Xu, Zhilu, Yeoh, Yun Kit, Tun, Hein, Fei, Na, Zhang, Jingwan, Morrison, Mark, Kamm, Michael, Yu, Jun, Chan, Francis K.L., and Ng, Siew C. (2024) Variation in the metagenomic analysis of fecal microbiome composition calls for a standardized operating approach. Microbiology Spectrum, 12 (12).

Preview

PDF (Published Version) - Published Version Available under License Creative Commons Attribution. Download (2MB) | Preview

Abstract

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 inflammatory bowel disease, colorectal cancer, and type 2 diabetes. Two different DNA 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 significant differences (PERMANOVA, P < 0.05) in gut microbiota composition across studies. The variations contributed by DNA extraction were primarily driven by different recovery efficiency of gram-positive bacteria, e.g., phyla Firmicutes and Actinobacteria. This was further confirmed by a parallel comparison of fecal samples from five healthy subjects and a standard mock community. In addition, the DNA extraction method influenced DNA biomass, quality, and the detection of specific lineage-associated diseases. Sample operating approach and batch effects should 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.

Item ID:	91183
Item Type:	Article (Research - C1)
ISSN:	2165-0497
Copyright Information:	© 2024 Xu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license
Date Deposited:	01 May 2026 02:56
FoR Codes:	32 BIOMEDICAL AND CLINICAL SCIENCES > 3207 Medical microbiology > 320799 Medical microbiology not elsewhere classified @ 100%
SEO Codes:	20 HEALTH > 2099 Other health > 209999 Other health not elsewhere classified @ 100%
Downloads:	Total: 1 Last 12 Months: 1
	More Statistics