Next Generation-Targeted Amplicon Sequencing (NG-TAS): an optimised protocol and computational pipeline for cost-effective profiling of circulating tumour DNA
Circulating tumour DNA (ctDNA) detection and monitoring have enormous potential clinical utility in oncology. We describe here a fast, flexible and cost-effective method to profile multiple genes simultaneously in low input cell-free DNA (cfDNA): Next Generation-Targeted Amplicon Sequencing (NG-TAS). We designed a panel of 377 amplicons spanning 20 cancer genes and tested the NG-TAS pipeline using cell-free DNA from two HapMap lymphoblastoid cell lines. NG-TAS consistently detected mutations in cfDNA when mutation allele fraction was > 1%. We applied NG-TAS to a clinical cohort of metastatic breast cancer patients, demonstrating its potential in monitoring the disease. The computational pipeline is available at https://github.com/cclab-brca/NGTAS_pipeline . ; This research was supported with funding from Cancer Research UK Cancer Research UK (C37096/A16673). MG has been supported by a Genentech research grant (CLL-010907) awarded to the Caldas Laboratory. MC has received funding from the European Union's Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement no. 660060