DNA and RNA metabarcoding Dotaz Zobrazit nápovědu
BACKGROUND: Environmental DNA and metabarcoding allow the identification of a mixture of species and launch a new era in bio- and eco-assessment. Many steps are required to obtain taxonomically assigned matrices from raw data. For most of these, a plethora of tools are available; each tool's execution parameters need to be tailored to reflect each experiment's idiosyncrasy. Adding to this complexity, the computation capacity of high-performance computing systems is frequently required for such analyses. To address the difficulties, bioinformatic pipelines need to combine state-of-the art technologies and algorithms with an easy to get-set-use framework, allowing researchers to tune each study. Software containerization technologies ease the sharing and running of software packages across operating systems; thus, they strongly facilitate pipeline development and usage. Likewise programming languages specialized for big data pipelines incorporate features like roll-back checkpoints and on-demand partial pipeline execution. FINDINGS: PEMA is a containerized assembly of key metabarcoding analysis tools that requires low effort in setting up, running, and customizing to researchers' needs. Based on third-party tools, PEMA performs read pre-processing, (molecular) operational taxonomic unit clustering, amplicon sequence variant inference, and taxonomy assignment for 16S and 18S ribosomal RNA, as well as ITS and COI marker gene data. Owing to its simplified parameterization and checkpoint support, PEMA allows users to explore alternative algorithms for specific steps of the pipeline without the need of a complete re-execution. PEMA was evaluated against both mock communities and previously published datasets and achieved results of comparable quality. CONCLUSIONS: A high-performance computing-based approach was used to develop PEMA; however, it can be used in personal computers as well. PEMA's time-efficient performance and good results will allow it to be used for accurate environmental DNA metabarcoding analysis, thus enhancing the applicability of next-generation biodiversity assessment studies.
- Klíčová slova
- Docker, HPC, container, eDNA, high performance computing, metabarcoding, pipeline, singularity,
- MeSH
- Archaea MeSH
- Bacteria MeSH
- environmentální DNA chemie genetika MeSH
- houby MeSH
- metagenomika metody normy MeSH
- referenční standardy MeSH
- respirační komplex IV genetika MeSH
- RNA ribozomální 16S genetika MeSH
- RNA ribozomální 18S genetika MeSH
- rostliny MeSH
- senzitivita a specificita MeSH
- software MeSH
- taxonomické DNA čárové kódování metody normy MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- environmentální DNA MeSH
- respirační komplex IV MeSH
- RNA ribozomální 16S MeSH
- RNA ribozomální 18S MeSH
Environmental DNA (eDNA) metabarcoding (parallel sequencing of DNA/RNA for identification of whole communities within a targeted group) is revolutionizing the field of aquatic biomonitoring. To date, most metabarcoding studies aiming to assess the ecological status of aquatic ecosystems have focused on water eDNA and macroinvertebrate bulk samples. However, the eDNA metabarcoding has also been applied to soft sediment samples, mainly for assessing microbial or meiofaunal biota. Compared to classical methodologies based on manual sorting and morphological identification of benthic taxa, eDNA metabarcoding offers potentially important advantages for assessing the environmental quality of sediments. The methods and protocols utilized for sediment eDNA metabarcoding can vary considerably among studies, and standardization efforts are needed to improve their robustness, comparability and use within regulatory frameworks. Here, we review the available information on eDNA metabarcoding applied to sediment samples, with a focus on sampling, preservation, and DNA extraction steps. We discuss challenges specific to sediment eDNA analysis, including the variety of different sources and states of eDNA and its persistence in the sediment. This paper aims to identify good-practice strategies and facilitate method harmonization for routine use of sediment eDNA in future benthic monitoring.
