Abstract Introduction Surgical biopsy or excisional resection is a standard practice in oncology diagnosis that requires a detectable tumor mass and can be highly invasive, limiting its role in the very early detection, as well as ongoing monitoring efforts, of genetic aberrations relevant to tumor biology. In contrast to surgical biopsies, liquid biopsies provide a readily accessible source of circulating tumor DNA, albeit in a highly diluted state that necessitates a very sensitive and robust method of detection. With recent advents in molecular barcoding and bioinformatics, next-generation sequencing (NGS) has emerged as a platform for probing this liquid oncology space. Herein, we evaluate the ability of the Oncomine™ cell-free DNA (cfDNA) Breast, Colon, and Lung NGS panels to reliably detect clinically relevant genomic alterations in a large background of non-mutated genetic material. Methods A total of 197 individual libraries were prepared across all three cfDNA panels according to the manufacturer protocols. Libraries were manually normalized, pooled, and loaded onto the ThermoFisher IonChef/S5xL system for bead-templating and sequencing. Samples included a mix of commercially available cfDNA controls, plasma samples, and cell lines spiked into healthy donor plasma, and were selected to cover a wide range of clinically relevant mutations and mimic the conditions inherent to real patient specimens. Isolation of cfDNA was performed on both Qiagen and Kingfisher platforms. Raw variant call files were generated using IonReporter and processed through a custom filtering pipeline to generate the final results. Results Nucleic acid isolations yields were comparable for cfDNA samples isolated using the Qiagen or Kingfisher extraction methods, with both yielding a 40-60% DNA recovery rate when known DNA quantities were spiked into plasma background. Library yield mapped to NGS panel size, and the greatest variability was observed for the Colon cfDNA panel. The sensitivity for all Oncomine™ cfDNA assays exceeded 92%, and was highly dependent on sample input, with robust detection at the 0.1% allele frequency requiring up to 50 ng of input cfDNA. A single false positive was present in one HapMap sample analyzed with the breast cfDNA panel. Conclusion The Oncomine™ cfDNA panels provide accurate detection of variants in cfDNA down to 0.1% variant allele frequency. However, increased DNA inputs are required to achieve this highly-sensitive level of detection. Although sensitivity is reduced at lower inputs, the ability to generate high-quality libraries from as little as 10 ng DNA ensures sufficient material can be obtained for sequencing from even low-yielding samples. Overall, these results indicate that the Oncomine™ cfDNA assays can aid in the very early diagnosis and monitoring of tumor populations via minimally-invasive blood-based diagnostics. Citation Format: Nathan Riccitelli, Nancy Valencia, Ashley Beams, Sarah Johnson, Reinhold Pollner. Progress toward non-invasive oncology diagnostics: Evaluation of cfDNA next-generation sequencing assays 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 2282.