Abstract 3533: Low-frequency variant detection in cell-free DNA by integrating double-stranded unique molecular identifiers with ultrafast amplicon-based library preparations

The use of liquid biopsy has seen tremendous growth in recent years as cell-free DNA (cfDNA) is a noninvasive and easily obtainable sample type with many diagnostics and prognostic values. While liquid biopsy can potentially enable new applications, such as early cancer detection, treatment monitoring, and drug resistance screening, it also presents challenges to accurate variant detection due to the low fraction of mutant DNA present in theses samples, which can be easily buried by the artifacts and background noise from systematic PCR and sequencing errors. While some success has been made using adapters with unique molecular identifiers (UMIs) in hybrid capture-based methods and performing deep sequencing, such approaches suffer from long, tricky, and tedious workflows and disappointingly low on-target rates. Additionally, the large amount of cfDNA required for hybrid capture workflows is not realistic to obtain from patients. We have developed the CleanPlex® UMI technology to provide a fast, simple, and reliable NGS solution for low-frequency variant detection. The technology features three simple steps to generate molecular-barcoded and target-enriched NGS libraries in under 4 hours. This amplicon-based method consists of a multiplex PCR reaction for molecular barcoding, followed by a biochemical reaction for removal of redundant PCR products, and finally a second round of PCR to add Illumina adapter sequences and sample indexes. To validate this new method, we designed an NGS panel targeting frequently mutated hotspots in 23 genes associated with lung cancer. NGS libraries were prepared with commercial reference cfDNA at 0.1% to 0.5% minor allele frequencies (mAF) and sequenced on an Illumina NextSeq®. Overall, we obtain high detection sensitivity for low-frequency alleles even at low DNA inputs. When including UMI in the analysis, we observe significant reductions in false positive calls. Nearly all known mutations covered by the CleanPlex UMI panel (12 out of 13) are detected at 0.1% mAF using 50ng of the reference cfDNA. At 0.25% mAF, all known mutants are detected with 100% PPV using 50ng of DNA. With only 20 ng of cfDNA, the method can achieve the same 100% detection at 0.5% mAF. The CleanPlex® UMI technology demonstrates high sensitivity with low false positive rate, for the detection of low-frequency alleles. Citation Format: Lucie S. Lee, Yang Lily Liu, Kathryn Pendleton, Jeffrey Liu, Lifeng Phil Lin, Guoying Liu, Zhitong Liu. Low-frequency variant detection in cell-free DNA by integrating double-stranded unique molecular identifiers with ultrafast amplicon-based library preparations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3533.