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

Meiling Gao†,
Maurizio Callari†,
Emma Beddowes,
Stephen-John Sammut,
Marta Grzelak,
Heather Biggs,
Linda Jones,
Abdelhamid Boumertit,
Sabine C. Linn,
Javier Cortes,
Mafalda Oliveira,
Richard Baird,
Suet-Feung ChinEmail author and
Carlos CaldasEmail author

†Contributed equally

Genome Medicine201911:1

https://doi.org/10.1186/s13073-018-0611-9

Received: 13 August 2018
Accepted: 17 December 2018
Published: 4 January 2019

Abstract

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.