Tumor heterogeneity

DNA samples extracted from solid tumors are rarely completely pure. Stromal or other normal cells and distinct subclonal tumor-cell populations are typically present in a sample, and can confound attempts to fit segmented log2 ratio values to absolute integer copy numbers.

CNVkit provides several points of integration with existing tools and methods for dealing with tumor heterogeneity and normal-cell contamination.

Inferring tumor purity and subclonal population fractions

The third-party program THetA2 can be used to estimate tumor cell content and infer integer copy number of tumor subclones in a sample. CNVkit provides wrappers for exporting segments to THetA2’s input format and importing THetA2’s result file as CNVkit’s segmented .cns files.

Using CNVkit with THetA2

THetA2’s input file is a BED-like file, typically with the extension .input, listing the read counts within each copy-number segment in a pair of tumor and normal samples. CNVkit can generate this file given the CNVkit-inferred tumor segmentation (.cns) and normal copy log2-ratios (.cnr) or copy number reference file (.cnn). This bypasses the initial step of THetA2, CreateExomeInput, which counts the reads in each sample’s BAM file.

After running the CNVkit Copy number calling pipeline on a sample, create the THetA2 input file:

# From a paired normal sample
cnvkit.py export theta Sample_Tumor.cns Sample_Normal.cnr -o Sample.theta2.input
# From an existing CNVkit reference
cnvkit.py export theta Sample_Tumor.cns reference.cnn -o Sample.theta2.input

Then, run THetA2 (assuming the program was unpacked at /path/to/theta2/):

# Generates Sample.theta2.BEST.results:
/path/to/theta2/bin/RunTHetA Sample.theta2.input
# Parameters for low-quality samples:
/path/to/theta2/python/RunTHetA.py Sample.theta2.input -n 2 -k 4 -m .90 --FORCE --NUM_PROCESSES `nproc`

Finally, import THetA2’s results back into CNVkit’s .cns format, matching the original segmentation (.cns) to the THetA2-inferred absolute copy number values.:

cnvkit.py import-theta Sample_Tumor.cns Sample.theta2.BEST.results

THetA2 adjusts the segment log2 values to the inferred cellularity of each detected subclone; this can result in one or two .cns files representing subclones if more than one clonal tumor cell population was detected. THetA2 also performs some significance testing of each segment representing a CNA, so there may be fewer segments derived from THetA2 than were originally found by CNVkit.

The segment values are still log2-transformed in the resulting .cns files, for convenience in plotting etc. with CNVkit. These files are also easily converted to other formats using the export command.

Adjusting copy ratios and segments for normal cell contamination

Alternatively, one can use an estimate of tumor fraction (from any source) to directly rescale segment log2 ratio values.

CNVkit has preliminary support for adjusting the copy number calls based on known tumor cell percentage and ploidy. This can be done in two different ways, currently.

Export integer copy numbers as BED

The freebayes export option emits integer copy number calls in a BED-like format that can be used with FreeBayes’s --cnv-map option. The --purity and --ploidy options work to rescale the segmented log2 ratio values under the assumption that some fraction of the sample’s cells have neutral copy number.

Example with tumor purity of 60% and a male reference:

cnvkit.py export freebayes Sample.cns --purity 0.6 -y -o Sample.cnvmap.bed

Copy-number-neutral regions are not shown in the output.

Rescale log2 ratios using cnvlib

To rescale the .cnr or .cns files as above, but without changing the file format, you can use a function in the Python library “cnvlib”, which implements the CNVkit command line options. In a Python script:

import cnvlib
from cnvlib.export import rescale_copy_ratios
my_array = cnvlib.read("MySample.cnr")
rescaled_array = rescale_copy_ratios(my_array, purity=0.6, is_reference_male=True)

Note that in this approach the output values are still log2-transformed, and are not rounded to integer copy number values. If rounding is needed, you can use the option round_to_integer (development version only):

rescaled_array = rescale_copy_ratios(my_array, purity=0.6, round_to_integer=True, is_reference_male=True)

This functionality is not directly available through the command line yet, but will be in a future release of CNVkit.