High-throughput DNA Metabarcoding Research Articles

First DNA metabarcoding diet assessment on the critically endangered Tricolour Langur, Presbytis chrysomelas cruciger

Presbytis chrysomelas cruciger or also known as the Tricolour langur—is rare, endemic to Sarawak and Kalimantan in Borneo and classified as a critically endangered subspecies. The current P. c. cruciger population size is uncertain because the numbers are continuously decreasing. At present, there is no comprehensive scientific report on P. c. cruciger in Sarawak, although this subspecies is known to inhabit Maludam area. Recent first sighting of P. c. cruciger in Jemoreng Sarawak presents a research opportunity to study its feeding from a molecular ecology perspective. Herein, we report the first findings on the dietary intake of P. c. cruciger using a high-throughput DNA metabarcoding approach. We emphasise the diet intake of P. c. cruciger from Jemoreng Protected Forest in Sarawak using DNA metabarcoding of the trnL region. Preliminary findings revealed 11 amplicon sequence variants (ASV) classified into one phylum, four classes, four orders, four families, three genera and three plant species. Fibraurea tinctoria (akar kuning; Family Menispermaceae), Poikilospermum suaveolens (akar jangkang; Family Urticaceae) and Litchi chinensis (lychee; Family Sapindaceae) were the three main plant species that were consumed by P. c. cruciger. Understanding the dietary intake of P. c. cruciger is of paramount importance for their conservation and management of the habitat areas where their population resides.

First DNA metabarcoding diet assessment on the critically endangered Tricolour Langur, Presbytis chrysomelas cruciger

Presbytis chrysomelas cruciger or also known as the Tricolour langur—is rare, endemic to Sarawak and Kalimantan in Borneo and classified as a critically endangered subspecies. The current P. c. cruciger population size is uncertain because the numbers are continuously decreasing. At present, there is no comprehensive scientific report on P. c. cruciger in Sarawak, although this subspecies is known to inhabit Maludam area. Recent first sighting of P. c. cruciger in Jemoreng Sarawak presents a research opportunity to study its feeding from a molecular ecology perspective. Herein, we report the first findings on the dietary intake of P. c. cruciger using a high-throughput DNA metabarcoding approach. We emphasise the diet intake of P. c. cruciger from Jemoreng Protected Forest in Sarawak using DNA metabarcoding of the trnL region. Preliminary findings revealed 11 amplicon sequence variants (ASV) classified into one phylum, four classes, four orders, four families, three genera and three plant species. Fibraurea tinctoria (akar kuning; Family Menispermaceae), Poikilospermum suaveolens (akar jangkang; Family Urticaceae) and Litchi chinensis (lychee; Family Sapindaceae) were the three main plant species that were consumed by P. c. cruciger. Understanding the dietary intake of P. c. cruciger is of paramount importance for their conservation and management of the habitat areas where their population resides.

Insights into freshwater ciliate diversity through high throughput DNA metabarcoding.

The freshwater bodies of India are highly biodiverse but still understudied, especially concerning ciliates. Ciliates constitute a significant portion of eukaryotic diversity and play crucial roles in microbial loops, nutrient recycling, and ecosystem maintenance. The present study aimed to elucidate ciliate diversity in three freshwater sites in the Delhi region of India: Okhla Bird Sanctuary (OBS), Sanjay Lake (SL), and Raj Ghat pond (RJ). This study represents the first investigation into the taxonomic diversity and richness of freshwater ciliates in India using a high-throughput DNA metabarcoding approach. For the analysis, total environmental DNA was extracted from the three freshwater samples, followed by sequencing of the 18S V4 barcode region and subsequent phylogenetic analyses. Operational taxonomic units (OTU) analyses revealed maximum species diversity in OBS (106), followed by SL (104) and RJ (99) sites. Ciliates from the classes Oligohymenophorea, Prostomatea, and Spirotrichea were dominant in the three sites. The study discusses the ability of the metabarcoding approach to uncover unknown and rare species. The study highlights the need for refined reference databases and cautious interpretation of the high-throughput sequencing-generated data while emphasizing the complementary nature of molecular and morphological approaches in studying ciliate diversity.

