A novel metabarcoding primer pair for environmental DNA analysis of Cephalopoda (Mollusca) targeting the nuclear 18S rRNA region.
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ABSTRACT:
SUBMITTER: de Jonge DSW
PROVIDER: S-EPMC8074623 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The assembly of eukaryotic ribosomes requires numerous factors that transiently associate with evolving pre-ribosomal particles. The Pumilio repeat-containing protein Nop9 briefly associates with the 90S pre-ribosome during its co-transcriptional assembly. Here, we show that Nop9 specifically binds an 11-nucleotide sequence of 18S rRNA that forms the 3΄ side of the central pseudoknot and helix 28 in the mature subunit. Crystal structures of Nop9 in the free and RNA-bound states reveal a new type of Pumilio repeat protein with a distinct structure, target sequence and RNA-binding mode. Nop9 contains 10 Pumilio repeats arranged into a U-shaped scaffold. The target RNA is recognized by two stretches of repeats in a bipartite manner, and three central bases are unrecognized as a result of the degeneracy of repeats 6 and 7. Our data suggest that Nop9 regulates the folding of 18S rRNA at early assembly stages of 90S.
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Project description:Freshwater ecosystems contain a large diversity of microeukaryotes that play important roles in maintaining their structure. Microeukaryote communities vary in composition and abundance on the basis of temporal and environmental variables and may serve as useful bioindicators of environmental changes. In the present study, 18S rRNA metabarcoding was employed to investigate the seasonal diversity of microeukaryote communities during four seasons in the Han River, Korea. In total, 882 unique operational taxonomic units (OTUs) were detected, including various diatoms, metazoans (e.g., arthropods and rotifers), chlorophytes, and fungi. Although alpha diversity revealed insignificant differences based on seasons, beta diversity exhibited a prominent variation in the community composition as per seasons. The analysis revealed that the diversity of microeukaryotes was primarily driven by seasonal changes in the prevailing conditions of environmental water temperature and dissolved oxygen. Moreover, potential indicator OTUs belonging to diatoms and chlorophytes were associated with seasonal and environmental factors. This analysis was a preliminary study that established a continuous monitoring system using metabarcoding. This approach could be an effective tool to manage the Han River along with other freshwater ecosystems in Korea.
| S-EPMC8793432 | biostudies-literature
Project description:DNA metabarcoding is a sophisticated molecular tool that can enhance biological surveys of freshwater plankton communities by providing broader taxonomic coverage and, for certain groups, higher taxonomic resolution compared to morphological methods. We conducted 18S rRNA gene metabarcoding analyses on 214 water samples collected over a four-month period from multiple sites within a freshwater reservoir. We detected 1,314 unique operational taxonomic units that included various metazoans, protists, chlorophytes, and fungi. Alpha diversity differed among sites, suggesting local habitat variation linked to differing species responses. Strong temporal variation was detected at both daily and monthly scales. Diversity and relative abundance patterns for several protist groups (including dinoflagellates, ciliates, and cryptophytes) differed from arthropods (e.g., cladocerans and copepods), a traditional focus of plankton surveys. This suggests that the protists respond to different environmental dimensions and may therefore provide additional information regarding ecosystem status. Comparison of the sequence-based population survey data to conventional-based data revealed similar trends for taxa that were ranked among the most abundant in both approaches, although some groups were missing in each data set. These results highlight the potential benefit of supplementing conventional biological survey approaches with metabarcoding to obtain a more comprehensive understanding of freshwater plankton community structure and dynamics.
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Project description:The potential of the 18S rRNA V9 metabarcoding approach for diet assessment was explored using MiSeq paired-end (PE; 2 × 150 bp) technology. To critically evaluate the method's performance with degraded/digested DNA, the diets of two zooplanktivorous fish species from the Bay of Biscay, European sardine (Sardina pilchardus) and European sprat (Sprattus sprattus), were analysed. The taxonomic resolution and quantitative potential of the 18S V9 metabarcoding was first assessed both in silico and with mock and field plankton samples. Our method was capable of discriminating species within the reference database in a reliable way providing there was at least one variable position in the 18S V9 region. Furthermore, it successfully discriminated diet between both fish species, including habitat and diel differences among sardines, overcoming some of the limitations of traditional visual-based diet analysis methods. The high sensitivity and semi-quantitative nature of the 18S V9 metabarcoding approach was supported by both visual microscopy and qPCR-based results. This molecular approach provides an alternative cost and time effective tool for food-web analysis.
