Comparing Microsporidia-targeting primers for environmental DNA sequencing.

Area of Science:

• Microbial Ecology
• Molecular Parasitology
• Bioinformatics

Background:

• Metabarcoding is crucial for detecting Microsporidia in environmental samples.
• Existing primers often miss diverse Microsporidia, particularly "long-branch" lineages.
• A comprehensive comparison of Microsporidia-targeting primer efficacy is lacking.

Purpose of the Study:

• To evaluate and compare the performance of different metabarcoding primer pairs for Microsporidia detection.
• To assess the ability of primers to capture Microsporidia diversity using in silico PCR.
• To identify primers that maximize Microsporidia detection and explore their taxonomic range.

Main Methods:

• In silico PCR simulations were performed using three short-read and two long-read primer pairs.
• Primer efficacy was tested against a diverse set of publicly available Microsporidia sensu lato small subunit ribosomal RNA (SSU-rRNA) gene sequences.
• The study analyzed differences in inferred richness and primer binding specificity.

Main Results:

• Primer pairs exhibited slight variations in their ability to infer Microsporidia richness.
• Some reverse primers demonstrated binding to Microsporidia taxa beyond the classical "long-branch" groups.
• Detected non-classical Microsporidia included metchnikovellidans, chytridiopsids, and "short-branch" Microsporidia.

Conclusions:

• The selection of primer pairs significantly influences the detected Microsporidia diversity in metabarcoding studies.
• Certain primers show potential for broader Microsporidia detection, including "short-branch" and related lineages.
• Further optimization of primer sets is needed for comprehensive Microsporidia surveillance in environmental DNA.