# Tumor analysis¶

CNVkit has been used most extensively on solid tumor samples sequenced with a target panel or whole-exome sequencing protocol. Several options and approaches are available to support this use case:

• If you have unpaired tumor samples, or no normal samples sequenced on the same platform, see the reference command for strategies.

• Use --drop-low-coverage to ignore bins with log2 normalized coverage values below -15. Virtually all tumor samples, even cancer cell lines, are not completely homogeneous. Even in regions of homozygous deletion in the largest tumor-cell clonal population, some sequencing reads will be obtained from contaminating normal cells without the deletion. Therefore, extremely low log2 copy ratio values do not indicate homozygous deletions but failed sequencing or mapping in all cells regardless of copy number status at that site, which are not informative for copy number. This option in the batch command applies to segmentation; the option is also available in the segment, metrics, segmetrics, gainloss and Tumor heterogeneity commands.

• Why -15? The null log2 value substituted for bins with zero coverage is -20 (about 1 millionth the average bin’s coverage), and the maximum positive shift that can be introduced by normalizing to the reference is 5 (for bins with 1/32 the average coverage; bins below this are masked out by the reference). In a .cnr file, any bins with log2 value below -15 are probably based on dummy values corresponding to zero-coverage (perhaps unmappable) bins, and not real observations.
• The batch command does not directly output integer copy number calls (see Tumor heterogeneity). Instead, use the --ploidy and --purity options in call to calculate copy number for each sample individually using known or estimated tumor-cell fractions. Also consider using --center median in highly aneuploid samples to shift the log2 value of true neutral regions closer to zero, as it may be slightly off initially.

• If SNV calls are available in VCF format, use the -v/--vcf option in the call and scatter commands to calculate or plot b-allele frequencies alongside each segment’s total copy number or log2 ratio. These values reveal allelic imbalance and loss of heterozygosity (LOH), supporting and extending the inferred CNVs.