Microbiota DNA isolation, 16S rRNA amplicon sequencing, and bioinformatic analysis for bacterial microbiome profiling of rodent fecal samples

Chloe J Love; Carolina Gubert; Saritha Kodikara; Geraldine Kong; Kim-Anh Lê Cao; Anthony J Hannan

doi:10.1016/j.xpro.2022.101772

Microbiota DNA isolation, 16S rRNA amplicon sequencing, and bioinformatic analysis for bacterial microbiome profiling of rodent fecal samples

STAR Protoc. 2022 Oct 21;3(4):101772. doi: 10.1016/j.xpro.2022.101772. eCollection 2022 Dec 16.

Authors

Chloe J Love¹, Carolina Gubert², Saritha Kodikara³, Geraldine Kong¹, Kim-Anh Lê Cao⁴, Anthony J Hannan⁵

Affiliations

¹ The Florey Institute of Neuroscience and Mental Health, University of Melbourne, Parkville, VIC 3010, Australia.
² The Florey Institute of Neuroscience and Mental Health, University of Melbourne, Parkville, VIC 3010, Australia. Electronic address: carolina.gubert@florey.edu.au.
³ Department of Anatomy and Physiology, University of Melbourne, Parkville, VIC 3010, Australia; Melbourne Integrative Genomics, School of Mathematics and Statistics, University of Melbourne, Parkville VIC, 3010, Australia.
⁴ Department of Anatomy and Physiology, University of Melbourne, Parkville, VIC 3010, Australia.
⁵ The Florey Institute of Neuroscience and Mental Health, University of Melbourne, Parkville, VIC 3010, Australia; Department of Anatomy and Physiology, University of Melbourne, Parkville, VIC 3010, Australia. Electronic address: anthony.hannan@florey.edu.au.

Abstract

Fecal samples are frequently used to characterize bacterial populations of the gastrointestinal tract. A protocol is provided to profile gut bacterial populations using rodent fecal samples. We describe the optimal procedures for collecting rodent fecal samples, isolating genomic DNA, 16S rRNA gene V4 region sequencing, and bioinformatic analyses. This protocol includes detailed instructions and example outputs to ensure accurate, reproducible results and data visualization. Comprehensive troubleshooting and limitation sections address technical and statistical issues that may arise when profiling microbiota. For complete details on the use and execution of this protocol, please refer to Gubert et al. (2022).

Keywords: Bioinformatics; Cancer; Genetics; Genomics; Health Sciences; High Throughput Screening; Microbiology; Model Organisms; Molecular Biology; RNAseq; Single Cell; Systems biology.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Bacteria / genetics
Computational Biology*
DNA
Microbiota*
RNA, Ribosomal, 16S / genetics
Rodentia / genetics

Substances

RNA, Ribosomal, 16S
DNA