Optimized bacterial DNA isolation method for microbiome analysis of human tissues.

Area of Science:

• Microbiology
• Genomics
• Molecular Biology

Background:

• Host DNA contamination in tissue samples hinders accurate microbiome analysis.
• Advances in microbiome sequencing reveal the microbiome's role in human health and disease.

Purpose of the Study:

• To develop and validate a DNA isolation protocol for tissue biopsies that reduces host DNA while preserving bacterial DNA integrity.
• To improve the analysis of host-associated bacteria in clinical samples.

Main Methods:

• Optimized lysis of human cells using saponin and Triton, combined with DNase treatment to remove host DNA.
• Evaluated bacterial DNA enrichment and preservation of relative abundance using qPCR.
• Validated the protocol with shotgun metagenomics on human colon biopsies from clinical studies.

Main Results:

• A saponin concentration of 0.0125% achieved a 4.5-fold enrichment of bacterial DNA.
• The protocol preserved the relative abundance of major bacterial phyla (Firmicutes, Bacteroidetes, γ-Proteobacteria, Actinobacteria).
• Metagenomic analysis of validated samples showed bacterial profiles and diversity metrics comparable to 16S rRNA sequencing.

Conclusions:

• The developed protocol efficiently enriches bacterial DNA from tissue biopsies, enabling comprehensive microbiome analysis.
• This method enhances the applicability of clinical tissue samples for microbiome research.
• Facilitates the study of a wide spectrum of bacteria in clinical settings.