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Project description:BackgroundZooplankton is an important component of aquatic organisms and has important biological and economical significance in freshwater ecosystems. However, traditional methods that rely on morphology to classify zooplankton require expert taxonomic skills. Moreover, traditional classification methods are time-consuming and labor-intensive, which is not practical for the design of conservation measures and ecological management tools based on zooplankton diversity assessment.MethodsWe used DNA metabarcoding technology with two different markers: the nuclear small subunit ribosomal RNA (18S rRNA) and mitochondrial cytochrome c oxidase (COI), to analyze 72 zooplankton samples collected in 4 seasons and 9 locations from the Sanmenxia Reservoir. We investigated seasonal changes in the zooplankton community and their relationship with water environmental factors.ResultsA total of 190 species of zooplankton were found, belonging to 12 phyla, 24 classes, 61 orders, 111 families, and 174 genera. Protozoa, especially ciliates, were the most diverse taxa. Richness and relative abundance of zooplankton showed significant seasonal changes. Both alpha and beta diversity showed seasonal trends: the diversity in summer and autumn was higher than that in winter and spring. The zooplankton diversity was most similar in winter and spring. By correlating metabarcoding data and water environmental factors, we proved that water temperature, chemical oxygen demand, total nitrogen and ammoniacal nitrogen were the main environmental factors driving the seasonal changes in zooplankton in the Sanmenxia Reservoir. Water temperature, followed by total nitrogen, were the most influential factors. This study highlights the advantages and some limitations of zooplankton molecular biodiversity assessment using two molecular markers.
| S-EPMC7983862 | biostudies-literature
Project description:Predator-prey relationships are important ecological interactions, affecting biotic community composition and energy flow through a system, and are of interest to ecologists and managers. Morphological diet analysis has been the primary method used to quantify the diets of predators, but emerging molecular techniques using genetic data can provide more accurate estimates of relative diet composition. This study used sequences from the 18S V9 rRNA barcoding region to identify prey items in the gastrointestinal (GI) tracts of predatory fishes. Predator GI samples were taken from the Black River, Cheboygan Co., MI, USA (n = 367 samples, 12 predator species) during periods of high prey availability, including the larval stage of regionally threatened lake sturgeon (Acipenser fulvescens Rafinesque 1817) in late May/early June of 2015 and of relatively lower prey availability in early July of 2015. DNA was extracted and sequenced from 355 samples (96.7%), and prey DNA was identified in 286 of the 355 samples (80.6%). Prey were grouped into 33 ecologically significant taxonomic groups based on the lowest taxonomic level sequences that could be identified using sequences available on GenBank. Changes in the makeup of diet composition, dietary overlap, and predator preference were analyzed comparing the periods of high and low prey abundance. Some predator species exhibited significant seasonal changes in diet composition. Dietary overlap was slightly but significantly higher during the period of high prey abundance; however, there was little change in predator preference. This suggests that change in prey availability was the driving factor in changing predator diet composition and dietary overlap. This study demonstrates the utility of molecular diet analysis and how temporal variability in community composition adds complexity to predator-prey interactions.
| S-EPMC6374664 | biostudies-literature
Project description:BackgroundMitochondria contain small genomes that are physically separate from those of nuclei. Their comparison serves as a model system for understanding the processes of genome evolution. Although complete mitochondrial genome sequences have been reported for more than 600 animals, the taxonomic sampling is highly biased toward vertebrates and arthropods, leaving much of the diversity yet uncharacterized.ResultsThe mitochondrial genome of the bellybutton nautilus, Nautilus macromphalus, a cephalopod mollusk, is 16,258 nts in length and 59.5% A+T, both values that are typical of animal mitochondrial genomes. It contains the 37 genes that are almost universally found in animal mtDNAs, with 15 on one DNA strand and 22 on the other. The arrangement of these genes can be derived from that of the distantly related Katharina tunicata (Mollusca: Polyplacophora) by a switch in position of two large blocks of genes and transpositions of four tRNA genes. There is strong skew in the distribution of nucleotides between the two strands, and analysis of this yields insight into modes of transcription and replication. There is an unusual number of non-coding regions and their function, if any, is not known; however, several of these demark abrupt shifts in nucleotide skew, and there are several identical sequence elements at these junctions, suggesting that they may play roles in transcription and/or replication. One of the non-coding regions contains multiple repeats of a tRNA-like sequence. Some of the tRNA genes appear to overlap on the same strand, but this could be resolved if the polycistron were cleaved at the beginning of the downstream gene, followed by polyadenylation of the product of the upstream gene to form a fully paired structure.ConclusionNautilus macromphalus mtDNA contains an expected gene content that has experienced few rearrangements since the evolutionary split between cephalopods and polyplacophorans. It contains an unusual number of non-coding regions, especially considering that these otherwise often are generated by the same processes that produce gene rearrangements. The skew in nucleotide composition between the two strands is strong and associated with the direction of transcription in various parts of the genomes, but a comparison with K. tunicata implies that mutational bias during replication also plays a role. This appears to be yet another case where polyadenylation of mitochondrial tRNAs restores what would otherwise be an incomplete structure.
