Identification of non-model mammal species using the MinION DNA sequencer from Oxford Nanopore.

Background: The Neotropics harbors the largest species richness of the planet; however, even in well-studied groups, there are potentially hundreds of species that lack a formal description, and likewise, many already described taxa are difficult to identify using morphology. Specifically in small mammals, complex morphological diagnoses have been facilitated by the use of molecular data, particularly from mitochondrial sequences, to obtain accurate species identifications. Obtaining mitochondrial markers implies the use of PCR and specific primers, which are largely absent for non-model organisms. Oxford Nanopore Technologies (ONT) is a new alternative for sequencing the entire mitochondrial genome without the need for specific primers. Only a limited number of studies have employed exclusively ONT long-reads to assemble mitochondrial genomes, and few studies have yet evaluated the usefulness of such reads in multiple non-model organisms. Methods: We implemented fieldwork to collect small mammals, including rodents, bats, and marsupials, in five localities in the northern extreme of the Cordillera Central of Colombia. DNA samples were sequenced using the MinION device and Flongle flow cells. Shotgun-sequenced data was used to reconstruct the mitochondrial genome of all the samples. In parallel, using a customized computational pipeline, species-level identifications were obtained based on sequencing raw reads (Whole Genome Sequencing). ONT-based identifications were corroborated using traditional morphological characters and phylogenetic analyses. Results: A total of 24 individuals from 18 species were collected, morphologically identified, and deposited in the biological collection of Universidad EAFIT. Our different computational pipelines were able to reconstruct mitochondrial genomes from exclusively ONT reads. We obtained three new mitochondrial genomes and eight new molecular mitochondrial sequences for six species. Our species identification pipeline was able to obtain accurate species identifications for up to 75% of the individuals in as little as 5 s. Finally, our phylogenetic analyses corroborated the identifications from our automated species identification pipeline and revealed important contributions to the knowledge of the diversity of Neotropical small mammals. Discussion: This study was able to evaluate different pipelines to reconstruct mitochondrial genomes from non-model organisms, using exclusively ONT reads, benchmarking these protocols on a multi-species dataset. The proposed methodology can be applied by non-expert taxonomists and has the potential to be implemented in real-time, without the need to euthanize the organisms and under field conditions. Therefore, it stands as a relevant tool to help increase the available data for non-model organisms, and the rate at which researchers can characterize life specially in highly biodiverse places as the Neotropics.

Uncovering new Firmicutes species in vertebrate hosts through metagenome-assembled genomes with potential for sporulation.

Metagenome-assembled genomes (MAGs) have contributed to identifying non-culturable microorganisms and understanding their ecological functions. MAGs offer an advantage in investigating sporulation-associated genes, especially given the difficulty of isolating many species residing in the gut microbiota of multiple hosts. Bacterial sporulation is a key survival mechanism with implications for pathogenicity and biotechnology. Here, we investigate MAGs from vertebrate hosts, emphasizing taxonomic identification and identifying sporulation-associated genes in potential novel species within the Firmicutes phylum. We identified potential new species in the classes Clostridia (Borkfalkiaceae, Lachnospiraceae, Monoglobaceae, and Oscillospiraceae families) and Bacilli (Bacillaceae and Erysipelotrichaceae families) through phylogenetic and functional pathway analyses, highlighting their sporulation potential. Our study covers 146 MAGs, 124 of them without refined taxonomic assignments at the family level. We found that Clostridia and Bacilli have unique sporulation gene profiles in the refined family MAGs for cattle, swine, poultry, and human hosts. The presence of genes related to Spo0A regulon, engulfment, and spore cortex in MAGs underscores fundamental mechanisms in sporulation processes in currently uncharacterized species with sporulation potential from metagenomic dark matter. Furthermore, genomic analyses predict sporulation potential based on gene presence, genome size, and metabolic pathways involved in spore formation. We emphasize MAGs covering families not yet characterized through the phylogenetic analysis, and with extensive potential for spore-forming bacteria within Clostridia, Bacilli, UBA4882, and UBA994 classes. These findings contribute to exploring spore-forming bacteria, which provides evidence for novel species diversity in multiple hosts, their adaptive strategies, and potential applications in biotechnology and host health.IMPORTANCESpores are essential for bacterial survival in harsh environments, facilitating their persistence and adaptation. Exploring sporulation-associated genes in metagenome-assembled genomes (MAGs) from different hosts contributes to clinical and biotechnological domains. Our study investigated the extent of genes associated with bacterial sporulation in MAGs from poultry, swine, cattle, and humans, revealing these genes in uncultivated bacteria. We identified potential novel Firmicutes species with sporulation capabilities through phylogenetic and functional analyses. Notably, MAGs belonging to Clostridia, Bacilli, and unknown classes, namely UBA4882 and UBA994, remained uncharacterized at the family level, which raises the hypothesis that sporulation would also be present in these genomes. These findings contribute to our understanding of microbial adaptation and have implications for microbial ecology, underlining the importance of sporulation in Firmicutes across different hosts. Further studies into novel species and their sporulation capability can contribute to bacterial maintenance mechanisms in various organisms and their applications in biotechnology studies.

