UnCoVar: a reproducible and scalable workflow for transparent and robust virus variant calling and lineage assignment using SARS-CoV-2 as an example.

BACKGROUND: At a global scale, the SARS-CoV-2 virus did not remain in its initial genotype for a long period of time, with the first global reports of variants of concern (VOCs) in late 2020. Subsequently, genome sequencing has become an indispensable tool for characterizing the ongoing pandemic, particularly for typing SARS-CoV-2 samples obtained from patients or environmental surveillance. For such SARS-CoV-2 typing, various in vitro and in silico workflows exist, yet to date, no systematic cross-platform validation has been reported. RESULTS: In this work, we present the first comprehensive cross-platform evaluation and validation of in silico SARS-CoV-2 typing workflows. The evaluation relies on a dataset of 54 patient-derived samples sequenced with several different in vitro approaches on all relevant state-of-the-art sequencing platforms. Moreover, we present UnCoVar, a robust, production-grade reproducible SARS-CoV-2 typing workflow that outperforms all other tested approaches in terms of precision and recall. CONCLUSIONS: In many ways, the SARS-CoV-2 pandemic has accelerated the development of techniques and analytical approaches. We believe that this can serve as a blueprint for dealing with future pandemics. Accordingly, UnCoVar is easily generalizable towards other viral pathogens and future pandemics. The fully automated workflow assembles virus genomes from patient samples, identifies existing lineages, and provides high-resolution insights into individual mutations. UnCoVar includes extensive quality control and automatically generates interactive visual reports. UnCoVar is implemented as a Snakemake workflow. The open-source code is available under a BSD 2-clause license at github.com/IKIM-Essen/uncovar.

Ultrasound- and Vortex-Assisted Dispersive Liquid-Liquid Microextraction of Parabens from Personal Care Products and Urine, Followed by High-Performance Liquid Chromatography.

Objectives: Parabens, which are p-hydroxybenzoic acid esters, are used as preservatives in personal care products, pharmaceuticals, and food because of their antimicrobial activity. However, they are also classified as suspected endocrine disruptors and carcinogens. In the present study, we aimed to optimize an ultrasound and vortex-assisted dispersive liquid-liquid microextraction (DLLME) procedure for the simultaneous extraction of methyl, ethyl, isopropyl, propyl, isobutyl, and butyl parabens from personal care products and urine. Materials and Methods: The extraction solvent type, extraction solvent volume, disperser solvent volume, sodium chloride concentration, ultrasonication time, and vortex application time were evaluated to obtain optimum recoveries by ultrasound and vortex-assisted DLLME. Parabens were detected using a validated high performanc-liquid chromatography (HPLC) method with fluorescence detection. Method validation was performed by examining linearity, the limit of detection, limit of quantification, accuracy, and precision. Results: The limits of detection and quantification of the HPLC method were between 0.09-0.18 µg/mL and 0.28-0.54 µg/mL, respectively. Precision was examined as the relative standard deviation, which was 0.22-1.81% and 1.12-2.03% for intra- and interday studies. Recovery percentages were higher than 96.00%. Samples of two paraben-free personal care products and synthetic urine were spiked with the analyses at 0.02 µg/mL and were successfully analyzed using the developed procedure with recovery values higher than 82.00%. Conclusion: The proposed procedure provided quantification of selected parabens at 20 ng/mL in analyzed personal care products and urine matrices with good precision and accuracy.

Development and Validation of a SPE-HPLC Method for Quantification of Rhein, Emodin, Chrysophanol and Physcion in Rhamnus petiolaris Boiss. & Balansa.

Anthraquinones exhibit a significant group of natural and synthetic quinone derivatives because of their biological activities and industrial applications. Rhamnaceae is one of the families known to contain different kinds of anthraquinones. In this study, it was aimed to quantify rhein, emodin, chrysophanol and physcion in fruits of Rhamnus petiolaris Boiss. & Balansa belonging to Rhamnaceae by solid phase extraction and high performance liquid chromatography with ultraviolet detection. The anthraquinones were separated using a C18 analytical column. Gradient elution was performed using a mobile phase consisted of 0.1% o-phosphoric acid solution and methanol. Analytes were detected at 254 nm. Calibration curves were prepared in the range of 0.25-5.00 µg/mL for rhein, chrysophanol, physcion, 1.00-50.00 µg/mL for emodin. Limits of detection and quantification were between 0.07-0.11 and 0.20-0.34 µg/mL, respectively. Relative standard deviations were ≤ 5.78% in repeatability and intermediate precision studies. Accuracy was determined as relative mean error (8.17-12.06%). Extraction was achieved by maceration with acetone and ethanol, followed by hydrophilic-lipophilic balance solid phase extraction. Recoveries were between 96.2 and 109.6%. The developed and validated method was successfully performed to quantify rhein, emodin, chrysophanol and physcion in R. petiolaris fruit extracts. Only physcion was not detected above limit of detection.

