Analytic attributes of the 16S rRNA gene sequencing methodology for human gut microbiota characterization

Gut microbiota is crucial for human health. While 16S rRNA gene sequencing is most used for characterizing this community, the validation and standardization of the technique are often overlooked. This study analyzes critical factors influencing the repeatability and intermediate precision of 16S rRNA gene sequencing methodology for human gut microbiota characterization, examining the impact of key analytical factors. Our investigation evaluated the effects of the DNA extraction protocol, sample homogenization, thawing, library preparation, and sequencing on measurements of precision. This established a standardized operating procedure (SOP) whose variability was assessed within a single laboratory (intermediate precision) by analyzing DNA extraction kit lot variations and the laboratory analyst handling the samples. We discovered that the DNA extraction protocol and sample thawing were the most significant drivers of variability in gut microbiota profiles. At the same time, the intermediate precision of the method was high. We determined the method's limit of quantification, revealing an impressive sensitivity down to just 11 to 18 rarefied read counts (with coefficients of variation of 30% and 20%, respectively). Beyond technical considerations, we also quantified the variation in gut microbiota profiles among individuals and over time. Our findings confirm substantial inter-individual differences while demonstrating that changes within individuals over a week are relatively small. This research illuminates some critical factors influencing the precision and consistency of 16S rRNA gene sequencing for gut microbiota analysis. By incorporating these insights into standardized protocols, we can significantly improve best practices in DNA sequencing methodologies, strengthening the reliability and comparability of human microbiome studies. KEY POINTS: • DNA extraction and sample thawing critically affect the method's precision. • We established an SOP with high repeatability, intermediate precision, and a specific limit of quantification. • Gut microbiota profiles substantially vary among individuals but remain stable over a week.