Plots and graphics

The scatter and heatmap plots can be used in two ways:

  1. Open the plot in an interactive window with zoom and other features. This is also compatible with Jupyter/IPython notebooks to render the plots inline.

  2. Generate a static image plot with the --output/-o option.

    • While PDF is a good choice to generate publication-quality figures that can be easily edited in Inkscape or Adobe Illustrator, other formats will work, indicated by the output filename extension – e.g. “-o myplot.png” to create PNG, or “-o myplot.svg” to create SVG.

(The diagram command can only generate a PDF file.)

As with any CNVkit command, the -h option will show the complete list of options available:

cnvkit.py scatter -h
cnvkit.py diagram -h
cnvkit.py heatmap -h

scatter

Plot bin-level log2 coverages and segmentation calls together. Without any further arguments, this plots the genome-wide copy number in a form familiar to those who have used array CGH.

cnvkit.py scatter Sample.cnr -s Sample.cns
# Shell shorthand
cnvkit.py scatter -s TR_95_T.cn{s,r}
_images/TR_95_T-scatter.png

The options --chromosome and --gene (or their single-letter equivalents) focus the plot on the specified region:

cnvkit.py scatter -s Sample.cn{s,r} -c chr7
cnvkit.py scatter -s Sample.cn{s,r} -c chr7:140434347-140624540
cnvkit.py scatter -s Sample.cn{s,r} -g BRAF

In the latter two cases, the genes in the specified region or with the specified names will be highlighted and labeled in the plot. The arguments -c and -g can be combined to e.g. highlight specific genes in a wider context:

# Show a chromosome arm, highlight one gene
cnvkit.py scatter -s Sample.cn{s,r} -c chr5:100-50000000 -g TERT
# Show the whole chromosome, highlight two genes
cnvkit.py scatter -s Sample.cn{s,r} -c chr7 -g BRAF,MET
# Highlight two genes in a specified range
cnvkit.py scatter -s TR_95_T.cn{s,r} -c chr12:50000000-80000000 -g CDK4,MDM2
_images/TR_95_T-CDK4-MDM2-scatter.png

When a chromosomal region is plotted with CNVkit’s “scatter” command , the size of the plotted datapoints is proportional to the weight of each point used in segmentation – a relatively small point indicates a less reliable bin. Therefore, if you see a cluster of smaller points in a short segment (or where you think there ought to be a segment, but there isn’t one), then you can cast some doubt on the copy number call in that region. The dispersion of points around the segmentation line also visually indicates the level of noise or uncertainty.

The bin-level log2 ratios or coverages can also be plotted without segmentation calls:

cnvkit.py scatter Sample.cnr

This can be useful for viewing the raw, un-corrected coverage depths when deciding which samples to use to build a profile, or simply to see the coverages without being helped/biased by the called segments.

The --trend option (-t) adds a smoothed trendline to the plot. This can be helpful if the segmentation is not available, or if you’re skeptical of the segmentation in a region.

Selection and highlighting

Chromosome-level views are controlled with the --chromosome/-c and --gene/-g options:

  • A gene name (e.g. -g TERT) or multiple gene names separated by commas (e.g. -g CDK4,MDM2) will plot the genomic around that gene, or genes, and highlight the gene or genes with a vertical gold stripe.

    • If multiple genes, they must all be on the same chromosome.
    • The --width/-w argument determines the size of the plotted genomic region, in terms of basepairs flanking the selected region.
    • Any other genes in the plotted region will not be shown unless also specified with -g.
  • A chromosome name alone (e.g. -c chr5) plots the whole chromosome. (No genes are highlighted.)

  • A region label with chromosome name and 1-based start and end coordinates (e.g. -c chr5:1000000-4000000) plots the specified region, with the start and end coordinates as the x-axis limits. All genes in this region (that are labeled in the input .cnr file) are highlighted and labeled.

    • If the start or end coordinate is left off (e.g. -c chr5:-4000000 or -c chr7:140000000-), the region is extended to the end of the chromosome in the direction of the open coordinate, i.e. it does what you’d think. If both are left off but - remains (e.g. -c chrY:-), the whole chromosome is shown, with all genes highlighted.
    • If -c is used, -w is ignored – only the specified genomic region will be shown, with no padding.
    • The -g option overrides the default behavior of showing all genes in the selection – only the genes specified with -g will be highlighted and labeled. To not show any genes, specify an empty string: -g ''
    • Special behavior occurs if there are no genes in the selected region: Instead, the selection itself is treated as a “gene”, highlighted and labeled with the string “Selection”, with padding controlled by -w. This behavior can be blocked by specifying an empty list of genes: -g '' – then the specified region will be plotted as usual, with nothing highlighted and no padding.

To create multiple region-specific plots at once, the regions of interest can be listed in a separate file and passed to the scatter command with the -l/--range-list option. This is equivalent to creating the plots separately with the -c option and then combining the plots into a single multi-page PDF.

Note

Only targeted genes can be highlighted and labeled; genes that are not included in the list of targets are not labeled in the .cnn or .cnr files and are therefore invisible to CNVkit.