Terrestrial-type nitrogen-fixing symbiosis between seagrass and a marine bacterium.

Symbiotic N2-fixing microorganisms have a crucial role in the assimilation of nitrogen by eukaryotes in nitrogen-limited environments1-3. Particularly among land plants, N2-fixing symbionts occur in a variety of distantly related plant lineages and often involve an intimate association between host and symbiont2,4. Descriptions of such intimate symbioses are lacking for seagrasses, which evolved around 100 million years ago from terrestrial flowering plants that migrated back to the sea5. Here we describe an N2-fixing symbiont, 'Candidatus Celerinatantimonas neptuna', that lives inside seagrass root tissue, where it provides ammonia and amino acids to its host in exchange for sugars. As such, this symbiosis is reminiscent of terrestrial N2-fixing plant symbioses. The symbiosis between Ca. C. neptuna and its host Posidonia oceanica enables highly productive seagrass meadows to thrive in the nitrogen-limited Mediterranean Sea. Relatives of Ca. C. neptuna occur worldwide in coastal ecosystems, in which they may form similar symbioses with other seagrasses and saltmarsh plants. Just like N2-fixing microorganisms might have aided the colonization of nitrogen-poor soils by early land plants6, the ancestors of Ca. C. neptuna and its relatives probably enabled flowering plants to invade nitrogen-poor marine habitats, where they formed extremely efficient blue carbon ecosystems7.

Proposal to reclassify the proteobacterial classes Deltaproteobacteria and Oligoflexia, and the phylum Thermodesulfobacteria into four phyla reflecting major functional capabilities.

The class Deltaproteobacteria comprises an ecologically and metabolically diverse group of bacteria best known for dissimilatory sulphate reduction and predatory behaviour. Although this lineage is the fourth described class of the phylum Proteobacteria, it rarely affiliates with other proteobacterial classes and is frequently not recovered as a monophyletic unit in phylogenetic analyses. Indeed, one branch of the class Deltaproteobacteria encompassing Bdellovibrio-like predators was recently reclassified into a separate proteobacterial class, the Oligoflexia. Here we systematically explore the phylogeny of taxa currently assigned to these classes using 120 conserved single-copy marker genes as well as rRNA genes. The overwhelming majority of markers reject the inclusion of the classes Deltaproteobacteria and Oligoflexia in the phylum Proteobacteria. Instead, the great majority of currently recognized members of the class Deltaproteobacteria are better classified into four novel phylum-level lineages. We propose the names Desulfobacterota phyl. nov. and Myxococcota phyl. nov. for two of these phyla, based on the oldest validly published names in each lineage, and retain the placeholder name SAR324 for the third phylum pending formal description of type material. Members of the class Oligoflexia represent a separate phylum for which we propose the name Bdellovibrionota phyl. nov. based on priority in the literature and general recognition of the genus Bdellovibrio. Desulfobacterota phyl. nov. includes the taxa previously classified in the phylum Thermodesulfobacteria, and these reclassifications imply that the ability of sulphate reduction was vertically inherited in the Thermodesulfobacteria rather than laterally acquired as previously inferred. Our analysis also indicates the independent acquisition of predatory behaviour in the phyla Myxococcota and Bdellovibrionota, which is consistent with their distinct modes of action. This work represents a stable reclassification of one of the most taxonomically challenging areas of the bacterial tree and provides a robust framework for future ecological and systematic studies.

Versatile cyanobacteria control the timing and extent of sulfide production in a Proterozoic analog microbial mat.