- Klíčová slova
- Aquatic ecosystems, Environmental DNA, Metabarcoding, Monitoring, Sediments,
- MeSH
- biodiverzita MeSH
- DNA genetika MeSH
- ekosystém MeSH
- environmentální DNA * MeSH
- monitorování životního prostředí metody MeSH
- taxonomické DNA čárové kódování MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
- Názvy látek
- DNA MeSH
- environmentální DNA * MeSH
Environmental DNA (eDNA) metabarcoding has gained growing attention as a strategy for monitoring biodiversity in ecology. However, taxa identifications produced through metabarcoding require sophisticated processing of high-throughput sequencing data from taxonomically informative DNA barcodes. Various sets of universal and taxon-specific primers have been developed, extending the usability of metabarcoding across archaea, bacteria and eukaryotes. Accordingly, a multitude of metabarcoding data analysis tools and pipelines have also been developed. Often, several developed workflows are designed to process the same amplicon sequencing data, making it somewhat puzzling to choose one among the plethora of existing pipelines. However, each pipeline has its own specific philosophy, strengths and limitations, which should be considered depending on the aims of any specific study, as well as the bioinformatics expertise of the user. In this review, we outline the input data requirements, supported operating systems and particular attributes of thirty-two amplicon processing pipelines with the goal of helping users to select a pipeline for their metabarcoding projects.
- Klíčová slova
- amplicon data analysis, bioinformatics, environmental DNA, metabarcoding, pipeline, review,
- MeSH
- analýza dat MeSH
- Archaea genetika klasifikace MeSH
- Bacteria genetika klasifikace MeSH
- environmentální DNA genetika MeSH
- Eukaryota genetika klasifikace MeSH
- metagenomika metody MeSH
- software * MeSH
- taxonomické DNA čárové kódování * metody MeSH
- výpočetní biologie * metody MeSH
- vysoce účinné nukleotidové sekvenování metody MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
- Názvy látek
- environmentální DNA MeSH
Rare and unknown actinobacteria from unexplored environments have the potential to produce new bioactive molecules. This study aimed to use 16 s rRNA metabarcoding to determine the composition of the actinobacterial community, particularly focusing on rare and undescribed species, in a nature reserve within the Brazilian Cerrado called Sete Cidades National Park. Since this is an inaccessible area without due legal authorization, it is understudied, and, therefore, its diversity and biotechnological potential are not yet fully understood, and it may harbor species with groundbreaking genetic potential. In total, 543 operational taxonomic units (OTUs) across 14 phyla were detected, with Actinobacteria (41.2%), Proteobacteria (26.5%), and Acidobacteria (14.3%) being the most abundant. Within Actinobacteria, 107 OTUs were found, primarily from the families Mycobacteriaceae, Pseudonocardiaceae, and Streptomycetaceae. Mycobacterium and Streptomyces were the predominant genera across all samples. Seventeen rare OTUs with relative abundance < 0.1% were identified, with 82.3% found in only one sample yet 25.5% detected in all units. Notable rare and transient genera included Salinibacterium, Nocardia, Actinomycetospora_01, Saccharopolyspora, Sporichthya, and Nonomuraea. The high diversity and distribution of Actinobacteria OTUs indicate the area's potential for discovering new rare species. Intensified prospection on underexplored environments and characterization of their actinobacterial diversity could lead to the discovery of new species capable of generating innovative natural products.
- Klíčová slova
- Abundance, OTUs, Preserved environments, Rare bacteria, Taxonomy,
- MeSH
- Actinobacteria * chemie klasifikace genetika izolace a purifikace MeSH
- biodiverzita MeSH
- metagenom MeSH
- půda chemie MeSH
- půdní mikrobiologie * MeSH
- RNA ribozomální 16S analýza MeSH
- taxonomické DNA čárové kódování MeSH
- veřejné parky MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Brazílie MeSH
- Názvy látek
- půda MeSH
- RNA ribozomální 16S MeSH
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.
