Improving Equivalency in Metagenomics: A Harmonized Process to Extract Fecal DNA

In the field of clinical chemistry, there is a major push to harmonize and standardize laboratory processes and materials to improve the equivalency of reported test results and reduce the risk of postanalytical errors. The impetus of harmonized results is expanding to the field of molecular pathology and is seen in the development of standardized reference materials for the measurement of pathogens in clinical samples, and the harmonization of PCR and fluorescence in situ hybridization methods for noninvasive prenatal diagnosis and cancer screening, respectively. Molecular methods are used to study microbial communities in the human body to assess contributions of microbiota to human health. Microbiota in the gastrointestinal tract comprise one of the best studied ecosystems owing to their large volume, high diversity, and relevance to several pathologies (e.g., diabetes, inflammatory bowel disease, and colorectal cancer). Until now, most studies in gut microbiota have used unique methodologies resulting in demographically distinct cohorts. This has limited the potential for interstudy comparisons and metaanalyses, as it is difficult to disentangle biological from methodological variation. To address the technical variation in metagenomics and more confidently assess contributions of microbiota to human health, a recent article by Costea et al.(1) suggests that a harmonized protocol outlined by the authors be used to extract DNA from feces. The authors tested 21 DNA extraction protocols on the same fecal samples and quantified the differences observed in the microbial community compositions. Procedural variables of library preparation and sample storage were contrasted with biological variations observed within the same specimen or within an individual over time. Extraction protocols were then ranked by the resulting DNA quantity and quality, estimates of recovered community diversity, and the ratio between gram-positive and gram-negative bacteria. Procedure reproducibility was tested within and across laboratories, and the accuracy of the top-performing DNA extraction methods was assessed using a mock community of bacterial species whose exact relative abundance was known. If only the ranks of the bacterial species of interest are required, most of the available protocols tested gave highly comparable results. However, for many applications, species-specific abundance information is required, and this information needs to be commensurable between methods. Using this metric, many of the protocols tested were not equivalent and instead introduced large batch effects. Of the species particularly affected by the extraction protocol, the majority were gram-positive, an unsurprising finding given the higher mechanical strength of gram-positive bacterial cell walls. In the selection of the final protocol, reproducibility, recovery of bacterial diversity, and automation were factors. In addition, the selected winning protocol accurately extracted DNA from the spiked bacteria in the test stool samples. As the literature regarding the harmonization of molecular methods in research or clinical laboratories expands, this article makes an important contribution. Variations in DNA extraction protocols can have large effects on the observed microbial composition of stool samples, and a harmonized method will improve the comparability of human gut microbiome studies and facilitate metaanalysis. Furthermore, because of its performance characteristics, the selected protocol should serve as a benchmark for new methods.

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