Metagenomic discovery of microbial eukaryotes in stool microbiomes

Area of Science:

• Microbiome Research
• Eukaryotic Genomics
• Metagenomics

Background:

• Host-associated microbiota include bacteria, archaea, viruses, and eukaryotes, but eukaryotes are understudied due to technical limitations.
• Microbial eukaryotes play significant roles in host health, yet current methods poorly recover their DNA, limiting comprehensive analysis.
• Existing research often overlooks microbial eukaryotes, focusing primarily on bacterial communities.

Purpose of the Study:

• To develop and validate a method for enhanced metagenomic discovery and analysis of host-associated microbial eukaryotes.
• To overcome technical limitations in unbiased exploration and DNA recovery of microbial eukaryotes from host samples.
• To improve the detection and characterization of microbial eukaryotes, including fungi, protists, and helminths.

Main Methods:

• Combined cell sorting, optimized eukaryotic cell lysis, and shotgun sequencing for metagenomic analysis.
• Developed a eukaryote-optimized cell lysis and DNA recovery protocol.
• Applied automated cell sorting to stool samples from healthy adults.

Main Results:

• The optimized lysis and DNA recovery method increased eukaryotic DNA yield by 38-fold in synthetic communities.
• Automated cell sorting increased microbial eukaryote reads by up to 28-fold compared to commercial kits.
• Identified novel eukaryotic taxa, assembled genomes from unknown eukaryotes, and predicted genes, with fungal read frequencies increasing 10,000-fold.

Conclusions:

• The developed method significantly enhances the representation and discovery of microbial eukaryotes in metagenomic studies.
• This approach enables unbiased inclusion of microbial eukaryotes in understanding host health and disease determinants.
• Facilitates the identification of new eukaryotic species and their functional genes within the host-associated microbiome.