Identifying group-specific primers for environmental Heterolobosa by high-throughput sequencing

Abstract

Diversity of Heterolobosea (Excavata) in environments is poorly understood despite their ecological occurrence and health-associated risk, partly because this group tends to be under-covered by most universal eukaryotic primers used for sequencing. To overcome the limits of the traditional morpho-taxonomy-based biomonitoring, we constructed a primer database listing existing and newly designed specific primer pairs that have been evaluated for Heterolobosea 18S rRNA sequencing. In silico taxonomy performance against the current SILVA SSU database allowed the selection of primer pairs that were next evaluated on reference culture amoebal strains. Two primer pairs were retained for monitoring the diversity of Heterolobosea in freshwater environments, using high-throughput sequencing. Results showed that one of the newly designed primer pairs allowed species-level identification of most heterolobosean sequences. Such primer pair could enable informative, cultivation-free assays for characterizing heterolobosean populations in various environments.

Fig. 1

Specificity of selected primer pairs for detection of the Heterolobosea. Agarose gel electrophoreses (2%) of PCR products using 6 different primer pairs for the environmental isolates of amoebae mentioned in Table S3. Ac: Acanthamoeba, Ec: Echinamoeba, Vr: Vermamoeba, Ng: Naegleria, Tt: Tetramitus, Vh: Vahlkampfia, Wi: Willaertia, Nu: Nuclearia, C: control PCR reaction with no template DNA. The numbers on the left correspond to the molecular weight (bp) of the BenchTop 100 bp DNA ladder (L). Results from PCR experiment conducted at 56°C.

Fig. 2

Sequence dissimilarity based on amplicons yielded by primer pair HF_1202/HR_1680, allows for a clear identification of heterolobosean genera (A). Each genus noted on the right side is colour‐coded. B) Dendrogram based on DNA distance matrix, depicting sequence divergence among the amplicon yielded by primer pair HF_1202/HR_1680 for Naegleria (B) and Tetramitus (C) genera. Scale bar represents the percentage of nucleotidic divergence, with each dotted line representing 1% divergence. [Colour figure can be viewed at wileyonlinelibrary.com]

Fig. 3

Taxonomic assignments of amplicons yielded by primer pairs HF_1198/HR_1680 and HF_1202/HR_1680, from 4 environmental samples. Relative abundances are shown for each sample, and sequences affiliated to the Heterolobosea clade are highlighted in red. [Colour figure can be viewed at wileyonlinelibrary.com]

Fig. 4

Taxonomic assignments of heterolobosean amplicons yielded by primer pairs HF_1198/HR_1680 and HF_1202/HR_1680, from 4 environmental sample. The conventional pipeline using VSEARCH and SILVA allows genus‐level taxonomic identification (A), while the improved pipeline described in this study allow for species‐level identification of most amplicons (B). [Colour figure can be viewed at wileyonlinelibrary.com]