Calling copy number gains and lossesΒΆ

The relationship between the observed copy ratio and the true underlying copy number depends on tumor cell fraction (purity), genome ploidy (which may be heterogeneous in a tissue sample), and the size of the subclonal population containing the CNA. Because of these ambiguities, CNVkit initially reports only the estimated log2 copy ratio, and supports several approaches to deriving absolute integer copy number or evaluating copy number gains and losses.

In a diploid genome, a single-copy gain in a perfectly pure, homogeneous sample has a copy ratio of 3/2. In log2 scale, this is log2(3/2) = 0.585, and a single-copy loss is log2(1/2) = -1.0.

In the diagram plot, for the sake of providing a clean visualization of confidently called CNAs, the default threshold to label genes is 0.5. This threshold will tend to display gene amplifications, fully clonal single-copy gains in fairly pure samples, most single-copy losses, and total (homozygous) deletions.

When using the gainloss command, choose a threshold to suit your needs depending on your knowledge of the sample’s purity, heterogeneity, and likely features of interest. As a starting point, try 0.1 or 0.2 if you are going to do your own filtering downstream, or 0.3 if not.

To evaluate the level of support for each segment, use the segmetrics command. In particular, the –ci option estimates the confidence interval of the segment mean: the difference between the upper and lower limits suggests the reliability of the estimate, and the value of the upper or lower limit suggests the minimum loss or gain value, respectively, supported by the bin-level log2 ratios in that segment.

The call command implements two simple methods to convert the log2 ratios in a segmented .cns file to absolute integer copy number values. Given known or estimated tumor purity and ploidy values, this command can also adjust for tumor heterogeneity.

A .cns file can be converted to BED or VCF format using the export command. These output styles display the inferred absolute integer copy number value of each segment. To first adjust for known purity and ploidy of the sample, or apply log2 thresholds for integer calls, use call before exporting.