- MeSH
- feces parazitologie MeSH
- genetická variace MeSH
- infekce hlísticemi řádu Strongylida genetika parazitologie MeSH
- koně genetika parazitologie MeSH
- nemoci koní genetika parazitologie MeSH
- rozptýlené repetitivní sekvence genetika MeSH
- Strongylida klasifikace genetika MeSH
- sympatrie MeSH
- taxonomické DNA čárové kódování * MeSH
- vysoce účinné nukleotidové sekvenování MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The world's oceans represent by far the largest biome, with great importance for the global ecosystem [1-4]. The vast majority of ocean biomass and biodiversity is composed of microscopic plankton. Recent results from the Tara Oceans metabarcoding study revealed that a significant part of the plankton in the upper sunlit layer of the ocean is represented by an understudied group of heterotrophic excavate flagellates called diplonemids [5, 6]. We have analyzed the diversity and distribution patterns of diplonemid populations on the extended set of Tara Oceans V9 18S rDNA metabarcodes amplified from 850 size- fractionated plankton communities sampled across 123 globally distributed locations, for the first time also including samples from the mesopelagic zone, which spans the depth from about 200 to 1,000 meters. Diplonemids separate into four major clades, with the vast majority falling into the deep-sea pelagic diplonemid clade. Remarkably, diversity of this clade inferred from metabarcoding data surpasses even that of dinoflagellates, metazoans, and rhizarians, qualifying diplonemids as possibly the most diverse group of marine planktonic eukaryotes. Diplonemids display strong vertical separation between the photic and mesopelagic layers, with the majority of their relative abundance and diversity occurring in deeper waters. Globally, diplonemids display no apparent biogeographic structuring, with a few hyperabundant cosmopolitan operational taxonomic units (OTUs) dominating their communities. Our results suggest that the planktonic diplonemids are among the key heterotrophic players in the largest ecosystem of our biosphere, yet their roles in this ecosystem remain unknown.
- Klíčová slova
- Tara Oceans, cosmopolitan, diplonemids, diversity, metabarcoding, plankton,
- MeSH
- biodiverzita * MeSH
- ekosystém * MeSH
- Euglenozoa klasifikace genetika MeSH
- oceány a moře MeSH
- plankton klasifikace genetika MeSH
- RNA protozoální genetika MeSH
- RNA ribozomální 18S genetika MeSH
- sekvenční analýza RNA MeSH
- taxonomické DNA čárové kódování MeSH
- vodní organismy fyziologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- oceány a moře MeSH
- Názvy látek
- RNA protozoální MeSH
- RNA ribozomální 18S MeSH
The comparison of the bacterial profile of intracellular (iDNA) and extracellular DNA (eDNA) isolated from cow rumen content stored under different conditions was conducted. The influence of rumen fluid treatment (cheesecloth squeezed, centrifuged, filtered), storage temperature (RT, -80 °C) and cryoprotectants (PBS-glycerol, ethanol) on quality and quantity parameters of extracted DNA was evaluated by bacterial DGGE analysis, real-time PCR quantification and metabarcoding approach using high-throughput sequencing. Samples clustered according to the type of extracted DNA due to considerable differences between iDNA and eDNA bacterial profiles, while storage temperature and cryoprotectants additives had little effect on sample clustering. The numbers of Firmicutes and Bacteroidetes were lower (P < 0.01) in eDNA samples. The qPCR indicated significantly higher amount of Firmicutes in iDNA sample frozen with glycerol (P < 0.01). Deep sequencing analysis of iDNA samples revealed the prevalence of Bacteroidetes and similarity of samples frozen with and without cryoprotectants, which differed from sample stored with ethanol at room temperature. Centrifugation and consequent filtration of rumen fluid subjected to the eDNA isolation procedure considerably changed the ratio of molecular operational taxonomic units (MOTUs) of Bacteroidetes and Firmicutes. Intracellular DNA extraction using bead-beating method from cheesecloth sieved rumen content mixed with PBS-glycerol and stored at -80 °C was found as the optimal method to study ruminal bacterial profile.
