Next Generation-Targeted Amplicon Sequencing (NG-TAS): an optimised protocol and computational pipeline for cost-effective profiling of circulating tumour DNA

• Published in: Genome medicine Vol. 11; no. 1; p. 1
• Main Authors: Gao, Meiling, Callari, Maurizio, Beddowes, Emma, Sammut, Stephen-John, Grzelak, Marta, Biggs, Heather, Jones, Linda, Boumertit, Abdelhamid, Linn, Sabine C, Cortes, Javier, Oliveira, Mafalda, Baird, Richard, Chin, Suet-Feung, Caldas, Carlos
• Format: Journal Article
• Language: English
• Published: England BioMed Central Ltd 04.01.2019; BioMed Central; BMC
• Subjects: Biological markers; Biomarkers, Tumor - blood; Biomarkers, Tumor - genetics; Breast cancer; Cancer genetics; Cancer metastasis; Cancer patients; Cell Line, Tumor; Circulating Tumor DNA - blood; Circulating Tumor DNA - genetics; Computer applications; Costs and Cost Analysis; ctDNA; Deep sequencing; Deoxyribonucleic acid; DNA; DNA fingerprinting; DNA sequencing; Female; Gene mutation; Genes; Genetic aspects; Genetic research; Genomes; Genomics; Health aspects; High-Throughput Nucleotide Sequencing - economics; High-Throughput Nucleotide Sequencing - methods; Humans; Liquid biopsy; Lymphoblastoid cell lines; Metastases; Metastasis; Method; Methods; Multiplexing; Mutation; NG-TAS; Oncology; Patients; Pipelines; Sequence Analysis, DNA - economics; Sequence Analysis, DNA - methods; Software; Tumors; Multiplexing; Heterogeneous; Deep sequencing; Liquid biopsy; NG-TAS; Mutation; Computational pipeline; ctDNA; Cancer
• ISSN: 1756-994X; 1756-994X
• DOI: 10.1186/s13073-018-0611-9

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 .