Obtaining genomes from uncultivated environmental microorganisms using FACS-based single-cell genomics

Area of Science:

• Microbiology
• Genomics
• Environmental Science

Background:

• Most environmental microorganisms are unculturable using standard techniques.
• Single-cell genomics offers a pathway to study these microbes.
• High-throughput methods are needed for efficient single-cell analysis.

Purpose of the Study:

• To develop a comprehensive protocol for obtaining genomes from uncultivated environmental microbes.
• To enable high-throughput single-cell isolation and genomic analysis.
• To facilitate the study of microbial genetic makeup in diverse environments.

Main Methods:

• Utilizing fluorescence-activated cell sorting (FACS) for high-throughput single-cell isolation.
• Implementing a protocol for sample preservation, cell lysis, and whole-genome amplification.
• Employing 16S rRNA sequencing for bacterial and archaeal cell identification.

Main Results:

• A comprehensive protocol for single-cell genome recovery from uncultivated microbes was established.
• The protocol yields up to 1 μg of genomic DNA per cell, suitable for downstream sequencing.
• Average genome completeness of recovered genomes was approximately 50%.

Conclusions:

• The developed FACS-based protocol provides a robust method for single-cell genomics of uncultivated environmental microbes.
• This approach expands the accessibility of microbial genomes for research.
• Further optimization may improve the completeness of recovered genomes.