DNA metabarcoding of cloacal swabs provides insight into diets of highly migratory sharks in the Mid-Atlantic Bight.

The abundances of migratory shark species observed throughout the Mid-Atlantic Bight (MAB) during productive summer months suggest that this region provides critical habitat and prey resources to these taxa. However, the principal prey assemblages sustaining migratory shark biomass in this region are poorly defined. We applied high-throughput DNA metabarcoding to shark feces derived from cloacal swabs across nine species of Carcharhinid and Lamnid sharks to (1) quantify the contribution of broad taxa (e.g., invertebrates, fishes) supporting shark biomass during seasonal residency in the MAB and (2) determine whether the species displayed distinct dietary preference indicative of resource partitioning. DNA metabarcoding resulted in high taxonomic (species-level) resolution of shark diets with actinopterygian and elasmobranch fishes as the dominant prey categories across the species. DNA metabarcoding identified several key prey groups consistent across shark taxa that are likely integral for sustaining their biomass in this region, including Atlantic menhaden (Brevoortia tyrannus), Atlantic mackerel (Scomber scombrus), and benthic elasmobranchs, including skates. Our results are consistent with previously published stomach content data for the shark species of similar size range in the Northwest Atlantic Ocean, supporting the efficacy of cloacal swab DNA metabarcoding as a minimally invasive diet reconstruction technique. The high reliance of several shark species on Atlantic menhaden could imply wasp-waist food-web conditions during the summer months, whereby high abundances of forage fishes sustain a diverse suite of migratory sharks within a complex, seasonal food web.

High-throughput DNA metabarcoding for determining the gut microbiome of captive critically endangered Malayan tiger (Pantheratigrisjacksoni) during fasting.

The Malayan tiger (Pantheratigrisjacksoni) is a critically endangered species native to the Malaysian Peninsula. To imitate wild conditions where tigers do not hunt every day, numerous wildlife sanctuaries do not feed their tigers daily. However, the effects of fasting on the gut microbiota of captive Malayan tigers remains unknown. This study aimed to characterise the gut microbiota of captive Malayan tigers by comparing their microbial communities during fasting versus normal feeding conditions. This study was conducted at the Melaka Zoo, Malaysian Peninsula and involved Malayan tigers fasted every Monday. In total, ten faecal samples of Malayan tiger, two of Bengal tiger (outgroup) and four of lion (outgroup) were collected and analysed for metabarcoding targeting the 16S rRNA V3-V4 region. In total, we determined 14 phyla, 87 families, 167 genera and 53 species of gut microbiome across Malayan tiger samples. The potentially harmful bacterial genera found in this study included Fusobacterium, Bacteroides, Clostridium sensu stricto 1, Solobacterium, Echerichiashigella, Ignatzschineria and Negativibacillus. The microbiome in the fasting phase had a higher composition and was more diverse than in the feeding phase. The present findings indicate a balanced ratio in the dominant phyla, reflecting a resetting of the imbalanced gut microbiota due to fasting. These findings can help authorities in how to best maintain and improve the husbandry and health of Malayan tigers in captivity and be used for monitoring in ex-situ veterinary care unit.

High-Throughput DNA Metabarcoding of Stem Sections from Trees with Cavities Describes Fungal Communities Associated with Variable Wood Decay, Position on Stem and Tree Species

The presence of unculturable fungi, variability in timing and frequency of fungal fruiting, hyper-rich fungal communities, and genetic and environmental variability explains the difficulty in adopting ideal sampling schemes and fungal identification approaches in studies of fungal communities in wood at variable stages of decay. Here, we use intensive within-tree sampling of five standing trees with cavities paired with high-throughput DNA metabarcoding, to study fungal communities in decayed and healthy wood of trees from two Populus species in British Columbia, Canada. The amplification of over 3000 fungal DNA sequence variants shows the presence of a hyper-rich wood fungal community that not only varied depending on PCR primers, tree species, tree stem portion and wood decay stage. but also included a large number of taxa unassignable to any known sub-kingdom taxonomic order based on published DNA sequences. Our data show that the use of two different primer sets greatly increases the power of the metabarcoding analysis. By testing three alternative models of fungal community composition, we identify the model that best explains fungal community by considering the position on the stem and distance from the cavity. We suggest this model may be used to design optimal sampling schemes to describe fungal communities in trees experiencing discrete decay pockets or cavities.