Cyanobacterial mats were hotspots of biogeochemical cycling during the Precambrian. However, mechanisms that controlled O2 release by these ecosystems are poorly understood. In an analog to Proterozoic coastal ecosystems, the Frasassi sulfidic springs mats, we studied the regulation of oxygenic and sulfide-driven anoxygenic photosynthesis (OP and AP) in versatile cyanobacteria, and interactions with sulfur reducing bacteria (SRB). Using microsensors and stable isotope probing we found that dissolved organic carbon (DOC) released by OP fuels sulfide production, likely by a specialized SRB population. Increased sulfide fluxes were only stimulated after the cyanobacteria switched from AP to OP. O2 production triggered migration of large sulfur-oxidizing bacteria from the surface to underneath the cyanobacterial layer. The resultant sulfide shield tempered AP and allowed OP to occur for a longer duration over a diel cycle. The lack of cyanobacterial DOC supply to SRB during AP therefore maximized O2 export. This mechanism is unique to benthic ecosystems because transitions between metabolisms occur on the same time scale as solute transport to functionally distinct layers, with the rearrangement of the system by migration of microorganisms exaggerating the effect. Overall, cyanobacterial versatility disrupts the synergistic relationship between sulfide production and AP, and thus enhances diel O2 production.

Role of Temperature and Growth Period in the Synthesis of Hydrothermally Grown TiO2 Nanorods.

Highly uniformed, surfactant free and vertically oriented titanium-di-oxide (TiO2) nanorods were grown on pre-treated fluorine doped tin oxide (FTO) using hydrothermal method through titanium tetra butoxide (Ti(OBu)4) as titanium source. Three different temperatures 130 °C, 150 °C and 180 °C were followed to grow the nanorods at a fixed reaction time of 4 h. The prepared TiO2 nanorods were annealed at the temperatures of 550 °C and 600 °C for 3 h. X-ray diffraction (XRD) analysis shows that obtained nanorods exhibit pure rutile phase. From scanning electron microscopy (SEM) analysis, it was found that increasing temperature led to decreasing the diameter of the nanorods. In addition to these, formation of hierarchical type TiO2 nanorods was also observed at 130 °C. UV-visible spectra analysis was carried out to find the influence of diameter of the nanorods on its optical properties. The plausible mechanism of the growth process is also discussed.

Improved taxonomic assignment of rumen bacterial 16S rRNA sequences using a revised SILVA taxonomic framework.

The taxonomy and associated nomenclature of many taxa of rumen bacteria are poorly defined within databases of 16S rRNA genes. This lack of resolution results in inadequate definition of microbial community structures, with large parts of the community designated as incertae sedis, unclassified, or uncultured within families, orders, or even classes. We have begun resolving these poorly-defined groups of rumen bacteria, based on our desire to name these for use in microbial community profiling. We used the previously-reported global rumen census (GRC) dataset consisting of >4.5 million partial bacterial 16S rRNA gene sequences amplified from 684 rumen samples and representing a wide range of animal hosts and diets. Representative sequences from the 8,985 largest operational units (groups of sequence sharing >97% sequence similarity, and covering 97.8% of all sequences in the GRC dataset) were used to identify 241 pre-defined clusters (mainly at genus or family level) of abundant rumen bacteria in the ARB SILVA 119 framework. A total of 99 of these clusters (containing 63.8% of all GRC sequences) had no unique or had inadequate taxonomic identifiers, and each was given a unique nomenclature. We assessed this improved framework by comparing taxonomic assignments of bacterial 16S rRNA gene sequence data in the GRC dataset with those made using the original SILVA 119 framework, and three other frameworks. The two SILVA frameworks performed best at assigning sequences to genus-level taxa. The SILVA 119 framework allowed 55.4% of the sequence data to be assigned to 751 uniquely identifiable genus-level groups. The improved framework increased this to 87.1% of all sequences being assigned to one of 871 uniquely identifiable genus-level groups. The new designations were included in the SILVA 123 release (https://www.arb-silva.de/documentation/release-123/) and will be perpetuated in future releases.

Meta-omics data and collection objects (MOD-CO): a conceptual schema and data model for processing sample data in meta-omics research.

