High-throughput Sequencing Metabarcoding Research Articles

High-throughput analysis using 16S rRNA gene of bacterial communities present in selected bivalves and gastropods species from Bayug Island, Iligan City, Philippines

Abstract. Albarido NA, Tabugo SR. 2024. High-throughput analysis using 16S rRNA gene of bacterial communities present in selected bivalves and gastropods species from Bayug Island, Iligan City, Philippines. Biodiversitas 25: 431-438. Seashells, which include bivalves and gastropods, have global recognition for their significant contributions to the economy, ecology, and medicine. They hold value as a food source and are highly regarded as effective biological indicators. The objective of this study is to identify the bacterial communities present in selected edible species of bivalves (Pinctada margaritifera Linnaeus, 1758 and Anadara granosa Linnaeus, 1758) and gastropods (Canarium urceus Linnaeus, 1758 and Conus stercusmuscarum Linnaeus, 1758), through high-throughput sequencing metabarcoding. Bacterial samples were collected via a swabbing technique on the surface and inside parts of selected mollusc species, which were then placed on sterilized seawater for DNA extraction. Genomic DNA was isolated from the samples, and the V3-V4 region of the 16S rRNA gene was amplified and sequenced using the Illumina MiSeq platform. Four amplicon libraries were generated, representing the two bivalve and two gastropod species in the study area. Data analysis was conducted using the Parallel Meta Suite software. Upon quality control and processing, 173,489 amplicon sequence variants (ASVs) were obtained. Within the bacterial community, the most abundant genera included Stenotrophomonas, Vibrio, Serratia, Photobacterium, and Shewanella. The assessment of alpha diversity, using the Shannon index, indicated a higher diversity in A. granosa. Furthermore, the analysis using the PICRUSt algorithm within the Parallel Meta Suite unveiled the involvement of specific bacteria found in the selected gastropod and bivalve species in various functions. These functions encompass protein production, xenobiotic metabolism, biodegradation, and other metabolism-related processes, supporting these organisms' ecological and physiological roles.

When phytoplankton do not bloom: the case of the dinoflagellate Lepidodinium chlorophorum in southern Brittany (France) assessed by environmental DNA

Green seawater discolorations caused by the marine dinoflagellate Lepidodinium chlorophorum are frequently observed during summer along southern Brittany coasts (NE Atlantic, France). Here, the ecology of L. chlorophorum is studied during a non-bloom period using high-throughput sequencing metabarcoding of environmental DNA (eDNA) samples for the detection of this species at low concentrations. Sediment samples (for metabarcoding and cyst analyses) were collected in January–February 2019 and water samples from two stations were collected at three water depths in September–March 2019–2020 and 2020–2021 (for metabarcoding and environmental parameters). The protistan community was dominated by dinoflagellates and was homogenous in the water column. Amplicon sequence variants (ASVs) associated with the genus Lepidodinium were detected in autumn–winter at low relative abundances (minimum: 0.01%). Increases in Lepidodinium abundance were positively correlated with pulses of ammonium re-suspended from bottom sediments. Although Lepidodinium eDNA (<1%) was detected in the sediments, no cyst morphotypes could be associated with Lepidodinium, and germination experiments revealed no Lepidodinium-like cells, leaving in doubt the existence of resting cysts of this species in the seed bank. It is hypothesised that temporary Lepidodinium cells remained present in the water column at low concentrations during the autumn–winter period, awaiting ammonium input from sediments to initiate growth, and that blooms develop when water column stratification and river input provide favourable environmental conditions for biomass increases.

A high-throughput amplicon sequencing approach for population-wide species diversity and composition survey.

