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Current challenges and best-practice protocols for microbiome analysis
Open accessAbstract
Analyzing the microbiome of diverse species and environments using next-generation sequencing techniques has significantly enhanced our understanding on metabolic, physiological and ecological roles of environmental microorganisms. However, the analysis of the microbiome is affected by experimental conditions (e.g. sequencing errors and genomic repeats) and computationally intensive and cumbersome downstream analysis (e.g. quality control, assembly, binning and statistical analyses). Moreover, the introduction of new sequencing technologies and protocols led to a flood of new methodologies, which also have an immediate effect on the results of the analyses. The aim of this work is to review the most important workflows for 16S rRNA sequencing and shotgun and long-read metagenomics, as well as to provide best-practice protocols on experimental design, sample processing, sequencing, assembly, binning, annotation and visualization. To simplify and standardize the computational analysis, we provide a set of best-practice workflows for 16S rRNA and metagenomic sequencing data (available at https://github.com/grimmlab/MicrobiomeBestPracticeReview).
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“In contrast to 16S rRNA sequencing, whole-genome shotgun metagenomic sequencing provides more comprehensive taxonomic and functional characterization, encompassing broader genomic content and enabling higher-resolution bacterial community profiling.”
From Microbiome meets immunotherapy: unlocking the hidden predictors of immune checkpoint inhibitors (2025) · cites this paper as [146]
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A mathematical framework to correct for compositionality in microbiome datasetsPreprint
Forry, S. P., Servetas, S. L., Kralj, J. G., Hunter, M. E., Dootz, J. N., & Jackson, S. A. (2025) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.1101/2025.05.29.656792
Metadata
- Authors
- Bharti, R., & Grimm, D. G.
- Affiliations
- Weihenstephan-Triesdorf University of Applied Sciences (DE)
- Corresponding author
- Grimm, Dominik G
- Venue
- Briefings in Bioinformatics
- Publisher
- Oxford University Press (OUP)
- Published
- 2019-12-18
- Volume
- 22
- Issue
- 1
- Pages
- 178-193
- ISSN
- 1467-5463 (ISSN-L)
- 1477-4054
- PMC
- PMC7820839
- OpenAlex
- W2995649573
- Cited by
- 592
- References
- 184
- Citations by year
- 2026: 91 · 2025: 129 · 2024: 101 · 2023: 107 · 2022: 80 · 2021: 74 · 2020: 10
- FWCI
- 3.61
- Type
- journal article
- Language
- en
- Open access
- hybrid
- doi.org
- Licence
- Locations
- Briefings in Bioinformatics (publishedVersion, cc-by)
- Europe PMC (PubMed Central) (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- mediaTUM – the media and publications repository of the Technical University Munich (Technical University Munich) (submittedVersion)
- mediaTUM – the media and publications repository of the Technical University Munich (Technical University Munich) (submittedVersion, other-oa)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Phylogenetic Studies
- Physical Sciences › Environmental Science › Ecology › Microbial Community Ecology and Physiology
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Gut microbiota and health
- Keywords
- microbiome analysis, 16S rRNA gene sequencing, shotgun metagenomics, long-read metagenomics, best practice protocol, sample processing, sequencing quality control, metagenome assembly, metagenome binning, functional annotation, data visualization, experimental design
- MeSH terms
- Animals, Humans, RNA, Ribosomal, 16S, Practice Guidelines as Topic, Sequence Analysis, DNA, Metagenomics, DNA Barcoding, Taxonomic, Microbiota
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