With the advent of advanced molecular meta-omics techniques and methods, a new era commenced for analysing and characterizing historic collection specimens, as well as recently collected environmental samples. Nucleic acid and protein sequencing-based analyses are increasingly applied to determine the origin, identity and traits of environmental (biological) objects and organisms. In this context, the need for new data structures is evident and former approaches for data processing need to be expanded according to the new meta-omics techniques and operational standards. Existing schemas and community standards in the biodiversity and molecular domain concentrate on terms important for data exchange and publication. Detailed operational aspects of origin and laboratory as well as object and data management issues are frequently neglected. Meta-omics Data and Collection Objects (MOD-CO) has therefore been set up as a new schema for meta-omics research, with a hierarchical organization of the concepts describing collection samples, as well as products and data objects being generated during operational workflows. It is focussed on object trait descriptions as well as on operational aspects and thereby may serve as a backbone for R&D laboratory information management systems with functions of an electronic laboratory notebook. The schema in its current version 1.0 includes 653 concepts and 1810 predefined concept values, being equivalent to descriptors and descriptor states, respectively. It is published in several representations, like a Semantic Media Wiki publication with 2463 interlinked Wiki pages for concepts and concept values, being grouped in 37 concept collections and subcollections. The SQL database application DiversityDescriptions, a generic tool for maintaining descriptive data and schemas, has been applied for setting up and testing MOD-CO and for concept mapping on elements of corresponding schemas.

The importance of designating type material for uncultured taxa.

Naming of uncultured Bacteria and Archaea is often inconsistent with the International Code of Nomenclature of Prokaryotes. The recent practice of proposing names for higher taxa without designation of lower ranks and nomenclature types is one of the most important inconsistencies that needs to be addressed to avoid nomenclatural instability. The Code requires names of higher taxa up to the rank of class to be derived from the type genus name, with a proposal pending to formalise this requirement for the rank of phylum. Designation of nomenclature types is crucial for providing priority to names and ensures their uniqueness and stability. However, only legitimate names proposed for axenic cultures can be used for this purpose. Candidatus names reserved for taxa lacking cultured representatives may be granted this right if recent proposals to use genome sequences as type material are endorsed, thereby allowing the Code to be fully applied to lineages represented by metagenome-assembled genomes (MAGs) or single amplified genomes (SAGs). Genome quality standards need to be considered to ensure unambiguous assignment of type material. Here, we illustrate the recommended practice by proposing nomenclature type material for four major uncultured prokaryotic lineages based on high-quality MAGs in accordance with the Code.

Refining the taxonomic structure of the phylum Acidobacteria.

The phylum Acidobacteria was created in 1997 in order to accommodate a large number of 16S rRNA gene sequences retrieved from various environments in cultivation-independent studies. At present, 26 major sequence clades or subdivisions (SDs) are recognized within this phylum, but only seven of them (SDs 1, 3, 4, 6, 8, 10 and 23) are commonly addressed as containing taxonomically described representatives. Here, we examined the currently explored diversity within the Acidobacteria using the candidate taxonomic unit circumscription system. Based on this analysis, 26 subdivisions were assigned to 15 class-level units, five of which contain described members. These include three earlier established classes Acidobacteriia, Blastocatellia and Holophagae, as well as two as-yet-undescribed groups defined by SDs 6 and 23, which we propose to name Vicinamibacteria classis nov. and Thermoanaerobaculia classis nov., respectively. The former assignment of Thermotomaculum hydrothermale to SD10 was found to be incorrect. This bacterium, therefore, was placed in the family Thermotomaculaceae fam. nov., order Thermotomaculales ord. nov. within the class Holophagae. We also propose establishing a number of high-level taxa to accommodate described representatives of SDs 3, 4, 6 and 23. The family Bryobacteraceae of SD3 Acidobacteria is placed in the order Bryobacterales ord. nov. within the taxonomic range of the class Acidobacteriia. The order Vicinamibacteriales ord. nov. is proposed to accommodate the family Vicinamibacteriaceae of SD6 Acidobacteria. Finally, the family Thermoanaerobaculaceae fam. nov., the order Thermoanaerobaculales ord. nov. are proposed to accommodate the only described representative of SD23, Thermoanaerobaculum aquaticum.

Adaptability as the key to success for the ubiquitous marine nitrite oxidizer Nitrococcus.

