Comparison of Lysis and Amplification Methodologies for Optimal 16S rRNA Gene Profiling for Human and Mouse Microbiome Studies

Rastegari, Farzaneh; Driscoll, Mark; Riordan, Jesse D.; Nadeau, Joseph H.; Johnson, Jethro S.; Weinstock, George M.

doi:10.3390/ijms26031180

This is an early access version, the complete PDF, HTML, and XML versions will be available soon.

Open AccessArticle

Comparison of Lysis and Amplification Methodologies for Optimal 16S rRNA Gene Profiling for Human and Mouse Microbiome Studies

by

Farzaneh Rastegari

^1,2,

Mark Driscoll

³,

Jesse D. Riordan

^4,5,

Joseph H. Nadeau

^5,6,

Jethro S. Johnson

^2,7,*

and

George M. Weinstock

^2,8

¹

Department of Computer Science and Engineering, University of Connecticut, 371 Fairfield Way, Storrs, CT 06269, USA

²

The Jackson Laboratory for Genomic Medicine, 10 Discovery Drive, Farmington, CT 06032, USA

³

Intus Biosciences, 400 Farmington Ave, Farmington, CT 06032, USA

⁴

Department of Anatomy & Cell Biology, Carver College of Medicine, The University of Iowa, Iowa City, IA 52242, USA

⁵

Pacific Northwest Research Institute, 720 Broadway, Seattle, WA 98122, USA

⁶

Center for Molecular Medicine, MaineHealth Institute for Research, Scarborough, ME 04074, USA

⁷

The Oxford Centre for Microbiome Studies, Kennedy Institute of Rheumatology, University of Oxford, Roosevelt Drive, Headington, Oxford OX3 7FY, UK

⁸

Genetics and Genome Science, University of Connecticut Health Center, 400 Farmington Ave, Farmington, CT 06030, USA

^*

Author to whom correspondence should be addressed.

Int. J. Mol. Sci. 2025, 26(3), 1180; https://doi.org/10.3390/ijms26031180

Submission received: 27 November 2024 / Revised: 21 January 2025 / Accepted: 22 January 2025 / Published: 29 January 2025

(This article belongs to the Section Molecular Genetics and Genomics)

Download Versions Notes

Abstract

When conducting sequence-based analysis of microbiome samples, it is important to accurately represent the bacterial communities present. The aim of this study was to compare two commercially available DNA isolation and PCR amplification approaches to determine their impact on the taxonomic composition of microbiome samples following 16S rRNA gene sequencing. A well-established 16S rRNA gene profiling approach, which was widely used in the Human Microbiome Project (HMP), was compared with a novel alkaline degenerative technique that utilizes alkaline cell lysis in combination with a degenerate pool of primers for nucleic acid extraction and PCR amplification. When comparing these different approaches for the microbiome profiling of human and mouse fecal samples, we found that the alkaline-based method was able to detect greater taxonomic diversity. An in silico analysis of predicted primer binding against a curated 16S rRNA gene reference database further suggested that this novel approach had the potential to reduce population bias found with traditional methods, thereby offering opportunities for improved microbial community profiling.

Keywords: DNA extraction; 16S rRNA gene sequencing; microbiome; alkaline-based lysis; bead-beating lysis; V1–V3 16S rRNA gene primers

Share and Cite

MDPI and ACS Style

Rastegari, F.; Driscoll, M.; Riordan, J.D.; Nadeau, J.H.; Johnson, J.S.; Weinstock, G.M. Comparison of Lysis and Amplification Methodologies for Optimal 16S rRNA Gene Profiling for Human and Mouse Microbiome Studies. Int. J. Mol. Sci. 2025, 26, 1180. https://doi.org/10.3390/ijms26031180

AMA Style

Rastegari F, Driscoll M, Riordan JD, Nadeau JH, Johnson JS, Weinstock GM. Comparison of Lysis and Amplification Methodologies for Optimal 16S rRNA Gene Profiling for Human and Mouse Microbiome Studies. International Journal of Molecular Sciences. 2025; 26(3):1180. https://doi.org/10.3390/ijms26031180

Chicago/Turabian Style

Rastegari, Farzaneh, Mark Driscoll, Jesse D. Riordan, Joseph H. Nadeau, Jethro S. Johnson, and George M. Weinstock. 2025. "Comparison of Lysis and Amplification Methodologies for Optimal 16S rRNA Gene Profiling for Human and Mouse Microbiome Studies" International Journal of Molecular Sciences 26, no. 3: 1180. https://doi.org/10.3390/ijms26031180

APA Style

Rastegari, F., Driscoll, M., Riordan, J. D., Nadeau, J. H., Johnson, J. S., & Weinstock, G. M. (2025). Comparison of Lysis and Amplification Methodologies for Optimal 16S rRNA Gene Profiling for Human and Mouse Microbiome Studies. International Journal of Molecular Sciences, 26(3), 1180. https://doi.org/10.3390/ijms26031180

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Menu

Comparison of Lysis and Amplification Methodologies for Optimal 16S rRNA Gene Profiling for Human and Mouse Microbiome Studies

Abstract

Share and Cite

Article Metrics

Article Access Statistics

Further Information

Guidelines

MDPI Initiatives

Follow MDPI