Genomic Analysis Research Articles

Superior target genes and pathways for RNAi-mediated pest control revealed by genome-wide analysis in the beetle Tribolium castaneum.

An increasing human population, the emergence of resistances against pesticides and their potential impact on the environment call for the development of new eco-friendly pest control strategies. RNA interference (RNAi)-based pesticides have emerged as a new option with the first products entering the market. Essentially, double-stranded RNAs targeting essential genes of pests are either expressed in the plants or sprayed on their surface. Upon feeding, pests mount an RNAi response and die. However, it has remained unclear whether RNAi-based insecticides should target the same pathways as classic pesticides or whether the different mode-of-action would favor other processes. Moreover, there is no consensus on the best genes to be targeted. We performed a genome-wide screen in the red flour beetle to identify 905 RNAi target genes. Based on a validation screen and clustering, we identified the 192 most effective target genes in that species. The transfer to oral application in other beetle pests revealed a list of 34 superior target genes, which are an excellent starting point for application in other pests. Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) analyses of our genome-wide dataset revealed that genes with high efficacy belonged mainly to basic cellular processes such as gene expression and protein homeostasis - processes not targeted by classic insecticides. Our work revealed the best target genes and target processes for RNAi-based pest control and we propose a procedure to transfer our short list of superior target genes to other pests. © 2024 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Genomic DNA extraction methods and phylogenetic analysis of Beauveria bassiana from Central Java, Indonesia, and its toxicity against the fall armyworm, Spodoptera frugiperda J.E. Smith (Lepidoptera: Noctuidae)

BackgroundControl techniques using biological control agents such as Beauveria bassiana (Balsamo) Vuillemin have the advantage of not showing any negative impacts on environmental health and safety issues. This study used isolates from B. bassiana collection from Laboratory of Pest Monitoring belonging to Indonesian Ministry of Agriculture (LPHP) in Central Java which showed potential in controlling target pest. The problems that still occur are the lack of facilities and infrastructure and the lack of quality testing of collection isolates in LPHP, so that the isolate identification process is still carried out in simple method, and bioassay testing on the fall armyworm (FAW), Spodoptera frugiperda J.E. Smith (Lepidoptera: Noctuidae) as a target pest, is not commonly conducted. The results of bioassay testing can be used to determine the potential of a biological agent to control target pest.ResultThe two DNA extraction methods showed different results regarding DNA concentration and purity values, but both methods were good and could be used to amplify DNA using PCR. The DNA band was amplified at 500–600 bp using primers ITS 1 and ITS 4. The results of molecular analysis showed that the four isolates of B. bassiana from Central Java were found in the same clade as B. bassiana from South Sumatra, Dhaka, and Oromia, where these isolates showed similar similarities descended from a common ancestor. Genetically, B. bassiana isolates from Central Java show more genetic similarities to B. bassiana isolates from South Sumatra, Indonesia. Quality testing was carried out by calculating the density and germination ability values for LPHP isolates from Sukoharjo (Sukoharjo isolate), Temanggung (Purworejo isolate), and Banyumas (Banyumas and Cilacap isolate), which showed varying results. The bioassay test used three isolates, namely B. bassiana from Sukoharjo, Banyumas, and Cilacap, which were selected based on density values, germination ability, and molecular analysis. The ability to cause death of the three isolates against S. frugiperda showed different results where the isolate from Sukoharjo, Banyumas, and Cilacap caused mortality of 60, 40, and 60%, and the LC50 value of each isolate was 3.3 × 106, 1.3 × 107, and 3.5 × 107 conidia ml−1, respectively.ConclusionMorphological identification by macroscopic, microscopic, and molecular analysis showed that the isolate from the LPHP collection in Central Java, Indonesia, was B. bassiana. Genetically, the four isolates showed similar characteristics to isolates from South Sumatra, Indonesia. B. bassiana isolates from collections from Central Java showed potentials as a biological control agent against S. frugiperda.

Chromosome-scale genome assembly of Apocynum pictum, a drought-tolerant medicinal plant from the Tarim Basin.