Nitrite-oxidizing bacteria (NOB) have conventionally been regarded as a highly specialized functional group responsible for the production of nitrate in the environment. However, recent culture-based studies suggest that they have the capacity to lead alternative lifestyles, but direct environmental evidence for the contribution of marine nitrite oxidizers to other processes has been lacking to date. We report on the alternative biogeochemical functions, worldwide distribution, and sometimes high abundance of the marine NOB Nitrococcus. These largely overlooked bacteria are capable of not only oxidizing nitrite but also reducing nitrate and producing nitrous oxide, an ozone-depleting agent and greenhouse gas. Furthermore, Nitrococcus can aerobically oxidize sulfide, thereby also engaging in the sulfur cycle. In the currently fast-changing global oceans, these findings highlight the potential functional switches these ubiquitous bacteria can perform in various biogeochemical cycles, each with distinct or even contrasting consequences.

SILVA tree viewer: interactive web browsing of the SILVA phylogenetic guide trees.

BACKGROUND: Phylogenetic trees are an important tool to study the evolutionary relationships among organisms. The huge amount of available taxa poses difficulties in their interactive visualization. This hampers the interaction with the users to provide feedback for the further improvement of the taxonomic framework. RESULTS: The SILVA Tree Viewer is a web application designed for visualizing large phylogenetic trees without requiring the download of any software tool or data files. The SILVA Tree Viewer is based on Web Geographic Information Systems (Web-GIS) technology with a PostgreSQL backend. It enables zoom and pan functionalities similar to Google Maps. The SILVA Tree Viewer enables access to two phylogenetic (guide) trees provided by the SILVA database: the SSU Ref NR99 inferred from high-quality, full-length small subunit sequences, clustered at 99% sequence identity and the LSU Ref inferred from high-quality, full-length large subunit sequences. CONCLUSIONS: The Tree Viewer provides tree navigation, search and browse tools as well as an interactive feedback system to collect any kinds of requests ranging from taxonomy to data curation and improving the tool itself.

Minimum information about a single amplified genome (MISAG) and a metagenome-assembled genome (MIMAG) of bacteria and archaea.

We present two standards developed by the Genomic Standards Consortium (GSC) for reporting bacterial and archaeal genome sequences. Both are extensions of the Minimum Information about Any (x) Sequence (MIxS). The standards are the Minimum Information about a Single Amplified Genome (MISAG) and the Minimum Information about a Metagenome-Assembled Genome (MIMAG), including, but not limited to, assembly quality, and estimates of genome completeness and contamination. These standards can be used in combination with other GSC checklists, including the Minimum Information about a Genome Sequence (MIGS), Minimum Information about a Metagenomic Sequence (MIMS), and Minimum Information about a Marker Gene Sequence (MIMARKS). Community-wide adoption of MISAG and MIMAG will facilitate more robust comparative genomic analyses of bacterial and archaeal diversity.

25 years of serving the community with ribosomal RNA gene reference databases and tools.

SILVA (lat. forest) is a comprehensive web resource, providing services around up to date, high-quality datasets of aligned ribosomal RNA gene (rDNA) sequences from the Bacteria, Archaea, and Eukaryota domains. SILVA dates back to the year 1991 when Dr. Wolfgang Ludwig from the Technical University Munich started the integrated software workbench ARB (lat. tree) to support high-quality phylogenetic inference and taxonomy based on the SSU and LSU rDNA marker genes. At that time, the ARB project maintained both, the sequence reference datasets and the software package for data analysis. In 2005, with the massive increase of DNA sequence data, the maintenance of the software system ARB and the corresponding rRNA databases SILVA was split between Munich and the Microbial Genomics and Bioinformatics Research Group in Bremen. ARB has been continuously developed to include new features and improve the usability of the workbench. Thousands of users worldwide appreciate the seamless integration of common analysis tools under a central graphical user interface, in combination with its versatility. The first SILVA release was deployed in February 2007 based on the EMBL-EBI/ENA release 89. Since then, full SILVA releases offering the database content in various flavours are published at least annually, complemented by intermediate web-releases where only the SILVA web dataset is updated. SILVA is the only rDNA database project worldwide where special emphasis is given to the consistent naming of clades of uncultivated (environmental) sequences, where no validly described cultivated representatives are available. Also exclusive for SILVA is the maintenance of both comprehensive aligned 16S/18S rDNA and 23S/28S rDNA sequence datasets. Furthermore, the SILVA alignments and trees were designed to include Eukaryota, another unique feature among rDNA databases. With the termination of the European Ribosomal RNA Database Project in 2007, the SILVA database has become the authoritative rDNA database project for Europe. The application spectrum of ARB and SILVA ranges from biodiversity analysis, medical diagnostics, to biotechnology and quality control for academia and industry.

