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.

• 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

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.

• 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

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.

• 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
• Geografické názvy
• oceány a moře 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.

• 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