Document Type

Article

Publication Date

1-29-2025

Publication Title

Int J Mol Sci

Keywords

JGM, RNA, Ribosomal, 16S, Humans, Animals, Mice, Microbiota, Feces, Bacteria, Polymerase Chain Reaction, DNA, Bacterial, Sequence Analysis, DNA

JAX Source

Int J Mol Sci. 2025;26(3):1180.

Volume

26

Issue

3

ISSN

1422-0067

PMID

39940948

DOI

https://doi.org/10.3390/ijms26031180

Grant

This research was funded in part by NIH, grant number HG012444

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.

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