Bacterial and yeast communities in raw milk from three dairy animal species in Nigeria.

The present study aimed to reveal the microbial (bacteria and yeast) composition of raw milk from dairy camel (n=10), cow (n=10) and goat (n=10) in North-western Nigeria. High-throughput DNA metabarcoding was used to compare microbial compositions in raw milk among the three species. Although the three species had similar dominant bacterial (Firmicutes and Proteobacteria) and yeast (Ascomycota and Basidiomycota) phyla, their microbial compositions at the genus level were noticeably different. The top differentially abundant bacterial and yeast genera (percentage abundance) were Lactobacillus (36%), Streptococcus (34%), Enterococcus (12%), Kluyveromyces (28%), Saccharomyces (24%), and Candida (18%), respectively. Principal coordinate analysis based on unweighted UniFrac values revealed significant differences in the structure of bacterial communities and no differences in yeast communities in milk samples from the three species. This study provides insight into the rich and diverse bacterial and yeast communities in raw animal milk consumed in Nigeria, which could play beneficial roles or pose health threats to consumers. However, further research on the economic significance of the microbial community in animal milk consumed in Nigeria is required.

High-Throughput DNA Metabarcoding as an Approach for Ichthyoplankton Survey in Oujiang River Estuary, China

High-throughput DNA metabarcoding of mitochondrial 12S rRNA and Cyt b gene sequences was coupled with a morphology-based identification tool to assess ichthyoplankton community structure in Oujiang River Estuary, China. The performances of 12S and Cyt b barcoding markers were compared in terms of taxonomic resolution, detection and coverage, and their suitability was established for use as a quick and powerful ichthyoplankton assessment tool. A total of 30,138 ichthyoplankton (2462 eggs and 27,676 larvae) samples were collected from April to August 2015 and identified to 145 taxa belonging to 57 families and 105 genera. June and July were the main spawning months. Ichthyoplankton were more abundant around Lingkun and Qidu Islands and the upper parts of Oujiang River Estuary. The 12S gene marker presented higher species coverage and detection rate than Cyt b. DNA metabarcoding exhibited more representative species identification power than morphology. The findings reported in this study provided a key attempt towards the development of time-efficient and cost-effective ichthyoplankton identification and assessment tool.

Determining the Dietary Preferences of Wild Asian Elephants (Elephas maximus) in Taman Negara National Park, Malaysia Based on Sex and Age using trnL DNA Metabarcoding Analysis.

The world's largest terrestrial mammal, Asian elephants, are known to have enormous feeding needs. Several factors such as season, sex, age, and daily activities influence the amount of food required by an individual. Generally, captive elephants have a limited choice of food on a daily basis compared with that of elephants in the wild. Elephants in captivity are fed according to a prepared feeding schedule, whereas wild elephants are free to choose the type of plants that they consume in their natural habitat. In the past, ecological observations have been widely used to determine the diet of wild elephants. However, the molecular approach has never been carried out. In the present study, we aimed to; 1) identify the plant diet of wild Asian elephants in Taman Negara National Park (TNNP) according to their sex and age using high-throughput DNA metabarcoding; and 2) determine the dietary formulation of captive elephants based on the generated plant metabarcoding database. DNA was extracted from 24 individual fecal samples collected using noninvasive sampling techniques from TNNP and the National Elephant Conservation Centre (NECC) Kuala Gandah. Seven pooled samples from male adult, female adult, male subadult, female subadult, male juvenile, female juvenile, and captive elephants were amplified and sequenced targeting the trnL region (50-150 base pairs). The CLC Genomic Workbench and PAST 4.02 software were used for data analysis. In total, 24 orders, 41 families, 233 genera, and 306 species of plants were successfully detected in the diet of the Asian elephants. The most abundant plant genera consumed were Sporobolus (21.88%), Musa (21.48%), and Ficus (10.80%). Plant variation was lower in samples from male elephants than in those from female elephants. The plant species identified were correlated with the nutrient benefits required by elephants. Adults and subadults consumed more plant species than were consumed by juvenile elephants. However, there was no significant difference between ages and sexes. The findings of this study can be used as guidance by the Department of Wildlife and National Parks for the management of captive elephants, especially in NECC Kuala Gandah.

