Poster Presentation GENEMAPPERS 2024

Using oxford nanopore technologies to study microbial cell-free DNA (mcfDNA) (#65)

Arielle Sulit 1 , Sarah Appleby 2 , John Pearson 3 , Rachel Purcell 1
  1. Department of Surgery and Critical Care, University of Otago, Christchurch, Christchurch, New Zealand
  2. Department of Medicine, University of Otago, Christchurch, Christchurch, New Zealand
  3. Biostatistics and Computational Biology Unit, University of Otago, Christchurch, Christchurch, New Zealand

Cell-free DNA (cfDNA) are DNA found in blood plasma, and is currently gaining traction as a screening alternative to more invasive methods (e.g. biopsies) in diseases. These include microbial cell-free DNA (mcfDNA) that could give information on the microbiome associated with diseases. Oxford Nanopore Technologies (ONT) offers more real-time capabilities, and more accessible equipment through their MinION platform, compared to technologies conventionally used to sequence cfDNA. However, ONT was initially optimised for long reads. We therefore aim to optimise its use for the shorter (40-100bp) mcfDNA fragments.

We fragmented ZymoBiomics Microbial Community DNA standards to simulate mcfDNA fragment sizes. We followed the recommended protocol for sequencing cfDNA on ONT MinION, specifically to allow for better retention of the shorter cfDNA fragments during bead cleanup, and to mitigate barcode cross-contamination. We compared results of this procedure to fragmented standards sequenced by a sequencing-by-synthesis (SBS) methodology (MGI technologies). Sequences were mapped to a microbial database using Kraken2 to obtain microbial profiles. We further explored the sensitivity of ONT MinION in detecting and identifying microbial profiles by sequencing different input concentrations of fragmented ZymoBiomics standards, and different preparations of the standards, including running these through a cfDNA extraction process, and spiking-in to plasma from volunteer participants.

For both platforms (ONT and MGI), we assigned 37.24% (SBS) and 34.75% (ONT) of ZymoBiomics reads to a species. SBS (36.12%) and ONT (30.44%) reads assigned to expected species from ZymoBiomics were comparable, though lower than theoretical proportions. Using ONT, we assigned expected taxonomies to all preparations of the standards, with comparable proportions.

ONT is comparable with SBS when sequencing standards, and shows sensitivity in sequencing low concentrations of the ZymoBiomics standards. We intend to determine its suitability in sequencing mcfDNA from colorectal cancer samples in future applications of the technology.