Microbial Bioactives
Microbial Bioactives | Online ISSN 2209-2161
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Volume 6 Number 1 2023
REVIEWS (Open Access)
Unmasking Bias in Microbiome Research: A Human-Centered Introduction to Methodological Pitfalls and Meta-Analytic Insights
Most Farhana Akter 1, Md. Robiul Islam 1, Shahadat Hossain 2*
Microbial Bioactives 6 (1) 1-8 https://doi.org/10.25163/microbbioacts.6110672
Submitted: 05 February 2023 Revised: 24 March 2023 Accepted: 06 April 2023 Published: 08 April 2023
Abstract
Microbiome research has transformed biological and biomedical sciences by enabling culture-independent exploration of complex microbial communities across diverse environments, from marine ecosystems to the human body. Despite these advances, microbiome studies remain highly vulnerable to systematic and random methodological biases that can substantially distort biological interpretation and limit reproducibility. This systematic review and meta-analysis synthesize evidence on how technical decisions across the microbiome research workflow influence observed microbial diversity and community structure. Specifically, it evaluates biases arising from study design, sampling strategies, DNA extraction methods, primer selection, sequencing platforms, and bioinformatic pipelines. Quantitative meta-analytic comparisons demonstrate that methodological choices frequently exert effect sizes comparable to, or greater than, true biological variables. Full-length 16S rRNA gene sequencing consistently reveals higher species richness than short-read approaches, while primer selection introduces pronounced taxon-specific dropouts, including clinically and ecologically important genera. Extraction kits display domain-dependent biases, often recovering bacterial diversity effectively while underestimating eukaryotic taxa. Case studies from marine biofilms, freshwater sediments, host-associated microbiomes, and pathogen surveillance illustrate how these biases propagate through downstream analyses, leading to underestimation of richness, misclassification of taxa, and erroneous ecological or clinical conclusions. Advances such as amplicon sequence variant inference and standardized reporting frameworks have improved resolution and transparency, yet database inconsistencies and low-biomass contamination remain persistent challenges. Collectively, the findings underscore that microbiome profiles are not neutral reflections of biological reality but products of methodological “lenses.” Addressing these biases through standardized protocols, informed methodological choices, and rigorous controls is essential for producing robust, comparable, and biologically meaningful microbiome research.
Keywords: Microbiome research; methodological bias; 16S rRNA sequencing; primer bias; DNA extraction; meta-analysis; microbial diversity; bioinformatics
References
Azam, F., & Worden, A. Z. (2004). Microbes, molecules, and marine ecosystems. Science, 303(5664), 1629–1630. https://doi.org/10.1126/science.1093892
Balvociute, M., & Huson, D. H. (2017). SILVA, RDP, Greengenes, NCBI and OTT—How do these taxonomies compare? BMC Genomics, 18, 114. https://doi.org/10.1186/s12864-017-3501-4
Bjerre, R. D., et al. (2019). Effects of sampling strategy and DNA extraction on human skin microbiome investigations. Scientific Reports, 9, 7619. https://doi.org/10.1038/s41598-019-53599-z
Callahan, B. J., et al. (2017). Exact sequence variants should replace operational taxonomic units. ISME Journal, 11, 2639–2643. https://doi.org/10.1038/ismej.2017.119
Costea, P. I., et al. (2017). Towards standards for human fecal sample processing in metagenomic studies. Nature Biotechnology, 35(11), 1069–1076. https://doi.org/10.1038/nbt.3960
Eren, A. M., et al. (2015). Minimum entropy decomposition: Unsupervised oligotyping. ISME Journal, 9, 968–979. https://doi.org/10.1038/ismej.2014.195
Folmer, O., et al. (1994). DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I. Molecular Marine Biology and Biotechnology, 3(5), 294–299. https://pubmed.ncbi.nlm.nih.gov/7881515/
Francioli, D., et al. (2021). DNA metabarcoding for the characterization of terrestrial microbiota—Pitfalls and solutions. Microorganisms, 9(2), 364. https://doi.org/10.3390/microorganisms9020361
Hebert, P. D. N., et al. (2003). Biological identifications through DNA barcodes. Proceedings of the Royal Society B, 270(1512), 313–321. https://doi.org/10.1098/rspb.2002.2218
Klindworth, A., et al. (2013). Evaluation of general 16S ribosomal RNA gene PCR primers. Nucleic Acids Research, 41(1), e1. https://doi.org/10.1093/nar/gks808
Mancabelli, L., et al. (2020). The impact of primer design on amplicon-based metagenomic profiling accuracy. Microorganisms, 8(1), 101. https://doi.org/10.3390/microorganisms8010101
Martínez, M., et al. (2023). Mitochondrial heteroplasmy and PCR amplification bias lead to wrong species delimitation. Genes, 14(4), 1098. https://doi.org/10.3390/genes14040998
Meisel, J. S., et al. (2016). Skin microbiome surveys are strongly influenced by experimental design. Journal of Investigative Dermatology, 136(5), 947–956. https://doi.org/10.1016/j.jid.2016.01.016
Na, H. S., et al. (2023). Comparative analysis of primers used for 16S rRNA gene sequencing in oral microbiome studies. Methods and Protocols, 6(4), 71. https://doi.org/10.3390/mps6040071
Quast, C., et al. (2013). The SILVA ribosomal RNA gene database project. Nucleic Acids Research, 41(D1), D590–D596. https://doi.org/10.1093/nar/gks1219
Santiago-Rodriguez, T. M., et al. (2023). The skin microbiome: Current techniques, challenges, and future directions. Microorganisms, 11(5), 512. https://doi.org/10.3390/microorganisms11051212
Shi, Z., et al. (2022). The effects of DNA extraction kits and primers on prokaryotic and eukaryotic microbial community profiling. Microorganisms, 10(6), 1213. https://doi.org/10.3390/microorganisms10061213
Wang, S., et al. (2022). Microbial richness of marine biofilms revealed by sequencing full-length 16S rRNA genes. Genes, 13(6), 1050. https://doi.org/10.3390/genes13061050
Yun, J. H., et al. (2022). Diet and exercise shape the gut microbiome: Mouse model insights. Journal of Microbiome Research, 5, 88–101.
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