Zooplankton diversity monitoring strategy for the urban coastal region using metabarcoding analysis

Marine ecosystems in urban coastal areas are exposed to many risks due to human activity. Thus, long-term and continuous monitoring of zooplankton diversity is necessary. High-throughput DNA metabarcoding has gained recognition as an efficient and highly sensitive approach to accurately describing the species diversity of marine zooplankton assemblages. In this study, we collected 30 zooplankton samples at about 2-week intervals for 1 year. Zooplankton diversity showing a typical four season pattern. Of the “total” and “common” zooplankton, we assigned 267 and 64 taxa. The cluster structure and seasonal diversity pattern were rough when only the “common” zooplankton was used. Our study examined how to maximize the benefits of metabarcoding for monitoring zooplankton diversity in urban coastal areas. The results suggest that to take full advantage of metabarcoding when monitoring a zooplankton community, it is necessary to carefully investigate potential ecosystem threats (non-indigenous species) through sufficient curation rather than disregarding low-abundance operational taxonomic units.

Arbuscular mycorrhizal fungal communities colonising the roots of indigenous legumes of South Africa as revealed by high-throughput DNA metabarcoding

The important ecological role of arbuscular mycorrhizal (AM) fungi on plant communities necessitates an understanding of the diversity and the factors that shape the community composition of AM fungi associated with plants. Therefore, this study investigated the diversity and composition of arbuscular mycorrhizal fungal (AMF) communities colonising the roots of eleven indigenous legumes in two South African provinces using high-throughput sequencing of the partial SSU rRNA gene. The analysis revealed 172 operational taxonomic units (OTUs, at 97% sequence similarity) belonging to eight genera. Arbuscular mycorrhizal fungal community composition varied among legumes within and between provinces, with the genus Glomus dominant. The AMF OTU richness and Shannon-Wiener index were significantly correlated with potassium, copper, manganese, zinc, and silt content. Canonical correspondence analysis indicated that nitrates, pH, manganese, and organic C were the principal soil properties significantly influencing AMF community composition. Overall, findings from this study elucidate that soil factors are pertinent environmental cues that shape legume-AMF symbiosis in semi-arid ecosystems.

Stem-inhabiting fungal communities differ between intact and snapped trees after hurricane Maria in a Puerto Rican tropical dry forest

Hurricanes impact forests by damaging trees and altering multiple ecosystem functions. As such, predicting which individuals are likely to be most affected has crucial economic importance as well as conservation value. Tree stem-inhabiting fungal communities, notably rot-causing agents, have been mentioned as a potential factor of tree predisposition to hurricane damage, but this assumption remains poorly explored. To examine this relationship, we sampled the stem wood of intact and damaged trees shortly after Hurricane Maria in a Puerto Rican dry tropical forest in 2017. We categorized samples depending on two types: trees with intact stems and trees in which stems were snapped. We extracted fungal environmental DNA of wood from 40 samples consisting of four different tree species. Fungal community taxonomic and functional richness and composition was assessed using high-throughput DNA metabarcoding. We found that snapped trees harbored significantly higher fungal operational taxonomic unit (OTU) richness than the intact trees and that the composition of the stem-inhabiting fungal communities diverged consistently between intact and snapped trees. On average, snapped trees’ fungal communities were relatively enriched in “other saprotrophs” guild category and depleted in endophytes. Conversely, intact trees had high relative abundances of Clonostachys, a mycoparasitic endophyte, suggesting that endophytic fungi might act as biocontrols in tree stems. Overall, our results support the hypothesis that stem-inhabiting fungal communities could represent a predisposition factor of tree damage caused by hurricanes in tropical dry forests.