Management of agricultural pests requires an understanding of pest species diversity, their interactions with beneficial insects and spatial-temporal patterns of pest abundance. Invasive and agriculturally important insect pests can build up very high populations, especially in cropping landscapes. Traditionally, sampling effort for species identification involves small sample sizes and is labour intensive. Here, we describe a multiprimer high throughput sequencing (HTS) metabarcoding method and associated analytical workflow for a rapid, intensive, high-volume survey of pest species compositions. We demonstrate our method using the taxonomically challenging Bemisia pest cryptic species complex as examples. The whiteflies Bemisia including the"tabaci" species are agriculturally important capable of vectoring diverse plant viruses that cause diseases and crop losses. Our multiprimer metabarcoding HTS amplicon approach simultaneously process high volumes of whitefly individuals, with efficiency to detect rare (i.e., 1%) test-species, while our improved whitefly primers for metabarcoding also detected beneficial hymenopteran parasitoid species from whitefly nymphs. Field-testing our redesigned Bemisia metabarcoding primer sets across the Tanzania, Uganda and Malawi cassava cultivation landscapes, we identified the sub-Saharan Africa 1 Bemisia putative species as the dominant pest species, with other cryptic Bemisia species being detected at various abundances. We also provide evidence that Bemisia species compositions can be affected by host crops and sampling techniques that target either nymphs or adults. Our multiprimer HTS metabarcoding method incorporated two overlapping amplicons of 472bp and 518bp that spanned the entire 657bp 3' barcoding region for Bemisia, and is particularly suitable to molecular diagnostic surveys of this highly cryptic insect pest species complex that also typically exhibited high population densities in heavy crop infestation episodes. Our approach can be adopted to understand species biodiversity across landscapes, with broad implications for improving transboundary biosecurity preparedness, thus contributing to molecular ecological knowledge and the development of control strategies for high-density, cryptic, pest-species complexes.

Novel universal primers for metabarcoding environmental DNA surveys of marine mammals and other marine vertebrates

AbstractMetabarcoding studies using environmental DNA (eDNA) and high‐throughput sequencing (HTS) are rapidly becoming an important tool for assessing and monitoring marine biodiversity, detecting invasive species, and supporting basic ecological research. Several barcode loci targeting teleost fish and elasmobranchs have previously been developed, but to date primer sets focusing on other marine megafauna, such as marine mammals, have received less attention. Similarly, there have been few attempts to identify potentially “universal” barcode loci which may be informative across multiple marine vertebrate orders. Here we describe the design and validation of two new sets of primers targeting hypervariable regions of the vertebrate mitochondrial 12S and 16S rRNA genes, which have conserved priming sites across virtually all cetaceans, pinnipeds, elasmobranchs, boney fish, sea turtles, and birds, and amplify fragments with consistently high levels of taxonomically diagnostic sequence variation. “In silico” validation using the OBITOOLS software showed our new barcode loci outperformed most existing vertebrate barcode loci for taxon detection and resolution. We also evaluated sequence diversity and taxonomic resolution of the new barcode loci in 680 complete marine mammal mitochondrial genomes demonstrating that they are effective at resolving amplicons for most taxa to the species level. Finally, we evaluated the performance of the primer sets with eDNA samples from aquarium communities with known species composition. These new primers will potentially allow surveys of complete marine vertebrate communities in single HTS metabarcoding assessments, simplifying workflows, reducing costs, and increasing accessibility to a wider range of investigators.

Annotated 18S and 28S rDNA reference sequences of taxa in the planktonic diatom family Chaetocerotaceae.

The species-rich diatom family Chaetocerotaceae is common in the coastal marine phytoplankton worldwide where it is responsible for a substantial part of the primary production. Despite its relevance for the global cycling of carbon and silica, many species are still described only morphologically, and numerous specimens do not fit any described taxa. Nowadays, studies to assess plankton biodiversity deploy high throughput sequencing metabarcoding of the 18S rDNA V4 region, but to translate the gathered metabarcodes into biologically meaningful taxa, there is a need for reference barcodes. However, 18S reference barcodes for this important family are still relatively scarce. We provide 18S rDNA and partial 28S rDNA reference sequences of 443 morphologically characterized chaetocerotacean strains. We gathered 164 of the 216 18S sequences and 244 of the 413 28S sequences of strains from the Gulf of Naples, Atlantic France, and Chile. Inferred phylogenies showed 84 terminal taxa in seven principal clades. Two of these clades included terminal taxa whose rDNA sequences contained spliceosomal and Group IC1 introns. Regarding the commonly used metabarcode markers in planktonic diversity studies, all terminal taxa can be discriminated with the 18S V4 hypervariable region; its primers fit their targets in all but two species, and the V4-tree topology is similar to that of the 18S. Hence V4-metabarcodes of unknown Chaetocerotaceae are assignable to the family. Regarding the V9 hypervariable region, most terminal taxa can be discriminated, but several contain introns in their primer targets. Moreover, poor phylogenetic resolution of the V9 region affects placement of metabarcodes of putative but unknown chaetocerotacean taxa, and hence, uncertainty in taxonomic assignment, even of higher taxa.