| S-EPMC1544340 | biostudies-literature
Project description:BACKGROUND:Several fungi-specific primers target the 18S rRNA gene sequence, one of the prominent markers for fungal classification. The design of most primers goes back to the last decades. Since then, the number of sequences in public databases increased leading to the discovery of new fungal groups and changes in fungal taxonomy. However, no reevaluation of primers was carried out and relevant information on most primers is missing. With this study, we aimed to develop an 18S rRNA gene sequence primer toolkit allowing an easy selection of the best primer pair appropriate for different sequencing platforms, research aims (biodiversity assessment versus isolate classification) and target groups. RESULTS:We performed an intensive literature research, reshuffled existing primers into new pairs, designed new Illumina-primers, and annealing blocking oligonucleotides. A final number of 439 primer pairs were subjected to in silico PCRs. Best primer pairs were selected and experimentally tested. The most promising primer pair with a small amplicon size, nu-SSU-1333-5'/nu-SSU-1647-3' (FF390/FR-1), was successful in describing fungal communities by Illumina sequencing. Results were confirmed by a simultaneous metagenomics and eukaryote-specific primer approach. Co-amplification occurred in all sample types but was effectively reduced by blocking oligonucleotides. CONCLUSIONS:The compiled data revealed the presence of an enormous diversity of fungal 18S rRNA gene primer pairs in terms of fungal coverage, phylum spectrum and co-amplification. Therefore, the primer pair has to be carefully selected to fulfill the requirements of the individual research projects. The presented primer toolkit offers comprehensive lists of 164 primers, 439 primer combinations, 4 blocking oligonucleotides, and top primer pairs holding all relevant information including primer's characteristics and performance to facilitate primer pair selection.
| S-EPMC6247509 | biostudies-literature
Project description:The nucleotide variation in the cytochrome c oxidase subunit I (COI) gene makes it ideal for assigning sequences to species. However, this variability also makes it difficult to design truly universal primers. Here, we present the forward primer "Sauron-S878," specifically designed to facilitate library preparation for metabarcoding. This primer is modified to improve the coverage of terrestrial species compared to the primer mCOIintF, optimized for aquatic systems, which raised the in silico coverage from 74.4% to 98.3% of available NCBI sequences (perfect match in 3' region, up to three mismatches in remaining primer). When paired with the reverse primer "jgHCO2198" (fragment length ~313 bp), these primers amplified 98.4% of 255 tested DNA extracts from various taxa, which are better than many other common COI barcoding primers. Furthermore, a single-tube protocol was developed, wherein these primers amplify the target gene, and attach MIDs and Illumina sequencing adapters in one reaction. This eliminates the need for re-amplification or enzymatic ligation during library preparation while keeping the flexibility to modularly combine primers and MIDs. Using the single-tube approach, three replicates of three mock samples were sequenced on a MiSeq platform with no adverse effects compared to commercial Nextera indexing kits. From this run, 75% of all included taxa could be recovered, with no considerable bias among taxonomic groups. Despite the fact that 98.4% of the extracts were confirmed to amplify in vitro, this number was lower than expected. A reason for this discrepancy was a clear link between the relative concentration of a specific DNA type in the template and the number of returned reads for this DNA. We would argue that such a bias may be especially problematic in metabarcoding where samples usually contain trace DNA in unknown amounts. However, how this affects the completeness of metabarcoding results has yet been poorly investigated.
| S-EPMC6308894 | biostudies-literature
Project description:BackgroundThe 18S rRNA gene is one of the most important molecular markers, used in diverse applications such as molecular phylogenetic analyses and biodiversity screening. The Mollusca is the second largest phylum within the animal kingdom and mollusks show an outstanding high diversity in body plans and ecological adaptations. Although an enormous amount of 18S data is available for higher mollusks, data on some early branching lineages are still limited. Despite of some partial success in obtaining these data from Solenogastres, by some regarded to be the most "basal" mollusks, this taxon still remained problematic due to contamination with food organisms and general amplification difficulties.ResultsWe report here the first authentic 18S genes of three Solenogastres species (Mollusca), each possessing a unique sequence composition with regions conspicuously rich in guanine and cytosine. For these GC-rich regions we calculated strong secondary structures. The observed high intra-molecular forces hamper standard amplification and appear to increase formation of chimerical sequences caused by contaminating foreign DNAs from potential prey organisms. In our analyses, contamination was avoided by using RNA as a template. Indication for contamination of previously published Solenogastres sequences is presented. Detailed phylogenetic analyses were conducted using RNA specific models that account for compensatory substitutions in stem regions.ConclusionsThe extreme morphological diversity of mollusks is mirrored in the molecular 18S data and shows elevated substitution rates mainly in three higher taxa: true limpets (Patellogastropoda), Cephalopoda and Solenogastres. Our phylogenetic tree based on 123 species, including representatives of all mollusk classes, shows limited resolution at the class level but illustrates the pitfalls of artificial groupings formed due to shared biased sequence composition.
| S-EPMC2841657 | biostudies-literature