Metabarcoding free‐living marine nematodes using curated 18S and CO1 reference sequence databases for species‐level taxonomic assignments

Abstract

High‐throughput sequencing has the potential to describe biological communities with high efficiency yet comprehensive assessment of diversity with species‐level resolution remains one of the most challenging aspects of metabarcoding studies. We investigated the utility of curated ribosomal and mitochondrial nematode reference sequence databases for determining phylum‐specific species‐level clustering thresholds. We compiled 438 ribosomal and 290 mitochondrial sequences which identified 99% and 94% as the species delineation clustering threshold, respectively. These thresholds were evaluated in HTS data from mock communities containing 39 nematode species as well as environmental samples from Vietnam. We compared the taxonomic description of the mocks generated by two read‐merging and two clustering algorithms and the cluster‐free Dada2 pipeline. Taxonomic assignment with the RDP classifier was assessed under different training sets. Our results showed that 36/39 mock nematode species were identified across the molecular markers (18S: 32, JB2: 19, JB3: 21) in UClust_ref OTUs at their respective clustering thresholds, outperforming UParse_denovo and the commonly used 97% similarity. Dada2 generated the most realistic number of ASVs (18S: 83, JB2: 75, JB3: 82), collectively identifying 30/39 mock species. The ribosomal marker outperformed the mitochondrial markers in terms of species and genus‐level detections for both OTUs and ASVs. The number of taxonomic assignments of OTUs/ASVs was highest when the smallest reference database containing only nematode sequences was used and when sequences were truncated to the respective amplicon length. Overall, OTUs generated more species‐level detections, which were, however, associated with higher error rates compared to ASVs. Genus‐level assignments using ASVs exhibited higher accuracy and lower error rates compared to species‐level assignments, suggesting that this is the most reliable pipeline for rapid assessment of alpha diversity from environmental samples.

A novel metabarcoding strategy for studying nematode communities

“…Most protocols for nematode metabarcoding include a nematode extraction step to reduce DNA contamination from other soil-living organisms [17][18][19]23] In a recent study, the SSU ribosomal DNA marker (SSU_04F/SSU_22R) outperformed the mitochondrial marker (JB3/JB5GED) in terms of nematode species and genus level detection [17,23]. However, in our study, important nematode species were not amplified and detected by the SSU primer set.…”

“…We selected four primer sets for amplicon sequencing of nematodes (S1 Table). The primer set SSU_04F/SSU_22R (SSU) amplifies the V1-V2 region of the 18S rRNA gene (Fig 1) and was recently used to describe assemblages of free-living soil nematodes using the MiSeq platform [17,23]. We designed a primer set, MMS (MMSF: 5′-GGTGCCAGCAGCCGCGGTA-3′, MMSR: 5′-CTTTAAGT TTCAGCTTTGC-3′) located in the variable region V4-V5 of the 18S rRNA gene (Fig 1).…”

Comparison of morphological, DNA barcoding, and metabarcoding characterizations of freshwater nematode communities

“…In our study, OTU clustering resulted in higher OTU numbers than obtained with the dada2 pipeline, although this was an unexpected result (as OTUs are basically ASVs clustered at a predefined cutoff). Recently, accurate results with the ASV‐approach using dada2 have been reported (Macheriotou et al, ). However, we found that, especially for the 18S gene region, the performance of dada2 was not satisfactory, as it led to the identification of only six ASVs.…”

“…As the true species richness could not be determined using barcoding and metabarcoding approaches, the validity of the comparison with the morphological sample was limited. A more reliable comparison would have been possible if the specimens had been identified prior to Sanger sequencing or metabarcoding, as was the case in other studies (Leasi et al, 2018;Macheriotou et al, 2019).…”

“…Metabarcoding has facilitated studies of small multicellular organisms, either whole communities or specific groups, with marine eukaryotes being a frequent focus (Brannock & Halanych, ; Dell'Anno, Carugati, Corinaldesi, Riccioni, & Danovaro, ; Haenel, Holovachov, Jondelius, Sundberg, & Bourlat, ). It can also be used in combination with morphological analyses, as demonstrated in studies of estuarine plankton (Abad et al, ; Harvey, Johnson, Fisher, Peterson, & Vrijenhoek, ; Leasi et al, ) and nematodes (Holovachov, ; Macheriotou et al, ) in marine habitats but also diatoms and other small organisms in freshwater habitats (Keck, Vasselon, Rimet, Bouchez, & Kahlert, ; Rimet, Vasselon, A.‐Keszte, & Bouchez, ). For nematodes in soil and marine habitats, however, combined microscopy and metabarcoding investigations have been carried out only at the family level (Darby, Todd, & Herman, ; Griffiths, Groot, Laros, Stone, & Geisen, ; Holovachov, Haenel, Bourlat, & Jondelius, ; Treonis et al, ), and direct comparisons of the performances of morphological identification, barcoding, and metabarcoding at the species level are still scarce (Leasi et al, ).…”