Detection of cell-free microbial DNA using a contaminant-controlled analysis framework

Area of Science:

• Microbiology
• Genomics
• Oncology

Background:

• The human microbiome is implicated in cancer development and progression.
• Microbiome-derived DNA in cell-free DNA (cfDNA) presents a potential non-invasive cancer biomarker.
• Low-biomass microbiome studies are susceptible to contamination, hindering biomarker development.

Purpose of the Study:

• To investigate the detectability of microbial cell-free DNA (cfmDNA) in metastatic melanoma patients.
• To establish a framework for accurate cfmDNA detection in plasma, accounting for contamination.
• To explore the potential of cfmDNA as a biomarker for cancer detection.

Main Methods:

• Utilized 16S-rRNA-gene sequencing and droplet digital PCR for cfmDNA detection.
• Employed rigorous experimental controls and negative controls to assess contamination.
• Applied an in silico decontamination strategy to filter contaminant microbial sequences.

Main Results:

• Detected low but significant levels of cfmDNA in patient plasma, above negative controls.
• Identified specific gut commensal bacteria (e.g., Faecalibacterium, Bacteroides) in plasma after decontamination.
• Demonstrated that plasma cfmDNA levels can be influenced by laboratory and reagent contaminants.

Conclusions:

• Plasma harbors detectable levels of cfmDNA, supporting its potential as a biomarker.
• An analytical decontamination framework is essential for accurate cfmDNA detection.
• This approach can be extended to differentiate cfmDNA profiles between healthy and cancer patients.