Whole-cell Reference Reagents Highlight Limitations and Biases in DNA Extraction of Human Gut Microbiome Samples

Despite advances in sequencing technologies, DNA extraction remains a key constraint in providing a true microbial profile due to its inherent tendency to introduce biases. Here, through a global study involving 22 labs, we analysed 44 different DNA extraction methodologies for the taxonomic profiling of a well-defined whole-cell reference reagent (RR) comprised of 20 common gut bacteria. We aimed to pinpoint the sources of bias and understand their effect on microbiome profiling accuracy. Through this study, Minimum Quality Criteria (MQC) were established and are used to evaluate extraction performance. We show there is a clear need for standardisation due to the lack of clearly defined procedural steps that consistently yield optimal outcomes in DNA extraction and challenge persistent controversies in the field regarding DNA extraction methodologies. This study presents a well-defined RR and MQC to combat DNA extraction bias and therefore moving the field towards reliable and comparable microbiome research.