@control_band = get_band(options[:control_chd])
|
|
832
|
+
#@expected_band = get_band(options[:expected_chd])
|
|
833
|
+
#calculate_signal
|
|
834
|
+
end
|
|
835
|
+
|
|
836
|
+
##returns an array of the names of the window threads in the control (heterophasic) band
|
|
837
|
+
#def control_band(control=0.5)
|
|
838
|
+
# puts "in control band with control = #{control}"
|
|
839
|
+
# @control_band ||= get_band(control)
|
|
840
|
+
#end
|
|
841
|
+
|
|
842
|
+
##returns an array of the names of the window threads in the expected (homophasic) band
|
|
843
|
+
#def expected_band(expected=1.0)
|
|
844
|
+
# @expected_band ||= get_band(expected)
|
|
845
|
+
#end
|
|
846
|
+
|
|
847
|
+
##gets an array of windows that cluster with a given window
|
|
848
|
+
public
|
|
849
|
+
def get_band(window=1.0)
|
|
850
|
+
##because of the weird step rounding error we need to find the internal name of the window.. so find it from the list from the name the user
|
|
851
|
+
##expects it to be, may give more than one passing window so keep only first one..
|
|
852
|
+
windows = find_window(window)
|
|
853
|
+
raise RuntimeError, "Couldnt find window #{window}, or window has no data to calculate: \n windows are #{self.densities.collect {|d| d.first} }" if windows.empty? ##if we have a window that is close enough to the specified window
|
|
854
|
+
idx = find_index(windows.first)
|
|
855
|
+
#find out which cluster the window is in
|
|
856
|
+
cluster = self.clusters[idx]
|
|
857
|
+
##get the other windows in the same cluster, ie the band...
|
|
858
|
+
band = []
|
|
859
|
+
self.clusters.each_index do |i|
|
|
860
|
+
if self.clusters[i] == cluster
|
|
861
|
+
band << self.densities[i].first
|
|
862
|
+
end
|
|
863
|
+
end
|
|
864
|
+
band
|
|
865
|
+
end
|
|
866
|
+
|
|
867
|
+
public
|
|
868
|
+
#Draws the contents of the @signal instance variable in a single PNG file +file+
|
|
869
|
+
def draw_signal(file="myfile.png", opts=@opts[:graphics]) #data.frame(bubs=data$bubbles_found,conf=data$bubbles_confirmed)
|
|
870
|
+
r = new_r
|
|
871
|
+
x_vals = self.densities[0][1]
|
|
872
|
+
r.eval "png('#{file}', width=#{opts[:width]}, height=#{opts[:height]})"
|
|
873
|
+
r.x_vals = x_vals
|
|
874
|
+
r.signal = self.signal
|
|
875
|
+
r.eval "plot(x_vals,signal, type=\"l\", xlab='position', ylab='ratio of signals (expected / control ~ homo / hetero)', main='#{file}' )"
|
|
876
|
+
r.eval "dev.off()"
|
|
877
|
+
end
|
|
878
|
+
|
|
879
|
+
private
|
|
880
|
+
def print_signal
|
|
881
|
+
end
|
|
882
|
+
|
|
883
|
+
public
|
|
884
|
+
#Returns the positions of the peaks in the signal curve calculated by Bio::Util::Gngm#get_peaks as an array
|
|
885
|
+
def peaks
|
|
886
|
+
@peak_indices.collect {|x| self.densities[0][1][x].to_f.floor}
|
|
887
|
+
end
|
|
888
|
+
|
|
889
|
+
public
|
|
890
|
+
#Draws the peaks calculated from the signal curve by the R function +Peaks+ in Bio::Util::Gngm#calculate_peaks. Adds boxes of width +:range+ to each peak and annotates the limits. Options are set in the global options hash +:peaks+.
|
|
891
|
+
#and relate to the Peaks function in R
|
|
892
|
+
def draw_peaks(file="myfile.png",opts=@opts[:graphics])
|
|
893
|
+
opts_a = @opts[:peaks]
|
|
894
|
+
opts_a.merge!(opts)
|
|
895
|
+
opts = opts_a ##sigh ...