Long-read metabarcoding of the eukaryotic rDNA operon to phylogenetically and taxonomically resolve environmental diversity.

High-throughput DNA metabarcoding of amplicon sizes below 500bp has revolutionized the analysis of environmental microbial diversity. However, these short regions contain limited phylogenetic signal, which makes it impractical to use environmental DNA in full phylogenetic inferences. This lesser phylogenetic resolution of short amplicons may be overcome by new long-read sequencing technologies. To test this idea, we amplified soil DNA and used PacBio Circular Consensus Sequencing (CCS) to obtain an ~4500-bp region spanning most of the eukaryotic small subunit (18S) and large subunit (28S) ribosomal DNA genes. We first treated the CCS reads with a novel curation workflow, generating 650 high-quality operational taxonomic units (OTUs) containing the physically linked 18S and 28S regions. To assign taxonomy to these OTUs, we developed a phylogeny-aware approach based on the 18S region that showed greater accuracy and sensitivity than similarity-based methods. The taxonomically annotated OTUs were then combined with available 18S and 28S reference sequences to infer a well-resolved phylogeny spanning all major groups of eukaryotes, allowing us to accurately derive the evolutionary origin of environmental diversity. A total of 1,019 sequences were included, of which a majority (58%) corresponded to the new long environmental OTUs. The long reads also allowed us to directly investigate the relationships among environmental sequences themselves, which represents a key advantage over the placement of short reads on a reference phylogeny. Together, our results show that long amplicons can be treated in a full phylogenetic framework to provide greater taxonomic resolution and a robust evolutionary perspective to environmental DNA.

Application of next generation sequencing for species identification in meat and poultry products: A DNA metabarcoding approach

Species substitution is one of the forms of food fraud for economic gain. The vast majority of existing DNA typing methods are targeted methods that developed for the determination of the specific species in food products. However, these methods are inappropriate for the analysis of unknown food products or a blend of more than one species. This study aimed to investigate the ability of high-throughput DNA metabarcoding to identify mammalian and avian species in mixed products. Next-generation sequencing of a short segment of the 16S ribosomal RNA (16S rRNA) mitochondrial gene was performed. Duplicate samples and two different databases produced very similar results. Although the relative abundance of reads obtained from each species could not make a quantitative assessment of the original species composition, this method still has the potential to determination of high and low contents of animal species in mixed products. Subsequently, we performed a market survey to identify animal species in 27 meat and poultry products sold in China using the developed DNA metabarcoding approach. The results indicated the presence of mislabeling of processed meat and poultry products.

Biodiversity assessments in the 21st century: the potential of insect traps to complement environmental samples for estimating eukaryotic and prokaryotic diversity using high-throughput DNA metabarcoding 1.

The rapid loss of biodiversity, coupled with difficulties in species identification, call for innovative approaches to assess biodiversity. Insects make up a substantial proportion of extant diversity and play fundamental roles in any given ecosystem. To complement morphological species identification, new techniques such as metabarcoding make it possible to quantify insect diversity and insect-ecosystem interactions through DNA sequencing. Here we examine the potential of bulk insect samples (i.e., containing many non-sorted specimens) to assess prokaryote and eukaryote biodiversity and to complement the taxonomic coverage of soil samples. We sampled 25 sites on three continents and in various ecosystems, collecting insects with SLAM traps (Brazil) and Malaise traps (South Africa and Sweden). We then compared our diversity estimates with the results obtained with biodiversity data from soil samples from the same localities. We found a largely different taxonomic composition between the soil and insect samples, testifying to the potential of bulk insect samples to complement soil samples. Finally, we found that non-destructive DNA extraction protocols, which preserve insect specimens for morphological studies, constitute a promising choice for cost-effective biodiversity assessments. We propose that the sampling and sequencing of insect samples should become a standard complement for biodiversity studies based on environmental DNA.