SNV b-allele frequencies

The allelic frequencies of heterozygous SNPs can be viewed alongside copy number by passing variants as a VCF file with the -v option. These allele frequences are rendered in a subplot below the CNV scatter plot.

cnvkit.py scatter Sample.cnr -s Sample.cns -v Sample.vcf

If only the VCF file is given by itself, just the allelic frequencies are plotted:

cnvkit.py scatter -v Sample.vcf

When given segments, the plot will show the mean b-allele frequency values above and below 0.5 of SNVs falling within each segment. Divergence from 0.5 indicates loss of heterozygosity (LOH) or allelic imbalance in the tumor sample.

cnvkit.py scatter -s Sample.cns -v Sample.vcf -i TumorID -n NormalID

Given a VCF with only the tumor sample called, it is difficult to focus on just the informative SNPs because it’s not known which SNVs are present and heterozygous in normal, germline cells. Better results can be had by giving CNVkit more information:

  • Call somatic mutations using paired tumor and normal samples. In the VCF, the somatic variants should be flagged in the INFO column with the string “SOMATIC”. (MuTect does this automatically.) Then CNVkit will skip these for plotting.
  • Add a “PEDIGREE” tag to the VCF header, listing the tumor sample as “Derived” and the normal as “Original”. (MuTect doesn’t do this, but it does add a nonstandard GATK header that CNVkit can extract the same information from.)
  • In lieu of a PEDIGREE tag, tell CNVkit which sample IDs are the tumor and normal using the -i and -n options, respectively.
  • If no paired normal sample is available, you can still filter for likely informative SNPs by intersecting your tumor VCF with a set of known SNPs such as 1000 Genomes, ESP6500, or ExAC. Drop the private SNVs that don’t appear in these databases to create a VCF more amenable to LOH detection.

diagram

Draw copy number (either individual bins (.cnn, .cnr) or segments (.cns)) on chromosomes as an ideogram. If both the bin-level log2 ratios and segmentation calls are given, show them side-by-side on each chromosome (segments on the left side, bins on the right side).

cnvkit.py diagram Sample.cnr
cnvkit.py diagram -s Sample.cns
cnvkit.py diagram -s Sample.cns Sample.cnr

If bin-level log2 ratios are provided (.cnr), genes with log2 ratio values beyond a fixed threshold will be labeled on the plot. This plot style works best with target panels of a few hundred genes at most; with whole-exome sequencing there are often so many genes affected by CNAs that the individual gene labels become difficult to read.

_images/TR_95_T-diagram.png

By default, the sex chromosomes X and Y are colorized relative to the expected ploidy, i.e. for female samples analyzed with a male reference, while the X chromosome has a copy ratio near +1.0 in the input .cnr and .cns files, in the output diagram it will be shown as neutral copy number (white or faint colors) rather than a gain (red), because the diploid X is expected. The sample sex can be specified with the -x/--sample-sex option, or will otherwise be guessed automatically (see Chromosomal sex). This correction is done by default, but can be disabled with the option --no-shift-xy.

heatmap

Draw copy number (either bins (.cnn, .cnr) or segments (.cns)) for multiple samples as a heatmap.

To get an overview of the larger-scale CNVs in a cohort, use the “heatmap” command on all .cns files:

cnvkit.py heatmap *.cns
_images/heatmap-tr-nod.png

The color range can be subtly rescaled with the -d option to de-emphasize low-amplitude segments, which are likely spurious CNAs:

cnvkit.py heatmap *.cns -d
_images/heatmap-tr.png

A heatmap can also be drawn from bin-level log2 coverages or copy ratios (.cnn, .cnr), but this can be slow to render at the genome-wide level. Consider doing this with a smaller number of samples and only for one chromosome or chromosomal region at a time, using the -c option:

cnvkit.py heatmap TR_9*T.cnr -c chr12
cnvkit.py heatmap TR_9*T.cnr -c chr7:125000000-145000000
_images/heatmap-tr-chr12.png

If an output file name is not specified with the -o option, an interactive matplotlib window will open, allowing you to select smaller regions, zoom in, and save the image as a PDF or PNG file.

The samples are shown in the order there’s given on the command line. If you use “*.cns” then the filenames might always be fetched alphabetically (depending on your operating system), but if you type them out in the order you like, it should keep that order. You can use the Unix shell to pull the names out of a file on the fly, e.g.:

cnvkit.py heatmap `cat filenames.txt`

As with diagram, the sex chromosomes X and Y are colorized relative to the expected ploidy, based on the sample and reference sex (see Chromosomal sex). This correction can be disabled with the option --no-shift-xy.

Customizing plots

The plots generated with the scatter and heatmap commands use the Python plotting library matplotlib.

To quickly adjust the displayed area of the genome in a plot, run either plotting command without the -o option to generate an interactive plot in a new window. You can then resize that plot up to the full size of your screen, use the plot window’s selection mode to select a smaller area of the genome, and use the plot window’s save button to save the plot in your preferred format.

You can customize font sizes and other aspects of the plots by configuring matplotlib. If you’re running CNVkit on the command line and not using it as a Python library, then you can just create a file in your home directory (or the same directory as cnvkit.py) called .matplotlibrc. For example, to shrink the font size of the x- and y-axis labels, put this line in the configuration file:

axes.labelsize      : small

For more control, in the Python intepreter (or a script, or a Jupyter notebook), import the cnvlib package module and call the do_scatter or do_heatmap function to create a plot. Then you can use matplotlib.pyplot to get the current axis and modify the plot elements, change font sizes, or anything else you like:

from glob import glob
from matplotlib import pyplot as plt
import cnvlib

segments = map(cnvlib.read, glob("*.cns"))
ax = cnvlib.do_heatmap(segments)
ax.set_title("All my samples")
plt.rcParams["font.size"] = 9.0
plt.show()