Analysis of Microbiomes from Ultra-Low Biomass Surfaces Using Novel Surface Sampling and Nanopore Sequencing

Area of Science:

• Microbiology
• Environmental Science
• Biotechnology

Background:

• Ultra-low biomass environments like cleanrooms require rapid microbiome assessment for human health and manufacturing.
• Current short-read sequencing methods are time-consuming and lack portability.

Purpose of the Study:

• To develop and validate a rapid, on-site nanopore sequencing approach for characterizing ultra-low biomass microbiomes.
• To assess the feasibility of using the Squeegee-Aspirator for Large Sampling Area (SALSA) device for this purpose.

Main Methods:

• A modified Oxford Nanopore Rapid PCR Barcoding Kit protocol was used.
• The Squeegee-Aspirator for Large Sampling Area (SALSA) surface sampling device was employed.
• Nanopore sequencing was performed on samples from a NASA Class 100K cleanroom.

Main Results:

• The novel approach achieved rapid microbiome characterization in approximately 24 hours.
• DNA amplification was 1-2 orders of magnitude higher than controls.
• Dominant species identified were *Paracoccus* and *Acinetobacter*.
• Negative controls revealed contamination from sampling reagents (*Cutibacerium acnes*).

Conclusions:

• The SALSA sampler combined with modified nanopore sequencing offers a novel, rapid method for low-biomass DNA profiling.
• Multiple negative controls and DNA-free techniques are crucial for accurate ultra-low biomass sample assessment.