- Klíčová slova
- Bacterial diversity, Bacteroidetes, Extracellular DNA, Firmicutes, Intracellular DNA, Metabarcoding, PCR-DGGE, Q-PCR, Rumen fluid, Storage conditions,
- MeSH
- bachor mikrobiologie MeSH
- Bacteroidetes klasifikace genetika izolace a purifikace MeSH
- denaturační gradientová gelová elektroforéza MeSH
- DNA bakterií izolace a purifikace MeSH
- fylogeneze MeSH
- grampozitivní bakterie klasifikace genetika izolace a purifikace MeSH
- kryoprezervace * MeSH
- kvantitativní polymerázová řetězová reakce MeSH
- odběr biologického vzorku metody MeSH
- skot MeSH
- taxonomické DNA čárové kódování * MeSH
- vysoce účinné nukleotidové sekvenování MeSH
- zvířata MeSH
- Check Tag
- skot MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- DNA bakterií MeSH
Fungi are key players in vital ecosystem services, spanning carbon cycling, decomposition, symbiotic associations with cultivated and wild plants and pathogenicity. The high importance of fungi in ecosystem processes contrasts with the incompleteness of our understanding of the patterns of fungal biogeography and the environmental factors that drive those patterns. To reduce this gap of knowledge, we collected and validated data published on the composition of soil fungal communities in terrestrial environments including soil and plant-associated habitats and made them publicly accessible through a user interface at https://globalfungi.com . The GlobalFungi database contains over 600 million observations of fungal sequences across > 17 000 samples with geographical locations and additional metadata contained in 178 original studies with millions of unique nucleotide sequences (sequence variants) of the fungal internal transcribed spacers (ITS) 1 and 2 representing fungal species and genera. The study represents the most comprehensive atlas of global fungal distribution, and it is framed in such a way that third-party data addition is possible.
The mangrove ecosystem is the world's fourth most productive ecosystem in terms of service value and offering rich biological resources. Microorganisms play vital roles in these ecological processes, thus researching the mangroves-microbiota is crucial for a deeper comprehension of mangroves dynamics. Amplicon sequencing that targeted V4 region of 16S rRNA gene was employed to profile the microbial diversities and community compositions of 19 soil samples, which were collected from the rhizosphere of 3 plant species (i.e., Avicennia marina, Ceriops tagal, and Rhizophora mucronata) in the mangrove forests of Lasbela coast, Pakistan. A total of 67 bacterial phyla were observed from three mangroves species, and these taxa were classified into 188 classes, 453 orders, 759 families, and 1327 genera. We found that Proteobacteria (34.9-38.4%) and Desulfobacteria (7.6-10.0%) were the dominant phyla followed by Chloroflexi (6.6-7.3%), Gemmatimonadota (5.4-6.8%), Bacteroidota (4.3-5.5%), Planctomycetota (4.4-4.9%) and Acidobacteriota (2.7-3.4%), Actinobacteriota (2.5-3.3%), and Crenarchaeota (2.5-3.3%). After considering the distribution of taxonomic groups, we prescribe that the distinctions in bacterial community composition and diversity are ascribed to the changes in physicochemical attributes of the soil samples (i.e., electrical conductivity (ECe), pH, total organic matter (OM), total organic carbon (OC), available phosphorus (P), and extractable potassium (CaCO3). The findings of this study indicated a high-level species diversity in Pakistani mangroves. The outcomes may also aid in the development of effective conservation policies for mangrove ecosystems, which have been hotspots for anthropogenic impacts in Pakistan. To our knowledge, this is the first microbial research from a Pakistani mangrove forest.
- Klíčová slova
- Illumina sequencing, Mangroves, Metabarcoding, Microbial diversity, Proteobacteria,
- MeSH
- Avicennia mikrobiologie MeSH
- Bacteria * klasifikace genetika izolace a purifikace MeSH
- biodiverzita * MeSH
- DNA bakterií genetika MeSH
- fylogeneze * MeSH
- mikrobiota * MeSH
- mokřady * MeSH
- půdní mikrobiologie * MeSH
- Rhizophoraceae mikrobiologie MeSH
- rhizosféra MeSH
- RNA ribozomální 16S * genetika MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Pákistán MeSH
- Názvy látek
- DNA bakterií MeSH
- RNA ribozomální 16S * MeSH
