Metabarcoding analysis of strongylid nematode diversity in two sympatric primate species

Abstract

Strongylid nematodes in large terrestrial herbivores such as great apes, equids, elephants, and humans tend to occur in complex communities. However, identification of all species within strongylid communities using traditional methods based on coproscopy or single nematode amplification and sequencing is virtually impossible. High-throughput sequencing (HTS) technologies provide opportunities to generate large amounts of sequence data and enable analyses of samples containing a mixture of DNA from multiple species/genotypes. We designed and tested an HTS approach for strain-level identification of gastrointestinal strongylids using ITS-2 metabarcoding at the MiSeq Illumina platform in samples from two free-ranging non-human primate species inhabiting the same environment, but differing significantly in their host traits and ecology. Although we observed overlapping of particular haplotypes, overall the studied primate species differed in their strongylid nematode community composition. Using HTS, we revealed hidden diversity in the strongylid nematode communities in non-human primates, more than one haplotype was found in more than 90% of samples and coinfections of more than one putative species occurred in 80% of samples. In conclusion, the HTS approach on strongylid nematodes, preferably using fecal samples, represents a time and cost-efficient way of studying strongylid communities and provides a resolution superior to traditional approaches.

Genetic diversity of primate strongylid nematodes: Do sympatric nonhuman primates and humans share their strongylid worms?

“…PCR amplification was performed on the second internal transcribed spacer (ITS‐2) of nuclear ribosomal DNA with forward primer Strongyl_ITS‐2_F (acg tct ggt tca ggg ttg) and reverse Strongyl_ITS‐2_R (atg ctt aag ttc agc ggg ta). We have already showed that these primers exhibit a low level of PCR bias and provide consistent community profiles (Pafčo et al, ), suggesting that they can be used for semi‐quantitative analyses of complex strongylid communities. HTS sequencing libraries were generated using a two‐step‐PCR approach following Fluidigm Access Array primer design (Pafčo et al, ).…”

“…We have already showed that these primers exhibit a low level of PCR bias and provide consistent community profiles (Pafčo et al, ), suggesting that they can be used for semi‐quantitative analyses of complex strongylid communities. HTS sequencing libraries were generated using a two‐step‐PCR approach following Fluidigm Access Array primer design (Pafčo et al, ). Analyses were carried out in two technical replicates with different tag primer barcodes.…”

“…Analyses were carried out in two technical replicates with different tag primer barcodes. DNA extraction from strongylid negative human faeces and water were used as negative controls and synthetic DNA templates as positive controls (Pafčo et al, ). The final library was sequenced using MiSeq Reagent Kit v2 (2 × 250 bp pair end reads 500 cycles) by Illumina MiSeq platform.…”

“…Next, low quality (expected error rate >1) reads were eliminated, and the filtered data set was denoised using dada2 (Callahan et al, ) in order to identify ITS‐2 haplotypes. To avoid inflation of strongylid diversity by PCR/sequencing artefacts that were not corrected by dada2, we considered only those ITS‐2 haplotypes that were consistently present in both technical duplicates for a given sample, (Pafčo et al, ). We excluded seven samples where the number of high‐quality sequences after all filtering steps was low (<500).…”

“…On the contrary, high‐throughput sequencing (HTS) provides a powerful tool for such analyses (Aivelo & Medlar, ; Avramenko et al, ; Lott, Hose, & Power, ; Tanaka et al, ). Recently, we applied a HTS approach relying on ITS‐2 metabarcoding on a two NHP host‐model system, and showed sufficient sensitivity for strain‐level profiling of strongylid nematodes and sufficient robustness against false positives (Pafčo et al, ).…”