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<h3>vcfR documentation</h3>
by
<br>
Brian J. Knaus and Niklaus J. Grünwald
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<div id="header">
<h1 class="title toc-ignore">Creating DNAbin objects</h1>
</div>
<p>Some applications require the conversion of variant data into
sequence data. We’ve provided a conversion from vcfR to a DNAbin object
which was created by the R package ape. The DNAbin object can be output
to the FASTA format, as well as several others, for conversion to
popular file formats recognized by other software. A number of decisions
will need to be made in order to facilitate the conversion from vcfR to
DNAbin. Here we attempt to address these decisions and convert our vcfR
object to a DNAbin object.</p>
<div id="data-import" class="section level2">
<h2>Data import</h2>
<p>We will use the example dataset provided by vcfR.</p>
<pre class="r"><code>library(vcfR)
data(vcfR_example)</code></pre>
</div>
<div id="creating-dnabin-objects" class="section level2">
<h2>Creating DNAbin objects</h2>
<p>The package <strong>ape</strong> handles sequence data using objects
of class <strong>DNAbin</strong>. The VCF file only contains information
on variant positions. Omitting invariant data provides for a more
efficient representation of the data than including the invariant sites.
Converting VCF data to sequence data presents a challenge in that these
invariant sites may need to be included. This means that these objects
can easily occupy large amounts of memory, and may exceed the physical
memory when long sequences with many samples are included. In order to
accomodate these issues, we’ve taken an approach which attempts to
create DNAbin objects from portions of a chomosome, such as a gene. This
means we’ll need a little more information than we’ve needed for other
conversions. First, we’ll need to locate and read in our VCF file, a
reference sequence and a gff file that has the coordinates for a
gene.</p>
<pre class="r"><code># Find the files.
vcf_file <- system.file("extdata", "pinf_sc50.vcf.gz", package = "pinfsc50")
dna_file <- system.file("extdata", "pinf_sc50.fasta", package = "pinfsc50")
gff_file <- system.file("extdata", "pinf_sc50.gff", package = "pinfsc50")
# Read in data.
vcf <- read.vcfR(vcf_file, verbose = FALSE)
dna <- ape::read.dna(dna_file, format="fasta")
gff <- read.table(gff_file, sep="\t", quote = "")</code></pre>
<p>We can use information from the annotation file (gff) to extract a
gene. Here we have specifically chosen one which has variants. We can
use IUPAC ambiguity codes to convert heterozygous sites into a one
character encoding. This results in a single sequence per individual.
Alternatively, we can create two haplotypes for each diploid sample,
resulting in two sequences per individual.</p>
<pre class="r"><code>record <- 130
# my_dnabin1 <- vcfR2DNAbin(vcf, consensus = TRUE, extract.haps = FALSE,
# gt.split='|', ref.seq=dna[,gff[record,4]:gff[record,5]],
# start.pos=gff[record,4], verbose=FALSE)
my_dnabin1 <- vcfR2DNAbin(vcf, consensus = TRUE, extract.haps = FALSE, ref.seq = dna[,
gff[record, 4]:gff[record, 5]], start.pos = gff[record, 4], verbose = FALSE)
my_dnabin1</code></pre>
<pre><code>## 18 DNA sequences in binary format stored in a matrix.
##
## All sequences of same length: 1043
##
## Labels:
## BL2009P4_us23
## DDR7602
## IN2009T1_us22
## LBUS5
## NL07434
## P10127
## ...
##
## Base composition:
## a c g t
## 0.238 0.295 0.272 0.194
## (Total: 18.77 kb)</code></pre>
<p>We can visualize the variable sites using tools from the package
‘ape.’</p>
<pre class="r"><code>par(mar=c(5,8,4,2))
ape::image.DNAbin(my_dnabin1[,ape::seg.sites(my_dnabin1)])</code></pre>
<p><img src="dnabin_files/figure-html/image_DNAbin1-1.png" width="672" style="display: block; margin: auto;" /></p>
<pre class="r"><code>par(mar=c(5,4,4,2))</code></pre>
<p>Here, the ambiguous sites are visualized as ‘other.’ While the DNAbin
object can include the ambiguity codes, not all downstream software
handle these codes well. So the user should excercise prudence when
using this option.</p>
<p>If we instead create two haplotypes for each diploid sample, it
results in a DNAbin object which includes only unambiguous
nucleotides(A, C, G and T). This typically requires the data to be
phased (I use <a
href="https://faculty.washington.edu/browning/beagle/beagle.html">beagle4</a>).
In VCF files this is indicated by delimiting the alleles of the genotype
with a pipe (‘|’) for phased data, while unphased data are delimited
with a forward slash (‘/’).</p>
<pre class="r"><code># my_dnabin1 <- vcfR2DNAbin(vcf, consensus=FALSE, extract.haps=TRUE,
# gt.split='|', ref.seq=dna[,gff[record,4]:gff[record,5]],
# start.pos=gff[record,4], verbose=FALSE)
my_dnabin1 <- vcfR2DNAbin(vcf, consensus = FALSE, extract.haps = TRUE, ref.seq = dna[,
gff[record, 4]:gff[record, 5]], start.pos = gff[record, 4], verbose = FALSE)</code></pre>
<pre class="r"><code>par(mar=c(5,8,4,2))
ape::image.DNAbin(my_dnabin1[,ape::seg.sites(my_dnabin1)])</code></pre>
<p><img src="dnabin_files/figure-html/image_DNAbin_2-1.png" width="672" style="display: block; margin: auto;" /></p>
<pre class="r"><code>par(mar=c(5,4,4,2))</code></pre>
<p>Once we have a DNAbin object, it can be analysed in a number of R
packages, such as ape and pegas. We can also output a fasta file for
other softwares to use.</p>
<pre class="r"><code>write.dna( my_dnabin1, file = 'my_gene.fasta', format = 'fasta' )
unlink('my_gene.fasta') # Clean up after we're done with the example.</code></pre>
<p>Also see:</p>
<ul>
<li>Heng Li’s <a href="https://github.com/lh3/seqtk">seqtk</a></li>
<li><a href="https://www.broadinstitute.org/gatk/">GATK’s</a>
FastaAlternateReferenceMaker</li>
</ul>
</div>
<center>
<hr class="style1">
<p>Copyright © 2017, 2018 Brian J. Knaus. All rights reserved.</p>
<p>USDA Agricultural Research Service, Horticultural Crops Research Lab.</p>
</center>
</div>
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// add bootstrap table styles to pandoc tables
function bootstrapStylePandocTables() {
$('tr.odd').parent('tbody').parent('table').addClass('table table-condensed');
}
$(document).ready(function () {
bootstrapStylePandocTables();
});
</script>
<!-- tabsets -->
<script>
$(document).ready(function () {
window.buildTabsets("TOC");
});
$(document).ready(function () {
$('.tabset-dropdown > .nav-tabs > li').click(function () {
$(this).parent().toggleClass('nav-tabs-open');
});
});
</script>
<!-- code folding -->
<!-- dynamically load mathjax for compatibility with self-contained -->
<script>
(function () {
var script = document.createElement("script");
script.type = "text/javascript";
script.src = "https://mathjax.rstudio.com/latest/MathJax.js?config=TeX-AMS-MML_HTMLorMML";
document.getElementsByTagName("head")[0].appendChild(script);
})();
</script>
</body>
</html>