|
|
896
|
+
#opts[:background] = opts[:background].to_s.upcase
|
|
897
|
+
#opts[:markov] = opts[:markov].to_s.upcase
|
|
898
|
+
self.get_peaks(opts)
|
|
899
|
+
r = new_r
|
|
900
|
+
#r.eval "suppressMessages ( library('Peaks') )"
|
|
901
|
+
r.signal = self.signal
|
|
902
|
+
r.x_vals = self.densities[0][1]
|
|
903
|
+
r.eval "png('#{file}', width=#{opts[:width]}, height=#{opts[:height]})"
|
|
904
|
+
#r.eval "spec = SpectrumSearch(signal,#{opts[:sigma]},threshold=#{opts[:threshold]},background=#{opts[:background]},iterations=#{opts[:iterations]},markov=#{opts[:markov]},window=#{opts[:window]})"
|
|
905
|
+
#peak_positions = r.pull "spec$pos"
|
|
906
|
+
#y = r.pull "spec$y"
|
|
907
|
+
r.y = @peak_y_values
|
|
908
|
+
r.pos = @peak_indices
|
|
909
|
+
r.eval "plot(x_vals,y, type=\"l\", xlab='position', ylab='Peaks', main='#{file}' )"
|
|
910
|
+
@peak_indices.each do |peak|
|
|
911
|
+
r.eval "rect(x_vals[#{peak}]-(#{opts[:range]/2}), 0, x_vals[#{peak}]+#{opts[:range]/2}, max(y), col=rgb(r=0,g=1,b=0, alpha=0.3) )"
|
|
912
|
+
r.eval "text(x_vals[#{peak}]-(#{opts[:range]/2}),max(y) + 0.05, floor(x_vals[#{peak}]-(#{opts[:range]/2})) )"
|
|
913
|
+
r.eval "text(x_vals[#{peak}]+(#{opts[:range]/2}), max(y) + 0.05, floor(x_vals[#{peak}]+(#{opts[:range]/2})) )"
|
|
914
|
+
end
|
|
915
|
+
r.eval "dev.off()"
|
|
916
|
+
r.quit
|
|
917
|
+
end
|
|
918
|
+
|
|
919
|
+
private
|
|
920
|
+
#Calculates the position of peaks in the signal curve
|
|
921
|
+
def get_peaks(opts=@opts[:peaks])
|
|
922
|
+
opts[:background] = opts[:background].to_s.upcase
|
|
923
|
+
opts[:markov] = opts[:markov].to_s.upcase
|
|
924
|
+
r = new_r
|
|
925
|
+
r.eval "suppressMessages ( library('Peaks') )"
|
|
926
|
+
r.signal = self.signal
|
|
927
|
+
r.x_vals = self.densities[0][1]
|
|
928
|
+
r.eval "spec = SpectrumSearch(signal,#{opts[:sigma]},threshold=#{opts[:threshold]},background=#{opts[:background]},iterations=#{opts[:iterations]},markov=#{opts[:markov]},window=#{opts[:window]})"
|
|
929
|
+
@peak_indices = r.pull "spec$pos"
|
|
930
|
+
if @peak_indices.instance_of?(Fixnum)
|
|
931
|
+
@peak_indices = [@peak_indices]
|
|
932
|
+
end
|
|
933
|
+
@peak_y_values = r.pull "spec$y"
|
|
934
|
+
r.quit
|
|
935
|
+
end
|
|
936
|
+
|
|
937
|
+
public
|
|
938
|
+
#Returns an array of polymorphisms in each thread/window <tt>[[window, polymorphism count] ]. Useful for sparse polymorphism counts or over small regions where small polymorphism counts can cause artificially large peaks in density curves.
|
|
939
|
+
def hit_count
|
|
940
|
+
arr = []
|
|
941
|
+
self.threads.each do |thread|
|
|
942
|
+
arr << [thread.first, thread.last.length]
|
|
943
|
+
end
|
|
944
|
+
arr
|
|
945
|
+
end
|
|
946
|
+
|
|
947
|
+
public
|
|
948
|
+
#Draws a barplot of the number of polymorphisms in each thread/window in a single PNG file +file+
|
|
949
|
+
def draw_hit_count(file="myfile.png",opts=@opts[:graphics])
|
|
950
|
+
r = new_r
|
|
951
|
+
wins = []
|
|
952
|
+
hits = []
|
|
953
|
+
self.threads.each do |thread|
|
|
954
|
+
wins << thread.first
|
|
955
|
+
if thread.last.empty?
