Variation in the metagenomic analysis of fecal microbiome composition calls for a standardized operating approach

Abstract

<p>The reproducibility in microbiome studies is limited due to the lack of one gold-standard operating procedure. The aim of this study was to examine the impact of protocol variations on microbiome composition using metagenomic data sets from a single center. We assessed the variation in a data set consisted of 2,722 subjects, including 9 subcohorts harboring healthy subjects and patients with various disorders, such as inflammatorybowel disease, colorectal cancer, and type 2 diabetes. Two differentDNA extraction kits, with or without lyticase, and two sample storage methods were compared. Our results indicated that DNA extraction had the largest impact on gut microbiota diversity among all host factors and sample operating procedures. Healthy subjects matched by age, body mass index, and sample operating methods exhibited reduced, yet significantdifferences(PERMANOVA, P < 0.05) in gut microbiota composition across studies. The variations contributed by DNA extraction were primarily driven by differentrecovery efficiencyof gram-positive bacteria, e.g., phyla Firmicutes and Actinobacteria. This was further confirmedby a parallel comparison of fecal samples from fivehealthy subjects and a standard mock community. In addition, the DNA extraction method influencedDNA biomass, quality, and the detection of specificlineage-associated diseases. Sample operating approach and batch effectsshould be considered for cohorts with large sample size or longitudinal cohorts to ensure that source data were appropriately generated and analyzed. Comparison between samples processed with inconsistent methods should be dealt with caution. This study will promote the establishment of a sample operating standard to enhance our understanding of microbiome and translating in clinical practice.</p>