Abstract
Circulating tumour DNA (ctDNA) detection and monitoring has 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.
Footnotes
mg390{at}cam.ac.uk
Maurizio.Callari{at}cruk.cam.ac.uk
Emma.Beddowes{at}cruk.cam.ac.uk
Stephen-John.Sammut{at}cruk.cam.ac.uk
Marta.Grzelak{at}cruk.cam.ac.uk
heather.biggs{at}addenbrookes.nhs.uk
linda.jones{at}addenbrookes.nhs.uk
ab2421{at}medschl.cam.ac.uk
s.linn{at}nki.nl
jacortes{at}vhio.net
moliveira{at}vhio.net
rdb39{at}medschl.cam.ac.uk
Suet-Feung.Chin{at}cruk.cam.ac.uk
Carlos.Caldas{at}cruk.cam.ac.uk
List of abbreviations
- NG-TAS
- Next Generation Targeted Amplicon Sequencing
- cfDNA
- Cell Free DNA
- ctDNA
- Circulating Tumour DNA
- dPCR
- Digital PCR
- TAm-Seq
- Targeted Amplicon Sequencing
- NGS
- Next Generation Sequencing
- FFPE
- Formalin Fixed Paraffin Embedded
- VAF
- Variant Allele frequency
- SNV
- Single Nucleotide Variant
- FP
- False Positive
- UDG
- Uracil DNA Glycosylase
- RECIST
- Response Evaluation Criteria in Solid Tumour
- CT
- Computed Tomography