A new technology for detection of ultra-low frequency mutations in both liquid biopsies and FFPE samples

Liquid biopsy is promising to revolutionise cancer testing, however its power and potential may not be fully realised until technologies advance to address technical limitations of current NGS library preparation technologies such as low sensitivity, low conversation efficiency, and complexity and duration of procedures.

We have developed a novel technology for preparing targeted NGS libraries that is highly resilient to inefficiencies associated with low integrity DNA, such as from liquid biopsies or FFPE. This technology called ATOM-seq (Adaptor Template Oligonucleotide Mediated Sequencing) fundamentally differs from the most frequently used library preparation techniques which use ligation/hybridisation capture or PCR amplicons. ATOM-Seq, which can be completed in less than 6 hours, is based on a novel method for adding unique molecular identifiers (UMIs) directly onto the 3’ end of starting material with very high efficiency, allowing for mutation hotspots to be interrogated with ultra-high sensitivity. Here we present technical data on the efficacy of the ATOM-Seq approach on a range of control samples, reference standards and clinical samples aimed to demonstrate that this technology is perfectly suited for clinical sample testing.

Sequenced libraries have very high genome mapping rates above 99% as well as high on-target rates at 90%. We detected reference material mutations from 1% (normal DNA 1 ng and FFPE DNA 10 ng input) down to 0.1% allele frequency (normal DNA 10ng and 20 ng input). Our protocol works with gDNA between 0.1-50 ng, with peak conversion efficiencies above 90% when only counting ‘biologically relevant’ UMI families. When used with clinical materials, we found that the ATOM-Seq technology is highly sensitive and robust when detecting low frequency mutations. These data demonstrate that ATOM-Seq is uniquely positioned for processing clinical material such as from liquid biopsies due to its high conversion rate and high sensitivity.