Apocynum pictum Schrenk is a semishrub of the Apocynaceae family with a wide distribution throughout the Tarim Basin that holds significant ecological, medicinal, and economic values. Here, we report the assembly of its chromosome-level reference genome using Nanopore long-read, Illumina HiSeq paired-end, and high-throughput chromosome conformation capture sequencing. The final assembly is 225.32 Mb in length with a scaffold N50 of 19.64 Mb. It contains 23,147 protein-coding genes across 11 chromosomes, 21,148 of which (91.36%) have protein functional annotations. Comparative genomics analysis revealed that A. pictum diverged from the closely related species Apocynum venetum approximately 2.2 million years ago and has not undergone additional polyploidizations after the core eudicot WGT-γ event. Karyotype evolution analysis was used to characterize interchromosomal rearrangements in representative Apocynaceae species and revealed that several A. pictum chromosomes were derived entirely from single chromosomes of the ancestral eudicot karyotype. Finally, we identified 50 members of the well-known stress-responsive WRKY transcription factor family and used transcriptomic data to document changes in their expression at 2 stages of drought stress, identifying a number of promising candidate genes. Overall, this study provides high-quality genomic resources for evolutionary and comparative genomics of the Apocynaceae, as well as initial molecular insights into the drought adaptation of this valuable desert plant.

REDalign: accurate RNA structural alignment using residual encoder-decoder network

BackgroundRNA secondary structural alignment serves as a foundational procedure in identifying conserved structural motifs among RNA sequences, crucially advancing our understanding of novel RNAs via comparative genomic analysis. While various computational strategies for RNA structural alignment exist, they often come with high computational complexity. Specifically, when addressing a set of RNAs with unknown structures, the task of simultaneously predicting their consensus secondary structure and determining the optimal sequence alignment requires an overwhelming computational effort of O(L6)\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$O(L^6)$$\\end{document} for each RNA pair. Such an extremely high computational complexity makes these methods impractical for large-scale analysis despite their accurate alignment capabilities.ResultsIn this paper, we introduce REDalign, an innovative approach based on deep learning for RNA secondary structural alignment. By utilizing a residual encoder-decoder network, REDalign can efficiently capture consensus structures and optimize structural alignments. In this learning model, the encoder network leverages a hierarchical pyramid to assimilate high-level structural features. Concurrently, the decoder network, enhanced with residual skip connections, integrates multi-level encoded features to learn detailed feature hierarchies with fewer parameter sets. REDalign significantly reduces computational complexity compared to Sankoff-style algorithms and effectively handles non-nested structures, including pseudoknots, which are challenging for traditional alignment methods. Extensive evaluations demonstrate that REDalign provides superior accuracy and substantial computational efficiency.ConclusionREDalign presents a significant advancement in RNA secondary structural alignment, balancing high alignment accuracy with lower computational demands. Its ability to handle complex RNA structures, including pseudoknots, makes it an effective tool for large-scale RNA analysis, with potential implications for accelerating discoveries in RNA research and comparative genomics.

Genomic insights into the cold adaptation and secondary metabolite potential of Pseudoalteromonas sp. WY3 from Antarctic krill

In the Antarctic marine ecosystem, krill play a pivotal role, yet the intricate microbial community intertwined with these diminutive crustaceans remains largely unmapped. In this study, we successfully isolated and characterized a unique bacterial strain, Pseudoalteromonas sp. WY3, from Antarctic krill. Genomic analysis revealed that WY3 harbors a multitude of genes associated with cold shock proteins, oxidoreductases, and enzymes involved in the osmotic stress response, equipping it with a robust molecular arsenal to withstand frigid Antarctic conditions. Furthermore, the presence of two distinct biosynthesis-related gene clusters suggests that WY3 has the potential to synthesize diverse secondary metabolites, including aryl polyenes and ribosomally synthesized and post-translationally modified peptides. Notably, the identification of genes encoding enzymes crucial for biological immunity pathways, such as apeH and ubiC, hints at a complex symbiotic relationship between WY3 and its krill host. This comprehensive study highlights the robust potential of WY3 for secondary metabolite production and its remarkable ability to thrive at extremely low temperatures in the Antarctic ecosystem, shedding light on the interplay between culturable microorganisms and their hosts in harsh environments, and providing insights into the underexplored microbial communities associated with Antarctic marine organisms and their role in environmental adaptation and biotechnological applications.