Biodiversity of phytoplankton cyst assemblages in surface sediments of the Black Sea based on metabarcoding

Resting stages are common for the life cycle of some phytoplankton species, including blooming and potentially toxic species. The “seed bank” accumulated in the sediments can initiate blooms in the water column and could be an early warning signal of harmful algal blooms (HABs). In order to identify the phytoplankton cyst assemblages, thirteen surface sediment samples were collected from different sites in the Black Sea. The diversity of the resting stages was assessed using high-throughput sequencing metabarcoding (V7-9 hypervariable region of the 18S rDNA). One hundred and eighty microalgal species were identified with high level of similarity to the reference sequences. Dinoflagellates were dominated by Biecheleria, Gymnodinium and Karlodinium. Within diatoms, Skeletonema, Chaetoceros and Thalassiosira were the most abundant genera. Sixteen of the detected operational taxonomic units (OTUs) were assigned to harmful microalgae (12 dinoflagellates species, 1 diatom, 1 haptophyte and 2 raphidophytes). No pattern of microalgal sequences depth distribution was discriminated. The results show that DNA metabarcoding has a great potential for assessment of the phytoplankton diversity in environmental sediments.

Assessment of Passive Traps Combined with High-Throughput Sequencing To Study Airborne Fungal Communities.

Techniques based on high-throughput sequencing (HTS) of environmental DNA have provided a new way of studying fungal diversity. However, these techniques suffer from a number of methodological biases which may appear at any of the steps involved in a metabarcoding study. Air is one of the most important environments where fungi can be found, because it is the primary medium of dispersal for many species. Looking ahead to future developments, it was decided to test 20 protocols, including different passive spore traps, spore recovery procedures, DNA extraction kits, and barcode loci. HTS was performed with the Illumina MiSeq platform targeting two subloci of the fungal internal transcribed spacer. Multivariate analysis and generalized linear models showed that the type of passive spore trap, the spore recovery procedure, and the barcode all impact the description of fungal communities in terms of richness and diversity when assessed by HTS metabarcoding. In contrast, DNA extraction kits did not significantly impact these results. Although passive traps may be used to describe airborne fungal communities, a study using specific real-time PCR and a mock community showed that these kinds of traps are affected by environmental conditions that may induce losses of biological material, impacting diversity and community composition results.IMPORTANCE The advent of high-throughput sequencing (HTS) methods, such as those offered by next-generation sequencing (NGS) techniques, has opened a new era in the study of fungal diversity in different environmental substrates. In this study, we show that an assessment of the diversity of airborne fungal communities can reliably be achieved by the use of simple and robust passive spore traps. However, a comparison of sample processing protocols showed that several methodological biases may impact the results of fungal diversity when assessed by metabarcoding. Our data suggest that identifying these biases is of paramount importance to enable a correct identification and relative quantification of community members.

Molecular Identification of Gambierdiscus and Fukuyoa (Dinophyceae) from Environmental Samples.

