Comparison of Oxford Nanopore and Sequencing-by-Synthesis Technologies for Sequencing cell-free bacterial DNA

The measurement of cell-free DNA is increasingly applied in clinical research, including for liquid biopsies for early disease detection and monitoring, as well as in personalised medicine. Cell-free DNA (cfDNA), typically ~ 150–200 bp, can be measured by PCR-based approaches and Next-Generation Sequencing. While cfDNA is routinely measured, cell-free bacterial DNA (cbDNA), the subset of cfDNA derived from bacteria, is not. Here, we compared Oxford Nanopore Technology (ONT) with sequencing-by-synthesis (SBS) for the measurement of cbDNA in 70 plasma samples and controls. We found that both platforms could successfully measure these low abundance fragments, closely matching the theoretical concentrations of control samples. However, platform-specific differences were observed in microbial taxonomic profiles from biological samples. Sequencing-by-synthesis generated substantially more total reads, although cbDNA read counts were comparable between platforms. ONT produced longer read lengths, SBS produced reads of higher quality. These results indicate that sequencing platform can influence the inferred microbial composition and diversity. Platform selection should be guided by study aims, required taxonomic resolution, and practical considerations such as sample volume availability, turnaround time and bioinformatic requirements.