Evaluation of metagenetic community analysis of planktonic copepods using Illumina MiSeq: Comparisons with morphological classification and metagenetic analysis using Roche 454

Abstract

Metagenetics is a rapid and taxonomically comprehensive method for revealing community structures within environmental samples, based on large amounts of sequence data produced by high-throughput sequencers. Because community structures of planktonic copepods are important in the ocean owing to their high diversity and abundance, a metagenetic analysis of the 28S D2 region using Roche 454 was previously developed. However, the Illumina MiSeq platform with a high sequence output is being used more frequently in metagenetics, and non-calanoid copepods have not previously been fully evaluated. Here, we evaluated an Illumina MiSeq-based metagenetic analysis using a mock community and field-collected samples that were examined in a previous study using Roche 454, and the community structure, including non-calanoid copepods, was compared among morphological and metagenetic analyses. We removed a singleton read and applied an appropriate abundance threshold to remove erroneous Molecular Operational Taxonomic Units (MOTUs) with low-abundance sequences in the MiSeq-based analysis. Results showed that the copepod community was successfully characterized using Illumina MiSeq. Higher-quality sequences were obtained using MiSeq than by Roche 454, which reduced the overestimation of diversity, especially at a strict 99% similarity threshold for MOTU clustering. Taxonomic compositions in terms of both biomass and presence/absence of species, including non-calanoids, were more appropriately represented in the MiSeq- than in Roche 454-based analysis. Our data showed that metagenetic analysis using Illumina MiSeq is more useful for revealing copepod communities than Roche 454, owing to the lower cost and higher quality.

Large-scale metabarcoding analysis of epipelagic and mesopelagic copepods in the Pacific

“…The metabarcoding analysis of copepods was mainly performed according to methods described in a previous paper [28]. Genomic DNA was extracted using the Gentra Puregene Cell and Tissue Kit (QIAGEN) from the 205 environmental zooplankton samples and two mock community samples containing morphologically identified copepod species (S1 Fig).…”

“…Genomic DNA was extracted using the Gentra Puregene Cell and Tissue Kit (QIAGEN) from the 205 environmental zooplankton samples and two mock community samples containing morphologically identified copepod species (S1 Fig). The 19 environmental samples at epipelagic layer in the tropical and subtropical Pacific were previously analyzed using Roche 454 or Illumina MiSeq in our previous studies [26, 28], and the same DNA or raw sequence data were used in this study. The DNA concentration of each sample was measured with a Qubit 2.0 Fluorometer (Life Technologies).…”

“…OTU clustering was performed using OptiClust [61]. Following Hirai et al [28] and the results of mock community analyses, we used a criterion of OTUs at the 98.5 % similarity threshold and a minimum sequence reads for OTUs of ≥ 8, to avoid overestimating diversity and to maintain an appropriate taxonomic resolution. The representative sequence of each copepod OTU was obtained based on the most abundant sequence reads.…”

“…This technique has already been applied to copepods using the Roche 454 high-throughput sequencer, revealing the broad-scale diversity and biogeography of copepods in the epipelagic layer of tropical and subtropical regions in the Pacific based on operational taxonomic units (OTUs) of 28S rRNA gene sequences [26, 27]. The metabarcoding method of copepods was further developed for use with the Illumina MiSeq platform, leading to low-cost and high-quality analyses of marine planktonic copepod diversity with high taxonomic resolutions [28]. In addition to high taxonomic resolution, metabarcoding analysis provides nucleotide sequences of OTUs, which are expected to provide insights into large-scale evolutionary processes and the reasons underlying patterns of copepod diversity.…”

Cryptic Zooplankton Diversity Revealed by a Metagenetic Approach to Monitoring Metazoan Communities in the Coastal Waters of the Okhotsk Sea, Northeastern Hokkaido

“…Although a high similarity threshold (e.g., 99%) is expected to be used when analyzing metazoan zooplankton (Mohrbeck et al, 2015), we instead used a comparatively low similarity threshold (97%), a technique recommended to prevent overestimations of diversity (Kunin et al, 2010) that is often used in metagenetic analyses of zooplankton using the 18S V9 region (Pearman et al, 2014;Casas et al, 2017). Additionally, we used a strict abundance threshold (eight minimum sequence reads), because rare MOTUs are not always reliable and lead to overestimations of diversity (Bokulich et al, 2013;Hirai et al, 2017b;Leray and Knowlton, 2017;Sommer et al, 2017). Indeed, an even stricter minimum threshold of 16 sequence reads would have identified 429 MOTUs, a number much higher than that detected morphologically.…”