A tool kit for quantifying eukaryotic rRNA gene sequences from human microbiome samples.

Area of Science:

• Microbiology
• Genomics
• Bioinformatics

Background:

• Eukaryotic microorganisms are crucial but under-explored components of the human microbiome.
• Accurate analysis of eukaryotic microbes in complex samples like stool is challenging.

Purpose of the Study:

• To present a comprehensive pipeline for the analysis of deep sequencing data from single-cell eukaryotes.
• To provide a validated toolkit for the sequence-based enumeration of eukaryotic organisms within human microbiome samples.

Main Methods:

• Design and testing of a novel 18S rRNA gene-specific PCR primer set.
• Comparison with a published rRNA gene internal transcribed spacer (ITS) primer set.
• Development and validation of a bioinformatics pipeline (BROCC) for taxonomic attribution of eukaryotic sequences.

Main Results:

• The developed primer sets and software pipeline were tested against defined eukaryotic specimens and human stool samples.
• The software pipeline demonstrated accurate taxonomic attribution on simulated and pyrosequence data.
• The study provides a robust toolkit for characterizing eukaryotic microbial communities.

Conclusions:

• The presented pipeline and primer sets offer a reliable method for studying eukaryotic microbes in the human microbiome.
• This work addresses a critical need for better tools in eukaryotic microbiome research.
• Enables deeper understanding of the role of eukaryotes in human health and disease.