Getting the guts right:Benchmarking faecal DNA extraction methods for comprehensive microbiota detection

Background: Faecal metagenomic sequencing is becoming an increasingly powerful tool in the context of gut diseases. A major limitation of the field is the lack of method standardisation in the nucleic acid extraction from faecal samples which can affect the detection of tough to lyse microorganisms (Fernández-Pato et al., 2024). This study aims to benchmark commonly used commercial faecal DNA extraction kits to determine their ability to recover the complete microbiota from samples.<br/><br/>Methods: Four commercial faecal DNA extraction kits and one non-kit based protocol were chosen for benchmarking in this study. Commercially available mock-community gut standards were used as positive controls. A tough-to-lyse microorganism mock-community was used to assess lysis efficiency and a donor microbiota positive control to assess correct detection of microbiota composition. Manufacturer recommended lysis methods were tested for each extraction kit.<br/><br/>The final DNA yield was quantified using the Qubit fluorometer and quality was checked with the Agilent Tapestation. Both Illumina and Oxford Nanopore Technologies whole-genome sequencing were used to sequence the resulting DNA via standard library preparation methods. Bioinformatic analysis was performed using Kraken2 for long read and AmpliSeq for short read data.<br/><br/>Results: The microorganisms detected and population proportions were compared to expected results from the control standards used. All methods detected the majority of organisms however some microorganisms were missed by some of the extraction methodologies. Full results will be reported.<br/><br/>Conclusions: Validation of DNA extraction methods is a crucial yet often overlooked step in faecal metagenomic studies. The rigorous evaluation of faecal DNA extraction methods demonstrates that failure to detect microorganisms is more often due to extraction limitations than true absence. Based on these findings, we propose a recommended extraction approach for faecal material in metagenomic research.