Ciguatera Fish Poisoning (CFP) is increasing across the Pacific and the distribution of the causative dinoflagellates appears to be expanding. Subtle differences in thecal plate morphology are used to distinguish dinoflagellate species, which are difficult to determine using light microscopy. For these reasons we sought to develop a Quantitative PCR assay that would detect all species from both Gambierdiscus and Fukuyoa genera in order to rapidly screen environmental samples for potentially toxic species. Additionally, a specific assay for F. paulensis was developed as this species is of concern in New Zealand coastal waters. Using the assays we analyzed 31 samples from three locations around New Zealand and the Kingdom of Tonga. Fourteen samples in total were positive for Gambierdiscus/Fukuyoa and two samples were also positive using the F. paulensis assay. Samples from the Kermadec Islands were further characterized using high-throughput sequencing metabarcoding. The majority of reads corresponded to Gambierdiscus species with three species identified at all sites (G. australes, G. honu and G. polynesiensis). This is the first confirmed identification of G. polynesiensis, a known ciguatoxin producer, in New Zealand waters. Other known toxin-producing genera were also detected, included Alexandrium, Amphidinium, Azadinium, Dinophysis, Ostreopsis, and Prorocentrum.

Application of high-throughput sequencing (HTS) metabarcoding to diatom biomonitoring: Do DNA extraction methods matter?

AbstractCurrent freshwater biomonitoring with diatoms is based on microscopic examination of the morphology of their silica skeleton. This standardized approach is time consuming and requires a high degree of taxonomic expertise. Metabarcoding combined with high-throughput sequencing (HTS) has great potential for next-generation biomonitoring applications but requires standardization. Molecular inventories are strongly influenced by the DNA extraction method used, but the effect of extraction protocols has not been tested to enable selection of the best DNA extraction method for HTS metabarcoding. We used 5 DNA extraction methods combining various types of cell lysis and DNA purification to extract DNA from 8 pure diatom cultures and 8 samples from streams and lakes with differing water quality. We compared the methods based on: 1) quality and purity of the extracted DNA, 2) community inventories obtained from HTS targeting the ribulose-1, 5-bisphosphate carboxylase (rbcL) barcode, and 3) similarity betwe...

Ground-truthing Phylotype Assignments for Antarctic Invertebrates

AbstractBiodiversity information from Antarctic terrestrial habitats helps conservation efforts, but the distribution and diversity particularly of microinvertebrates remains poorly understood. Springtails, mites, tardigrades, nematodes and rotifers are difficult to identify using morphological features, hence DNA-based metabarcoding methods are well suited for their study. We compared taxonomy assignments of a high throughput sequencing metabarcoding approach using one ribosomal DNA (18S rDNA) and one mitochondrial DNA (cytochrome c oxidase subunit I - COI) marker with morphological reference data. Specifically, we compared metabarcoding or morphological taxonomic assignments on multiple taxonomic levels in an artificial DNA blend containing Australian invertebrates, and in seven extracts of Antarctic soils containing known micro-faunal taxa. Avoiding arbitrary application of metabarcoding analysis parameters, we calibrated those parameters with metabarcoding data from non-Antarctic soils. Metabarcoding approaches employing 18S rDNA and COI markers enabled detection of small and cryptic Antarctic invertebrates, and on low taxonomic ranks 18S data outperformed COI data in this respect. Morphological taxonomy determination did not outperform metabarcoding approaches. Our study demonstrates how barcoding markers can be tested prior to their application to specific taxonomic groups, and that taxonomy fidelity of markers needs to be validated in relation to environment, taxa, and available reference information.

Arthropod and oligochaete assemblages from grasslands of the southern Kenai Peninsula, Alaska.

BackgroundBy the end of this century, the potential climate-biome of the southern Kenai Peninsula is forecasted to change from transitional boreal forest to prairie and grasslands, a scenario that may already be playing out in the Caribou Hills region. Here, spruce (Picea × lutzii Little [glauca × sitchensis]) forests were heavily thinned by an outbreak of the spruce bark beetle (Dendroctonus rufipennis (Kirby, 1837)) and replaced by the native but invasive grass species, Calamagrostis canadensis (Michx.) P. Beauv. As part of a project designed to delimit and characterize potentially expanding grasslands in this region, we sought to characterize the arthropod and earthworm communities of these grasslands.We also used this sampling effort as a trial of applying high-throughput sequencing metabarcoding methods to a real-world inventory of terrestrial arthropods.New informationWe documented 131 occurrences of 67 native arthropod species at ten sites, characterizing the arthropod fauna of these grasslands as being dominated by Hemiptera (60% of total reads) and Diptera (38% of total reads). We found a single exotic earthworm species, Dendrobaena octaedra (Savigny, 1826), at 30% of sites and one unidentified enchytraeid at a single site. The utility of high-throughput sequencing metabarcoding as a tool for bioassessment of terrestrial arthropod assemblages was confirmed.