Emerging threat of artemisinin partial resistance markers (pfk13 mutations) in Plasmodium falciparum parasite populations in multiple geographical locations in high transmission regions of Uganda

BackgroundArtemisinin-based combination therapy (ACT) is currently recommended for treatment of uncomplicated malaria. However, the emergence and spread of partial artemisinin resistance threatens their effectiveness for malaria treatment in sub-Saharan Africa where the burden of malaria is highest. Early detection and reporting of validated molecular markers (pfk13 mutations) in Plasmodium falciparum is useful for tracking the emergence and spread of partial artemisinin resistance to inform containment efforts.MethodsGenomic surveillance was conducted at 50 surveillance sites across four regions of Uganda in Karamoja, Lango, Acholi and West Nile from June 2021 to August 2023. Symptomatic malaria suspected patients were recruited and screened for presence of parasites. In addition, dried blood spots (DBS) were collected for parasite genomic analysis with PCR and sequencing. Out of 563 available dried blood spots (DBS), a random subset of 240 P. falciparum mono-infections, confirmed by a multiplex PCR were selected and used for detecting the pfk13 mutations by Sanger sequencing using Big Dye Terminator method. Regional variations in the proportions of pfk13 mutations were assessed using the chi square or Fisher’s exact tests while Kruskal–Wallis test was used to compare absolute parasite DNA levels between wild type and mutant parasites.ResultsOverall, 238/240 samples (99.2%) contained sufficient DNA and were successfully sequenced. Three mutations were identified within the sequenced samples; pfk13 C469Y in 32/238 (13.5%) samples, pfk13 A675V in 14/238 (5.9%) and pfk13 S522C in (1/238 (0.42%) samples across the four surveyed regions. The prevalence of pfk13 C469Y mutation was significantly higher in Karamoja region (23.3%) compared to other regions, P = 0.007. The majority of parasite isolates circulating in West Nile are of wild type (98.3), P = 0.002. Relative parasite DNA quantity did not differ in samples carrying the wild type, C469Y and A675V alleles (Kruskal–Wallis test, P = 0.6373).ConclusionDetection of validated molecular markers of artemisinin partial resistance in multiple geographical locations in this setting provides additional evidence of emerging threat of artemisinin partial resistance in Uganda. In view of these findings, periodic genomic surveillance is recommended to detect and monitor levels of pfk13 mutations in other regions in parallel with TES to assess potential implication on delayed parasite clearance and associated treatment failure in this setting. Future studies should consider identification of potential drivers of artemisinin partial resistance in the different malaria transmission settings in Uganda.

Genomic Characterization and Comparative Analysis of Streptococcus zhangguiae sp. nov. Isolated from the Respiratory Tract of Marmota Himalayana.

Two Gram-stain-positive, oxidase-negative, non-motile, facultative anaerobic, α-hemolytic, coccus-shaped bacteria (zg-86T and zg-70) were isolated from the respiratory tracts of marmots (Marmota Himalayana) on the Qinghai-Tibet Plateau of China. Phylogenetic analysis of the 16S rRNA gene and 545 core genes revealed that these two strains belong to the Streptococcus genus. These strains were most closely related to Streptococcus respiraculi HTS25T, Streptococcus cuniculi CCUG 65085T, and Streptococcus marmotae HTS5T. The average nucleotide identity (ANI) and digital DNA‒DNA hybridization (dDDH) were below the threshold for species delineation. The predominant cellular fatty acids (CFAs) in this novel species were C16:0, C18:0, and C18:1ω9c, whereas the primary polar lipids were phosphatidylglycerol (PG), phosphatidylethanolamine (PE) and an unknown phosphoglycolipid (PGL). The optimal growth conditions for the strains were 37°C, pH 7.0, and 0.5% (w/v) NaCl on brain-heart infusion (BHI) agar supplemented with 5% defibrinated sheep blood. Comparative genomics analyses revealed the potential pathogenicity of strain zg-86T through comparisons with suis subclade strains in terms of virulence factors, pathogen-host interactions (PHIs) and mobile genetic factors (MGEs). Based on the phenotypic characteristics and phylogenetic analyses, we propose that these two isolates represent novel species in the genus Streptococcus, for which the names Streptococcus zhangguiae sp. nov. (the type strain zg-86T=GDMCC 1.1758T=JCM 34273T) is proposed.