Detection of Genes Related to Antibiotic Resistance in Leptospira.

Leptospirosis is a disease caused by the bacteria of the Leptospira genus, which can usually be acquired by humans through contact with urine from infected animals; it is also possible for this urine to contaminate soils and bodies of water. The disease can have deadly consequences in some extreme cases. Fortunately, until now, patients with leptospirosis have responded adequately to treatment with doxycycline and azithromycin, and no cases of antibiotic resistance have been reported. However, with the extensive use of such medications, more bacteria, such as Staphylococci and Enterococci, are becoming resistant. The purpose of this study is to determine the presence of genes related to antibiotic resistance in the Leptospira genus using bioinformatic tools, which have not been undertaken in the past. Whole genomes from the 69 described Leptospira species were downloaded from NCBI's GeneBank and analyzed using CARD (The Comprehensive Antibiotic Resistant Database) and RAST (Rapid Annotations using Subsystem Technology). After a detailed genomic search, 12 genes associated with four mechanisms were found: resistance to beta-lactamases, vancomycin, aminoglycoside adenylyltransferases, as well as multiple drug efflux pumps. Some of these genes are highly polymorphic among different species, and some of them are present in multiple copies in the same species. In conclusion, this study provides evidence of the presence of genes related to antibiotic resistance in the genomes of some species of the genus Leptospira, and it is the starting point for future experimental evaluation to determine whether these genes are transcriptionally active in some species and serovars.

Draft genome sequence of rhamnolipid-producing bacteria Thermoanaerobacter thermocopriae strain CM-CNRG TB177 isolated from an oil reservoir in Mexico.

The draft genome of Thermoanaerobacter thermocopriae CM-CNRG TB177 isolated from an oil reservoir in Mexico was determined and annotated. The organism is a thermophilic and strict anaerobe bacterium that produces rhamnolipids, using glucose as a carbon source. The predicted genome size is 2,496,169 bp and 2,550 genes.

Genome sequences of three Bartonella schoenbuchensis strains from Czechia.

Bartonella schoenbuchensis causes bacteremia in ruminants and is transmitted by deer keds. Here, we report the complete genome sequences of three B. schoenbuchensis strains (L2, L19, and L24) recently isolated from deer keds (Lipoptena fortisetosa) in Czechia.

Non-B DNA in plant genomes: prediction, mapping, and emerging roles.

Regulating gene expression in plant development and environmental responses is vital for mitigating the effects of climate change on crop growth and productivity. The eukaryotic genome largely shows the canonical B-DNA structure that is organized into nucleosomes with histone modifications shaping the epigenome. Nuclear proteins and RNA interactions influence chromatin conformations and dynamically modulate gene activity. Non-B DNA conformations and their transitions introduce novel aspects to gene expression modulation, particularly in response to environmental shifts. We explore the current understanding of non-B DNA structures in plant genomes, their interplay with epigenomics and gene expression, and advances in methods for their mapping and characterization. The exploration of so far uncharacterized non-B DNA structures remains an intriguing area in plant chromatin research and offers insights into their potential role in gene regulation.

Whole-genome sequences of Helicobacter pylori isolated from patients with high risk of gastric cancer in the Andes of Nariño, Colombia.

We sequenced the complete genomes of 16 Helicobacter pylori isolates obtained from patients residing in Nariño, Colombia. These isolates were collected from patients presenting various gastric lesions classified according to the Correa cascade classification. The genomic characterization of these isolates provides valuable insights into the structure, composition, and pathogenicity.

