October 2020—An increase in the number of copies of a gene, or amplification, is regarded as the most common gain-of-function alteration across various cancer types. The authors developed a bioinformatics tool (Amplicon Architect) to identify extrachromosomal oncogene (ecDNA) amplification from whole genome sequencing (WGS) data based on three characteristic features: circularity of ecDNA, absence of a centromere, and high levels of amplification. The tool was validated in 44 cancer-derived cell lines known to have ecDNA. A combination of centromeric and noncentromeric FISH probes was used to identify extrachromosomal DNA, and the tool was able to classify 83 percent of these signals as representing circular ecDNA amplicons. Interestingly, some of these cases revealed the presence of concurrent extrachromosomal and intrachromosomal signals, suggesting that some ecDNA had reintegrated into the genome.