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. 2023:30:52.
doi: 10.1051/parasite/2023056. Epub 2023 Nov 28.

Comparing Microsporidia-targeting primers for environmental DNA sequencing

Annemie Doliwa  1 , Daniel Grabner  1 , Bernd Sures  2 , Micah Dunthorn  3
Affiliations

Comparing Microsporidia-targeting primers for environmental DNA sequencing

Annemie Doliwa et al. Parasite. 2023.

Abstract
in English, French

Metabarcoding is a powerful tool to detect classical, and well-known "long-branch" Microsporidia in environmental samples. Several primer pairs were developed to target these unique microbial parasites, the majority of which remain undetected when using general metabarcoding primers. Most of these Microsporidia-targeting primer pairs amplify fragments of different length of the small subunit ribosomal RNA (SSU-rRNA) gene. However, we lack a broad comparison of the efficacy of those primers. Here, we conducted in silico PCRs with three short-read (which amplify a few-hundred base pairs) and two long-read (which amplify over a thousand base pairs) metabarcoding primer pairs on a variety of publicly available Microsporidia sensu lato SSU-rRNA gene sequences to test which primers capture most of the Microsporidia diversity. Our results indicate that the primer pairs do result in slight differences in inferred richness. Furthermore, some of the reverse primers are also able to bind to microsporidian subtaxa beyond the classical Microsporidia, which include the metchnikovellidan Amphiamblys spp., the chytridiopsid Chytridiopsis typographi and the "short-branch" microsporidian Mitosporidium daphniae.

Title: Comparaison des amorces ciblant les Microsporidies pour le séquençage de l’ADN environnemental.

Abstract: Le métabarcoding est un outil puissant pour détecter les microsporidies classiques et bien connues à « longues branches » dans les échantillons environnementaux. Plusieurs paires d’amorces ont été développées pour cibler ces parasites microscopiques exceptionnels, dont la majorité restent indétectables lors de l’utilisation d’amorces générales de métabarcoding. La plupart de ces paires d’amorces ciblant les microsporidies amplifient des fragments de différentes longueurs du gène de la petite sous-unité de l’ARN ribosomal (SSU-rRNA). Cependant, nous manquons d’une comparaison générale de l’efficacité de ces amorces. Ici, pour tester quelles amorces capturent la plus grande partie de la diversité des microsporidies, nous avons réalisé des PCR in silico avec trois paires d’amorces de métabarcoding à lecture courte (qui amplifient quelques centaines de paires de bases) et deux paires d’amorces de métabarcoding à lecture longue (qui amplifient plus d’un millier de bases), sur une variété de séquences du gène SSU-rRNA de Microsporidia sensu lato accessibles au public. Nos résultats indiquent que les paires d’amorces entraînent de légères différences dans la richesse déduite. En outre, certaines des amorces inverses sont également capables de se lier à des sous-taxons de microsporidies au-delà des Microsporidia classiques, notamment les Metchnikovellidae Amphiamblys spp., le Chytridiopsida Chytridiopsis typographi et la microsporidie à « branches courtes » Mitosporidium daphniae.

Keywords: Barcoding; Diversity; Metabarcoding; Microsporidia; Parasites; Protists.

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Figures

Figure 1
Figure 1
Number of microsporidian species in silico amplified by the different primer pair combinations, according to the threshold of accepted mismatches (from none to a maximum of three). The color scheme indicates the corresponding microsporidian subtaxa. The “All sequences”-bar serves as a guidance as it depicts all reference sequences on which the primer pairs were tested.
Figure 2
Figure 2
Number of microsporidian species to which each single primer in silico bound, according to the threshold of accepted mismatches (from none to a maximum of three). The color scheme indicates the corresponding microsporidian subtaxa. The “All sequences”-bar serves as a guidance and depicts all reference sequences on which the primers were tested.
See this image and copyright information in PMC

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