Comparative genomics of plant growth promoting phosphobacteria isolated from acidic soils.

Despite being one of the most abundant elements in soil, phosphorus (P) often becomes a limiting macronutrient for plants due to its low bioavailability, primarily locked away in insoluble organic and inorganic forms. Phosphate solubilizing and mineralizing bacteria, also called phosphobacteria, isolated from P-deficient soils have emerged as a promising biofertilizer alternative, capable of converting these recalcitrant P forms into plant-available phosphates. Three such phosphobacteria strains-Serratia sp. RJAL6, Klebsiella sp. RCJ4, and Enterobacter sp. 198-previously demonstrated their particular strength as plant growth promoters for wheat, ryegrass, or avocado under abiotic stresses and P deficiency. Comparative genomic analysis of their draft genomes revealed several genes encoding key functionalities, including alkaline phosphatases, isonitrile secondary metabolites, enterobactin biosynthesis and genes associated to the production of indole-3-acetic acid (IAA) and gluconic acid. Moreover, overall genome relatedness indexes (OGRIs) revealed substantial divergence between Serratia sp. RJAL6 and its closest phylogenetic neighbours, Serratia nematodiphila and Serratia bockelmanii. This compelling evidence suggests that RJAL6 merits classification as a novel species. This in silico genomic analysis provides vital insights into the plant growth-promoting capabilities and provenance of these promising PSRB strains. Notably, it paves the way for further characterization and potential application of the newly identified Serratia species as a powerful bioinoculant in future agricultural settings.

Bioinformatic Approaches for Comparative Analysis of Viruses.

The field of viral genomic studies has experienced an unprecedented increase in data volume. New strains of known viruses are constantly being added to the GenBank database and so are completely new species with little or no resemblance to our databases of sequences. In addition to this, metagenomic techniques have the potential to further increase the number and rate of sequenced genomes. Besides, it is important to consider that viruses have a set of unique features that often break down molecular biology dogmas, e.g., the flux of information from RNA to DNA in retroviruses and the use of RNA molecules as genomes. As a result, extracting meaningful information from viral genomes remains a challenge and standard methods for comparing the unknown and our databases of characterized sequences may need adaptations. Thus, several bioinformatic approaches and tools have been created to address the challenge of analyzing viral data. This chapter offers descriptions and protocols of some of the most important bioinformatic techniques for comparative analysis of viruses. The authors also provide comments and discussion on how viruses' unique features can affect standard analyses and how to overcome some of the major sources of problems. Protocols and topics emphasize online tools (which are more accessible to users) and give the real experience of what most bioinformaticians do in day-by-day work with command-line pipelines. The topics discussed include (1) clustering related genomes, (2) whole genome multiple sequence alignments for small RNA viruses, (3) protein alignment for marker genes and species affiliation, (4) variant calling and annotation, and (5) virome analyses and pathogen identification.

Metagenome-assembled genomes (MAGs) suggest an acetate-driven protective role in gut microbiota disrupted by Clostridioides difficile.

Clostridioides difficile may have a negative impact on gut microbiota composition in terms of diversity and abundance, thereby triggering functional changes supported by the differential presence of genes involved in significant metabolic pathways, such as short-chain fatty acids (SCFA). This work has evaluated shotgun metagenomics data regarding 48 samples from four groups classified according to diarrhea acquisition site (community- and healthcare facility-onset) and positive or negative Clostridioides difficile infection (CDI) result. The metagenomic-assembled genomes (MAGs) obtained from each sample were taxonomically assigned for preliminary comparative analysis concerning differences in composition among groups. The predicted genes involved in metabolism, transport, and signaling remained constant in microbiota members; characteristic patterns were observed in MAGs and genes involved in SCFA butyrate and acetate metabolic pathways for each study group. A decrease in genera and species, as well as relative MAG abundance with the presence of the acetate metabolism-related gene, was evident in the HCFO/- group. Increased antibiotic resistance markers (ARM) were observed in MAGs along with the genes involved in acetate metabolism. The results highlight the need to explore the role of acetate in greater depth as a potential protector of the imbalances produced by CDI, as occurs in other inflammatory intestinal diseases.

Comparative analysis of transposable elements dynamics in fish with different sex chromosome systems.