Age-related environmental gradients influence invertebrate distribution in the Prince Charles Mountains, East Antarctica.

The potential impact of environmental change on terrestrial Antarctic ecosystems can be explored by inspecting biodiversity patterns across large-scale gradients. Unfortunately, morphology-based surveys of Antarctic invertebrates are time-consuming and limited by the cryptic nature of many taxa. We used biodiversity information derived from high-throughput sequencing (HTS) to elucidate the relationship between soil properties and invertebrate biodiversity in the Prince Charles Mountains, East Antarctica. Across 136 analysed soil samples collected from Mount Menzies, Mawson Escarpment and Lake Terrasovoje, we found invertebrate distribution in the Prince Charles Mountains significantly influenced by soil salinity and/or sulfur content. Phyla Tardigrada and Arachnida occurred predominantly in low-salinity substrates with abundant nutrients, whereas Bdelloidea (Rotifera) and Chromadorea (Nematoda) were more common in highly saline substrates. A significant correlation between invertebrate occurrence, soil salinity and time since deglaciation indicates that terrain age indirectly influences Antarctic terrestrial biodiversity, with more recently deglaciated areas supporting greater diversity. Our study demonstrates the value of HTS metabarcoding to investigate environmental constraints on inconspicuous soil biodiversity across large spatial scales.

A primer to metabarcoding surveys of Antarctic terrestrial biodiversity

AbstractIce-free regions of Antarctica are concentrated along the coastal margins but are scarce throughout the continental interior. Environmental changes, including the introduction of non-indigenous species, increasingly threaten these unique habitats. At the same time, the unique biotic communities subsisting in isolation across the continent are difficult to survey due to logistical constraints, sampling challenges and problems related to the identification of small and cryptic taxa. Baseline biodiversity data from remote Antarctic habitats are still missing for many parts of the continent but are critical to the detection of community changes over time, including newly introduced species. Here we review the potential of standardized (non-specialist) sampling in the field (e.g. from soil, vegetation or water) combined with high-throughput sequencing (HTS) of bulk DNA as a possible solution to overcome some of these problems. In particular, HTS metabarcoding approaches benefit from being able to process many samples in parallel, while workflow and data structure can stay highly uniform. Such approaches have quickly gained recognition and we show that HTS metabarcoding surveys are likely to play an important role in continent-wide biomonitoring of all Antarctic terrestrial habitats.

Stepwise implementation of high-throughput sequencing metabarcoding to estuarine macrobenthic communities

This work was supported by FEDER through POFC-COMPETE by national funds from ‘Fundacao para a Ciencia e a Tecnologia (FCT)’ in the scope of the grant FCOMP-01-0124-FEDER-015429 and also by the strategic programme UID/BIA/04050/2013 (POCI-01- 0145-FEDER-007569) also funded by national funds through the FCT I.P. and by the ERDF through the COMPETE2020 - Programa Operacional Competitividade e Internacionalizacao (POCI). Jorge Lobo was supported by a PhD fellowship (SFRH/BD/69750/2010) from FCT.

Metabarcoding approach for nonindigenous species surveillance in marine coastal waters

In this study, high-throughput sequencing (HTS) metabarcoding was applied for the surveillance of plankton communities within the southeastern (SE) Baltic Sea coastal zone. These results were compared with those from routine monitoring survey and morphological analyses. Four of five nonindigenous species found in the samples were identified exclusively by metabarcoding. All of them are considered as invasive in the Baltic Sea with reported impact on the ecosystem and biodiversity. This study indicates that, despite some current limitations, HTS metabarcoding can provide information on the presence of exotic species and advantageously complement conventional approaches, only requiring the same monitoring effort as before. Even in the currently immature status of HTS, this combination of HTS metabarcoding and observational records is recommended in the early detection of marine pests and delivery of the environmental status metrics of nonindigenous species.