Comprehensive genomic analysis and evaluation of in vivo and in vitro safety of Heyndrickxia coagulans BC99

This study aimed to evaluate the safety profile and beneficial effects of Heyndrickxia coagulans strain BC99 (BC99) for potential use in functional foods and pharmaceuticals. We began with whole genome sequencing of BC99, followed by a comprehensive safety assessment comprising genome analysis, hemolysis, cytotoxicity, antibiotic susceptibility tests, and cell adhesion and tolerance studies, along with acute and subacute oral toxicity studies in animal models. BC99 was isolated from a well-characterized collection originating from the feces of a healthy infant. Our results indicated no hemolytic activity on Columbia blood agar plates and broad antibiotic sensitivity, including to gentamicin, ampicillin, chloramphenicol, ciprofloxacin, and others. Cytotoxicity testing confirmed no adverse effects on HT-29 cells and significant adhesive properties to intestinal epithelial cells. Tolerance tests demonstrated over 90% viability of BC99 under simulated gastrointestinal conditions. In vivo studies in mice and rats confirmed the absence of adverse effects following oral administration. Collectively, these findings support BC99’s robust tolerance to gastrointestinal environments, strong adhesion capabilities, and a broad spectrum of antibiotic resistance, underlining its potential as a safe and effective agent for gut microbiota modulation and host health enhancement.

Identification of Genes and Long Non-Coding RNAs Putatively Related to Portunus trituberculatus Sex Determination and Differentiation Using Oxford Nanopore Technology Full-Length Transcriptome Sequencing

The swimming crab (Portunus trituberculatus) is an economically important species in China, and its growth traits show obvious sexual dimorphism. Thus, it is important to study the mechanism of sex determination and differentiation in this species. Herein, we identified 2138 differentially expressed genes and 132 differentially expressed long non-coding RNAs (lncRNAs) using Oxford Nanopore Technology full-length transcriptome sequencing. We predicted 561 target genes of the differentially expressed lncRNAs according to their location and base pair complimentary principles. Furthermore, pathways related to sex determination, differentiation, and reproduction were enriched for lncRNAs according to gene ontology and Kyoto Encyclopedia of Genes and Genomes analyses. This indicated that lncRNAs might play regulatory roles in these pathways. Our results could form the basis for future studies of sex determination and differentiation in P. trituberculatus.

Assembly and comparative analysis of the multichromosomal mitochondrial genome of globally endangered seagrass Halophila beccarii

BackgroundHalophila beccarii is one of the oldest two generations of seagrass plants and one of the 10 species of seagrass currently at risk of extinction worldwide. Therefore, how to effectively protect the H. beccarii resources from extinction is a huge challenge. Molecular biology research can provide a scientific basis for species conservation. So far, there has been no detailed analysis of the mitochondrial genome of the genus Halophila.ResultsThe mitochondrial genome of H. beccarii was assembled into 28 circular chromosomes, ranging in length from 41,738 bp to 104,744 bp, with a total length of 1,964,072 bp and a GC content of 46.71%. It contains 39 genes, including 26 protein coding genes, 10 tRNA genes, and 3 rRNA genes. Repeat sequence analysis and prediction of RNA editing sites revealed a total of 850 dispersed repeats, 1,205 simple repeats, 61 tandem repeats, and 120 RNA editing sites. Analysis of codon usage indicates that codons ending in A/U are preferred. Gene migration between the mitochondrial genome and the chloroplast genome was observed through homologous fragment detection. In addition, Ka/Ks analysis showed that most protein coding genes in the mitochondrial genome experienced negative selection, while only the nad3 gene experienced potential positive selection in most Alismatales. Nucleotide polymorphism analysis revealed variations in each gene, with rpl10 being the most significant. In addition, comparative analysis shows that the GC content is conserved, but there are significant differences in the size and structure of mitochondrial genomes among different species of Alismatales. The phylogenetic analysis based on the mitochondrial genome reflects the exact evolutionary and taxonomic status of H. beccarii.ConclusionIn this study, we sequenced and annotated the mitochondrial genome of H. beccarii, and compared it with the mitochondrial genomes of other plants in Alismatales. Our findings enrich the mitogenome database of seagrass plants and highlight the potential for mitochondrial genes to help decipher plant evolutionary history.

