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

## TL;DR

This study compares two DNA extraction and amplification methods for 16S rRNA gene sequencing to determine which better captures microbial diversity in human and mouse fecal samples.

## Contribution

The novel alkaline-based method was shown to detect greater taxonomic diversity and reduce population bias compared to traditional approaches.

## Key findings

- The alkaline-based method detected greater taxonomic diversity in microbiome samples.
- In silico analysis suggested the novel method reduces population bias compared to traditional methods.

## 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.