|
|
956
|
+
hits << 0.01 ##pseudovalue gets around the case where a thread has no hits... which messes up barplot in R
|
|
957
|
+
else
|
|
958
|
+
hits << thread.last.length
|
|
959
|
+
end
|
|
960
|
+
end
|
|
961
|
+
r.wins = wins
|
|
962
|
+
r.hits = hits
|
|
963
|
+
r.eval "png('#{file}', width=#{opts[:width]}, height=#{opts[:height]})"
|
|
964
|
+
r.eval "barplot(hits, names.arg=c(wins), xlab='window', log='y', ylab='number of hits', main='Number of Polymorphisms #{file}', col=rgb(r=0,g=1,b=1, alpha=0.3), na.rm = TRUE)"
|
|
965
|
+
r.eval "dev.off()"
|
|
966
|
+
end
|
|
967
|
+
|
|
968
|
+
public
|
|
969
|
+
#Returns an array of values representing the ratio of average of the expected threads/windows to the control threads/windows. Sets @signal, the signal curve.
|
|
970
|
+
def calculate_signal
|
|
971
|
+
r = new_r
|
|
972
|
+
name = "a"
|
|
973
|
+
control_names = []
|
|
974
|
+
expected_names = []
|
|
975
|
+
self.densities.each do |d|
|
|
976
|
+
if @control_band.include?(d.first)
|
|
977
|
+
density_array = d.last
|
|
978
|
+
r.assign name, density_array ##although windows go in in numeric order, r wont allow numbers as names in data frames so we need a proxy
|
|
979
|
+
control_names << "#{name}=#{name}"
|
|
980
|
+
elsif @expected_band.include?(d.first)
|
|
981
|
+
density_array = d.last
|
|
982
|
+
r.assign name, density_array
|
|
983
|
+
expected_names << "#{name}=#{name}"
|
|
984
|
+
end
|
|
985
|
+
name = name.next
|
|
986
|
+
end
|
|
987
|
+
data_frame_command = "control = data.frame(" + control_names.join(",") + ")"
|
|
988
|
+
r.eval data_frame_command
|
|
989
|
+
r.eval "control_mean = apply(control, 1, function(ecks) mean((as.numeric(ecks))) )"
|
|
990
|
+
data_frame_command = "expected = data.frame(" + expected_names.join(",") + ")"
|
|
991
|
+
r.eval data_frame_command
|
|
992
|
+
r.eval "expected_mean = apply(expected, 1, function(ecks) mean((as.numeric(ecks))) )"
|
|
993
|
+
r.eval "signal = expected_mean / control_mean"
|
|
994
|
+
signal = r.pull "signal"
|
|
995
|
+
r.quit
|
|
996
|
+
@signal = signal
|
|
997
|
+
end
|
|
998
|
+
|
|
999
|
+
public
|
|
1000
|
+
def signal
|
|
1001
|
+
@signal ||= calculate_signal
|
|
1002
|
+
end
|
|
1003
|
+
|
|
1004
|
+
|
|
1005
|
+
|
|
1006
|
+
##finds the index of a window in the densties array
|
|
1007
|
+
private
|
|
1008
|
+
def find_index(window)
|
|
1009
|
+
self.densities.index {|x| x.first == window}
|
|
1010
|
+
end
|
|
1011
|
+
|
|
1012
|
+
private
|
|
1013
|
+
#finds the windows internal name, taking into account the Ruby rounding error
|
|
1014
|
+
def find_window(number)
|
|
1015
|
+
self.densities.collect {|d| d.first if d.first == number or (d.first >= number - ERROR_MARGIN and d.first <= number + ERROR_MARGIN) }.compact
|
|
1016
|
+
end
|
|
1017
|
+
|
|
1018
|
+
private
|
|
1019
|
+
#Returns a new rinruby object
|
|
1020
|
+
def new_r
|
|
1021
|
+
r = RinRuby.new(echo = false, interactive = false)
|
|
1022
|
+
r.eval "options(warn=-1)"
|
|
1023
|
+
return r
|
|
1024
|
+
end
|
|
1025
|
+
|
|
1026
|
+
|
|
1027
|
+
end
|
|
1028
|
+
end
|
|
1029
|
+
end
|