Transposable elements (TEs) are widespread genomic components with substantial roles in genome evolution and sex chromosome differentiation. In this study, we compared the TE composition of three closely related fish with different sex chromosome systems: Megaleporinus elongatus (Z1Z1Z2Z2/Z1W1Z2W2), Megaleporinus macrocephalus (ZZ/ZW) (both with highly differentiated W sex chromosomes), and Leporinus friderici (without heteromorphic sex chromosomes). We created custom TE libraries for each species using clustering methods and manual annotation and prediction, and we predicted TE temporal dynamics through divergence-based analysis. The TE abundance ranged from 16% to 21% in the three mobilomes, with L. friderici having the lowest overall. Despite the recent amplification of TEs in all three species, we observed differing expansion activities, particularly between the two genera. Both Megaleporinus recently experienced high retrotransposon activity, with a reduction in DNA TEs, which could have implications in sex chromosome composition. In contrast, L. friderici showed the opposite pattern. Therefore, despite having similar TE compositions, Megaleporinus and Leporinus exhibit distinct TE histories that likely evolved after their separation, highlighting a rapid TE expansion over short evolutionary periods.

Plant genome information facilitates plant functional genomics.

MAIN CONCLUSION: In this review, we give an overview of plant sequencing efforts and how this impacts plant functional genomics research. Plant genome sequence information greatly facilitates the studies of plant biology, functional genomics, evolution of genomes and genes, domestication processes, phylogenetic relationships, among many others. More than two decades of sequencing efforts have boosted the number of available sequenced plant genomes. The first plant genome, of Arabidopsis, was published in the year 2000 and currently, 4604 plant genomes from 1482 plant species have been published. Various large sequence initiatives are running, which are planning to produce tens of thousands of sequenced plant genomes in the near future. In this review, we give an overview on the status of sequenced plant genomes and on the use of genome information in different research areas.

Unveiling microbial guilds and symbiotic relationships in Antarctic sponge microbiomes.

Marine sponges host diverse microbial communities. Although we know many of its ecological patterns, a deeper understanding of the polar sponge holobiont is still needed. We combine high-throughput sequencing of ribosomal genes, including the largest taxonomic repertoire of Antarctic sponge species analyzed to date, functional metagenomics, and metagenome-assembled genomes (MAGs). Our findings show that sponges harbor more exclusive bacterial and archaeal communities than seawater, while microbial eukaryotes are mostly shared. Furthermore, bacteria in Antarctic sponge holobionts establish more cooperative interactions than in sponge holobionts from other environments. The bacterial classes that established more positive relations were Bacteroidia, Gamma- and Alphaproteobacteria. Antarctic sponge microbiomes contain microbial guilds that encompass ammonia-oxidizing archaea, ammonia-oxidizing bacteria, nitrite-oxidizing bacteria, and sulfur-oxidizing bacteria. The retrieved MAGs showed a high level of novelty and streamlining signals and belong to the most abundant members of the main microbial guilds in the Antarctic sponge holobiont. Moreover, the genomes of these symbiotic bacteria contain highly abundant functions related to their adaptation to the cold environment, vitamin production, and symbiotic lifestyle, helping the holobiont survive in this extreme environment.

Draft genome sequence of a triple hybrid Escherichia coli strain isolated from a healthy donor feces.

Herein is reported the draft genome sequence of a triple hybrid Escherichia coli strain isolated from a healthy donor feces. The assembly is 5.2 Mbp, composed of 247 contigs, with a N50 of 77, 241 bp, presenting a GC content of 50.8%.

Whole-genome analysis reveals the diversification of Galapagos rail (Aves: Rallidae) and confirms the success of goat eradication programs.

Similar to other insular birds around the world, the Galapagos rail (Laterallus spilonota Gould, 1841) exhibits reduced flight capacity following its colonization of the archipelago ~1.2 mya. Despite their short evolutionary history, rails have colonized seven different islands spanning the entire width of the archipelago. Galapagos rails were once common on islands with sufficiently high altitudes to support shrubs in humid habitats. After humans introduced goats, this habitat was severely reduced due to overgrazing. Habitat loss devastated some rail populations, with less than 50 individuals surviving, rendering the genetic diversity of Galapagos rail a pressing conservation concern. Additionally, one enigma is the reappearance of rails on the island of Pinta after they were considered extirpated. Our approach was to investigate the evolutionary history and geographic distribution of Galapagos rails as well as examine the genome-wide effects of historical population bottlenecks using 39 whole genomes across different island populations. We recovered an early divergence of rail ancestors leading to the isolated populations on Pinta and a second clade comprising the rest of the islands, historically forming a single landmass. Subsequently, the separation of the landmass ~900 kya may have led to the isolation of the Isabela population with more panmictic populations found on Santa Cruz and Santiago islands. We found that rails genomes contain long runs of homozygosity (>2 Mb) that could be related to the introduction of goats. Finally, our findings show that the modern eradication of goats was critical to avoiding episodes of inbreeding in most populations.