Core bacterial community of soy-daddawa: Insights from high-throughput DNA metabarcoding

High-throughput sequence analysis of microbial communities in food matrices provides unprecedented insights into species richness and potential safety of foods. Here, the bacterial community of soy-daddawa, a fermented indigenous food condiment, was investigated for the first time using high-throughput DNA metabarcoding (16S rRNA gene). Analyses of 1 246 692 high-quality 16S rRNA gene sequences obtained from seven soy-daddawa samples/producers yielded 1520 operational taxonomic units (OTUs). Beta-diversity analysis of soy-daddawa bacterial communities suggested differences between samples, and in some cases, clustering according to geographical proximity of producers. Sixty-five OTUs (taxonomically assigned to 13 phylotypes) comprised the core (100% threshold) bacterial community of soy-daddawa and included Bacillus amyloliquefaciens, B. lichenformis, B. mojavensis, B. pumilus, B. subtilis, B. subtilis subsp. spizizenii, B. tequilensis, Exiguobacterium sp., Staphylococcus epidermidis and S. pseudintermedius. However, genera with known pathogenic species such as Atopostipes, Enterococcus, Granulicatella, Nosocomicoccus, Proteus, Pseudomonas and Vagoccoccus constituted at least 1% (relative abundance) in at least one sample. Insights gained from this study are useful for the development of a starter culture technology for the controlled fermentation of soy-daddawa. Also, the presence of potentially pathogenic species corroborates established global consensus on the microbiological risks associated with spontaneously fermented indigenous foods.

Sensitivity and accuracy of high-throughput metabarcoding methods for early detection of invasive fish species

High-throughput DNA metabarcoding has gained recognition as a potentially powerful tool for biomonitoring, including early detection of aquatic invasive species (AIS). DNA based techniques are advancing, but our understanding of the limits to detection for metabarcoding complex samples is inadequate. For detecting AIS at an early stage of invasion when the species is rare, accuracy at low detection limits is key. To evaluate the utility of metabarcoding in future fish community monitoring programs, we conducted several experiments to determine the sensitivity and accuracy of routine metabarcoding methods. Experimental mixes used larval fish tissue from multiple “common” species spiked with varying proportions of tissue from an additional “rare” species. Pyrosequencing of genetic marker, COI (cytochrome c oxidase subunit I) and subsequent sequence data analysis provided experimental evidence of low-level detection of the target “rare” species at biomass percentages as low as 0.02% of total sample biomass. Limits to detection varied interspecifically and were susceptible to amplification bias. Moreover, results showed some data processing methods can skew sequence-based biodiversity measurements from corresponding relative biomass abundances and increase false absences. We suggest caution in interpreting presence/absence and relative abundance in larval fish assemblages until metabarcoding methods are optimized for accuracy and precision.

Extracellular DNA extraction is a fast, cheap and reliable alternative for multi-taxa surveys based on soil DNA

DNA metabarcoding on soil samples is increasingly used for large-scale and multi-taxa biodiversity studies. However, DNA extraction may be a major bottleneck for such wide uses. It should be cost/time effective and allow dealing with large sample volumes so as to maximise the representativeness of both micro- and macro-organisms diversity. Here, we compared the performances of a fast and cheap extracellular DNA extraction protocol with a total DNA extraction method in retrieving bacterial, eukaryotic and plant diversity from tropical soil samples of ca. 10 g. The total DNA extraction protocol yielded more high-quality DNA. Yet, the extracellular DNA protocol provided similar diversity assessments although it presented some differences in clades relative abundance and undersampling biases. We argue that extracellular DNA is a good compromise between cost, labor, and accuracy for high-throughput DNA metabarcoding studies of soil biodiversity.