Abstract Liquid biopsy testing utilising Next Generation Sequencing (NGS) is rapidly moving towards clinical adoption for personalised oncology. However, before NGS can fulfil its potential any novel testing approach must identify ways of reducing errors, allowing for separating true low-frequency mutations from procedural artefacts, and be designed to improve upon current technologies while also avoiding their limitations. Popular NGS technologies typically utilise two approaches; PCR and ligation, which have known limitations and seem to have reached a development plateau with only small, stepwise improvements being made. To maximise the ultimate utility of liquid biopsy testing we have developed a highly versatile approach to NGS: Adaptor Template Oligo Mediated Sequencing (ATOM-Seq). ATOM-Seq’s strengths and versatility avoid the major limitations of both PCR- and ligation-based approaches. This technology is ligation free, simple, efficient, flexible, and streamlined, and offers novel advantages that make it perfectly suited for use on highly challenging clinical material. Using reference materials, we demonstrate detection of known SNVs at allele frequencies of 0.1% using as little as 20 ng of DNA, as well as the ability to detect fusions from RNA. We illustrate ATOM-Seq’s suitability for clinical testing by showing high concordance rates between paired cfDNA and FFPE clinical samples.

Adaptor Template Oligo-Mediated Sequencing (ATOM-Seq): A versatile and ultra-sensitive UMI-based NGS library preparation technology, for use with cfDNA and cfRNA