Breast cancer genomic analyses reveal genes, mutations, and signaling networks.

Breast cancer (BC) is the most commonly diagnosed cancer and the predominant cause of death in women. BC is a complex disorder, and the exploration of several types of BC omic data, highlighting genes, perturbations, signaling and cellular mechanisms, is needed. We collected mutational data from 9,555 BC samples using cBioPortal. We classified 1174 BC genes (mutated ≥ 40 samples) into five tiers (BCtier_I-V) and subjected them to pathway and protein‒protein network analyses using EnrichR and STRING 11, respectively. BCtier_I possesses 12 BC genes with mutational frequencies > 5%, with only 5 genes possessing > 10% frequencies, namely, PIK3CA (35.7%), TP53 (34.3%), GATA3 (11.5%), CDH1 (11.4%) and MUC16 (11%), and the next seven BC genes are KMT2C (8.8%), TTN (8%), MAP3K1 (8%), SYNE1 (7.2%), AHNAK2 (7%), USH2A (5.5%), and RYR2 (5.4%). Our pathway analyses revealed that the five top BC pathways were the PI3K-AKT, TP53, NOTCH, HIPPO, and RAS pathways. We found that BC panels share only seven genes. These findings show that BC arises from genetic disruptions evident in BC signaling and protein networks.

Cultivation of deep-sea bacteria from the Northwest Pacific Ocean and characterization of Limnobacter profundi sp. nov., a phenol-degrading bacterium

Despite previous culture-independent studies highlighting the prevalence of the order Burkholderiales in deep-sea environments, the cultivation and characterization of deep-sea Burkholderiales have been infrequent. A total of 243 deep-sea bacterial strains were isolated from various depths in the Northwest Pacific Ocean, with 33 isolates (13.6%) from a depth of 4000 m classified into Burkholderiales. Herein, we report the isolation and genome characteristics of strain SAORIC-580T, from a depth of 4000 m in the Northwest Pacific Ocean. The strain showed a close phylogenetic relationship with Limnobacter thiooxidans CS-K2T, sharing 99.9% 16S rRNA gene sequence identity. The complete whole-genome sequence of strain SAORIC-580T comprised 3.3 Mbp with a DNA G+C content of 52.5%. Comparative genomic analysis revealed average nucleotide identities between 79.4–85.7% and digital DNA-DNA hybridization values of 19.9–29.5% when compared to other Limnobacter genomes, indicating that the strain represents a novel species within the genus. Genomic analysis revealed unique adaptations to deep-sea conditions, including genes associated with phenol degradation, stress responses, cold adaptation, heavy metal resistance, signal transduction, and carbohydrate metabolism. The SAORIC-580T genome was found to be more abundant in the deep sea than at the surface in the trenches of the Northwest Pacific Ocean, suggesting adaptations to the deep-sea environment. Phenotypic characterization highlighted distinct differences from other Limnobacter species, including variations in growth conditions, enzyme activities, and phenol degradation capabilities. Chemotaxonomic markers of the strain included ubiquinone-10, major fatty acids such as C16:0, C16:1, and C18:1, and major polar lipids including phosphatidylethanolamine, phosphatidylglycerol, and diphosphatidylglycerol. Based on the polyphasic taxonomic data, it is concluded that strain SAORIC-580T (= KACC 21440T = NBRC 114111T) represents a novel species, for which the name Limnobacter profundi sp. nov. is proposed.