A phylogenetic framework for the kingdom Fungi based on 18S rRNA gene sequences.

The usage of molecular phylogenetic approaches is critical to advance the understanding of systematics and community processes in the kingdom Fungi. Among the possible phylogenetic markers (or combinations of them), the 18S rRNA gene appears currently as the most prominent candidate due to its large availability in public databases and informative content. The purpose of this work was the creation of a reference phylogenetic framework that can serve as ready-to-use package for its application on fungal classification and community analysis. The current database contains 9329 representative 18S rRNA gene sequences covering the whole fungal kingdom, a manually curated alignment, an annotated and revised phylogenetic tree with all the sequence entries, updated information on current taxonomy, and recommendations of use. Out of 201 total fungal taxa with more than two sequences in the dataset, 179 were monophyletic. From another perspective, 66% of the entries had a tree-derived classification identical to that obtained from the NCBI taxonomy, whereas 34% differed in one or the other rank. Most of the differences were associated to missing taxonomic assignments in NCBI taxonomy, or the unexpected position of sequences that positioned out of their theoretically corresponding clades. The strong correlation observed with current fungal taxonomy evidences that 18S rRNA gene sequence-based phylogenies are adequate to reflect genealogy of Fungi at the levels of order and above, and justify their further usage and exploration.

SPE-HPLC Determination of Chlorogenic and Phenolic Acids in Coffee.

A new solid phase extraction-high-performance liquid chromatography method with ultraviolet detection was developed and validated for the determination of three chlorogenic acids (5-O-caffeoylquinic, 3-O-caffeoylquinic and 4-O-caffeoylquinic acids) and six phenolic acids (caffeic, ferulic, sinapic, protocatechuic, p-hydroxybenzoic acid (PHBA) and vanillic acid (VA)) in coffee bean samples. Extraction was performed using hydrophilic-lipophilic balance cartridges. Separations were accomplished using a C18 guard column (10 × 2.1 mm, 3 µm) and a C18 analytical column (50 × 2.1 mm, 3 µm). o-Phosphoric acid solution (0.08%) and methanol/water/acetonitrile (85:10:5) solution were used as mobile phase with a gradient system. A UV detector was used at 325 nm for 5-O-caffeoylquinic, caffeic, 3-O-caffeoylquinic, 4-O-caffeoylquinic, ferulic, sinapic acids, and 215 nm for protocatechuic, PHBA and VA. Calibration equations and coefficients of determination were determined by least-squares method with weighting factor. Limits of detection and quantification were in the range of 0.15-0.69 and 0.46-2.09 mg/L, respectively. Precision and accuracy of the proposed method were investigated with coffee sample spiked at low, medium and high concentrations. The developed and validated method was applied for the determination of nine phenolic compounds in seven coffee bean samples from different origins with high accuracy and repeatability.

UniEuk: Time to Speak a Common Language in Protistology!

Universal taxonomic frameworks have been critical tools to structure the fields of botany, zoology, mycology, and bacteriology as well as their large research communities. Animals, plants, and fungi have relatively solid, stable morpho-taxonomies built over the last three centuries, while bacteria have been classified for the last three decades under a coherent molecular taxonomic framework. By contrast, no such common language exists for microbial eukaryotes, even though environmental '-omics' surveys suggest that protists make up most of the organismal and genetic complexity of our planet's ecosystems! With the current deluge of eukaryotic meta-omics data, we urgently need to build up a universal eukaryotic taxonomy bridging the protist -omics age to the fragile, centuries-old body of classical knowledge that has effectively linked protist taxa to morphological, physiological, and ecological information. UniEuk is an open, inclusive, community-based and expert-driven international initiative to build a flexible, adaptive universal taxonomic framework for eukaryotes. It unites three complementary modules, EukRef, EukBank, and EukMap, which use phylogenetic markers, environmental metabarcoding surveys, and expert knowledge to inform the taxonomic framework. The UniEuk taxonomy is directly implemented in the European Nucleotide Archive at EMBL-EBI, ensuring its broad use and long-term preservation as a reference taxonomy for eukaryotes.