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
listing the read counts within each copy-number segment in a pair of tumor and
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
# 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¶
CNVkit’s call command converts the segmented log2 ratio estimates to absolute integer copy numbers.
clonal method in this command uses an estimate of tumor fraction (from
any source) to directly rescale segment log2 ratio values and round them to the
nearest integer copy number. Example with tumor purity of 60% and a male
cnvkit.py call -m clonal Sample.cns --purity 0.6 -y -o Sample.call-clonal.cns
Alternatively, if the tumor cell fraction is not known, hard thresholds can be used instead:
cnvkit.py call -m threshold Sample.cns -y -o Sample.call-threshold.cns