Table 2.

Biological phenomena that affect the detection of copy number gains using panel NGS data

Biological phenomena	Why relevant?	How does it affect detection of copy number gains?
Neoplastic cell content	Measurements are obtained from a mixture of tumor-derived and non-neoplastic gDNA	(i) The actual detected increase in coverage/deviation in BAF increases with neoplastic cell content (ii) Influences the estimation of the allele copy number (iii) Determines assay sensitivity (in combination with thresholds used to identify statistically significant gains)
Allele copy number/magnitude of amplification	Clinical consequences are based on cutoffs in allele copy number of gene amplification	(i) The detected increase in coverage/deviation in BAF increases with allele copy number (ii) Assay sensitivity should match the clinically relevant cutoffs in alllele copy number
Aneuploidy	Can affect normalization and allele copy number estimation	(i) Results in underestimation or overestimation depending on the nature and extend of aneuploidy and the number of genomic regions that are included in the gene panel (ii) High-level copy number gains are likely less affected compared to low copy number alterations