Modeling within and between Sub-Genomes Epistasis of Synthetic Hexaploid Wheat for Genome-Enabled Prediction of Diseases.

Common wheat (Triticum aestivum) is a hexaploid crop comprising three diploid sub-genomes labeled A, B, and D. The objective of this study is to investigate whether there is a discernible influence pattern from the D sub-genome with epistasis in genomic models for wheat diseases. Four genomic statistical models were employed; two models considered the linear genomic relationship of the lines. The first model (G) utilized all molecular markers, while the second model (ABD) utilized three matrices representing the A, B, and D sub-genomes. The remaining two models incorporated epistasis, one (GI) using all markers and the other (ABDI) considering markers in sub-genomes A, B, and D, including inter- and intra-sub-genome interactions. The data utilized pertained to three diseases: tan spot (TS), septoria nodorum blotch (SNB), and spot blotch (SB), for synthetic hexaploid wheat (SHW) lines. The results (variance components) indicate that epistasis makes a substantial contribution to explaining genomic variation, accounting for approximately 50% in SNB and SB and only 29% for TS. In this contribution of epistasis, the influence of intra- and inter-sub-genome interactions of the D sub-genome is crucial, being close to 50% in TS and higher in SNB (60%) and SB (60%). This increase in explaining genomic variation is reflected in an enhancement of predictive ability from the G model (additive) to the ABDI model (additive and epistasis) by 9%, 5%, and 1% for SNB, SB, and TS, respectively. These results, in line with other studies, underscore the significance of the D sub-genome in disease traits and suggest a potential application to be explored in the future regarding the selection of parental crosses based on sub-genomes.

Draft genomes of two phylotype I and II Ralstonia solanacearum species complex (RSSC) isolates causing bacterial wilt in tomato plants from Costa Rica.

Bacteria from RSSC hold agricultural significance as they are the causal agents of bacterial wilt. Here, we report the draft genomes of two bacteria extracted from vascular tissues of infected tomato plants. Isolate RALF-MA was classified as Ralstonia pseudosolanacearum (phylotype I) and RALSA-MA as Ralstonia solanacearum (phylotype II).

Genomes of nine biofilm-forming filamentous strains of Cyanobacteria (genera Jaaginema, Scytonema, and Karukerafilum gen. nov.) isolated from mangrove habitats of Guadeloupe (Lesser Antilles).

Biofilm-forming cyanobacteria are abundant in mangrove ecosystems, colonizing various niches including sediment surface and periphyton where they can cover large areas, yet have received limited attention. Several filamentous isolates were recently isolated from Guadeloupe, illustrating the diversity and novelty present in these biofilms. In this study, nine strains belonging to three novel lineages found abundantly in Guadeloupe biofilms are characterized by genome sequencing, morphological and ultrastructural examination, metabolome fingerprinting and searched for secondary metabolites biosynthesis pathways. Assignation of two lineages to known genera is confirmed, namely Scytonema and Jaaginema. The third lineage corresponds to a new Coleofasciculales genus herein described as Karukerafilum gen. nov. The four strains belonging to this genus group into two subclades, one of which displays genes necessary for nitrogen fixation as well as the complete pathway for geosmin production. This study gives new insights into the diversity of mangrove biofilm-forming cyanobacteria, including genome-based description of a new genus and the first genome sequence available for the genus Jaaginema.

Closed genomes of four multidrug resistance Salmonella enterica serotype Infantis isolated in Costa Rica.

Here, we report the complete genome of four S. enterica Infantis isolated in Costa Rica from human, poultry rinse, and raw chicken meat from 2017 to 2019. All genomes belonged to ST32 and carried a 310-kb plasmid with many antimicrobial resistance genes including the bla CTX-M65 gene.