Table 1.

Technical considerations for detection of copy number gains (gene amplifications) using panel NGS data

Technical issue	Why relevant?	Considerations
Panel content	Panel size and selection of genomic loci can affect detection of copy number gains	(i) Contains amplicons/probes sufficiently spread throughout the genome (ii) Includes loci likely to not be affected by copy number variation in tumor of interest (iii) Minimal number of amplicons/probes per gene, preferably throughout gene locus (iv) For BAF, include sufficient number of heterogeneous loci for sufficient “SNP-density”
Normalization	Required to correct for differences in gDNA input quality/quantity	Choose method that is not/minimally affected by copy number variation
Reference pool	Is required to detect coverage outliers indicative of copy number gains	(i) Internal and/or external reference pool (ii) Includes samples without copy number variation (e.g., normal tissue) (iii) Processed using identical protocols
Thresholds	Required to distinguish genuine copy number gains from technical noise	(i) Validated by positive/negative controls using other methods (ii) Includes minimal coverage thresholds to prevent false positive calls from poor quality gDNA (iii) Include positive and negative controls on a regular basis, to ensure assay stability and test validated thresholds
Sensitivity	Awareness of assay limitations is critical for routine diagnostics	(i) Affected by thresholds and neoplastic cell percentage (ii) Should be included in clinical report