Design of a Targeted Sequencing Assay to Detect Rare Mutations in Circulating Tumor DNA

Background: Qualitative and quantitative detection of circulating tumor DNA (ctDNA) is a liquid biopsy technology used for early cancer diagnosis. However, the plasma ctDNA content is extremely low, so it is difficult to detect somatic mutations of tumors using conventional sequencing methods. Target region sequencing (TRS) technology, through enrichment of the target genomic region followed by next generation sequencing, overcomes this challenge and has been widely used in ctDNA sequencing. Methods: We designed a ctDNA sequencing panel to capture 128 tumor genes, and tested the performance of the panel by running TRS for ctDNA of a clear cell renal cell carcinoma (ccRCC) patient and 12 breast cancer patients. Results: TRS using the new ctDNA panel at more than 500 x coverage depth achieved almost the same accuracy as traditional whole-exome sequencing (WES). PBRM1 p.L641V was detected in the plasma sample of the ccRCC patient with an allele frequency of 0.2%. The ctDNA of 12 breast cancer patients was sequenced at a depth of 500-fold, achieving 99.89% coverage; 34 genes were detected with mutations, including the drug target genes BRCA2, PTEN, TP53, APC, KDR, and NOTCH2. Conclusions: This TRS new ctDNA panel can be used to detect mutations in cell-free DNA from multiple types of cancer.