Genome-Based Classification of 'Haloarcula aidinensis' and Description of Three Novel Halophilic Archaea Isolated from an Inland Saline Lake, Coastal Saline Soil, and a Marine Solar Saltern.

'Haloarcula aidinensis' was described by a pioneer Chinese scientist focused on halophilic archaea thirty years ago, and the type strain of 'Haloarcula aidinensis' was recently classified based on phylogenetic, phylogenomic, and comparative genomic analyses. Other three novel halophilic archaeal strains, CK38T, DT43T, and SYNS111T, isolated from diverse saline environments in China, were simultaneously subjected to polyphasic classification. 'Haloarcula aidinensis' A5 was found to be related to Haloarcula amylolytica, while strains CK38T, DT43T, and SYNS111T represented three novel species of Haloarcula based on phylogenetic, phylogenomic, and comparative genomic analyses. These strains can be distinguished from other species within the genus Haloarcula based on multiple phenotypic characteristics. The major phospholipids, phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, and phosphatidylglycerol sulfate can be detected in these strains, while the glycolipid profiles of these strains are diverse. Strains A5 and DT43T contained glucosyl mannosyl glucosyl diether and a diglycosyl diether, while other strains CK38T and SYNS111T had sulfated mannosyl glucosyl diether and mannosyl glucosyl diether. Thus, strain A5 should be a reference strain of Har. amylolytica and three novel species of Haloarcula, Haloarcula sediminis sp. nov., Haloarcula brevis sp. nov., and Haloarcula regularis sp. nov. are proposed to accommodate strains CK38T (= CGMCC 1.62732T = JCM 36675T), DT43T (= CGMCC 1.18924T = JCM 36146T), and SYNS111T (= CGMCC 1.62601T = JCM 36149T).

Genomic insights into key mechanisms for carbon, nitrogen, and phosphate assimilation by the acidophilic, halotolerant genus Acidihalobacter members.

In-depth comparative genomic analysis was conducted to predict carbon, nitrogen, and phosphate assimilation pathways in the halotolerant, acidophilic genus Acidihalobacter. The study primarily aimed to understand how the metabolic capabilities of each species can determine their roles and effects on the microbial ecology of their unique saline and acidic environments, as well as in their potential application to saline water bioleaching systems. All four genomes encoded the genes for the complete tricarboxylic acid cycle, including 2-oxoglutarate dehydrogenase, a key enzyme absent in obligate chemolithotrophic acidophiles. Genes for a unique carboxysome shell protein, csoS1D, typically found in halotolerant bacteria but not in acidophiles, were identified. All genomes contained lactate and malate utilization genes, but only Ac. ferrooxydans DSM 14175T contained genes for the metabolism of propionate. Genes for phosphate assimilation were present, though organized differently across species. Only Ac. prosperus DSM 5130T and Ac. aeolianus DSM 14174T genomes contained nitrogen fixation genes, while Ac. ferrooxydans DSM 14175T and Ac. yilgarnensis DSM 105917T possessed genes for urease transporters and respiratory nitrate reductases, respectively. The findings suggest that all species can fix carbon dioxide but can also potentially utilize exogenous carbon sources and that the non-nitrogen-fixing species rely on alternative nitrogen assimilation mechanisms.

Chromosome-scale genome assembly of Codonopsis pilosula and comparative genomic analyses shed light on its genome evolution

IntroductionCodonopsis pilosula is a significant plant in traditional Chinese medicine, valued for its edible and medicinal properties. However, the lack of available genomic resources has hindered further research.MethodsThis study presents the first chromosome-scale genome assembly of C. pilosula using PacBio CLR reads and Hi-C scaffolding technology. Additionally, Ks analysis and syntenic depth analysis were performed to elucidate its evolutionary history.ResultsThe final assembly yielded a high-quality genome of 679.20 Mb, which was anchored to 8 pseudo-chromosomes with an anchoring rate of 96.5% and a scaffold N50 of 80.50 Mb. The genome assembly showed a high completeness of 97.6% based on Benchmarking with Universal Single-Copy Orthologs (BUSCO) analysis. Repetitive elements constituted approximately 76.8% of the genome, with long terminal repeat retrotransposons (LTRs) accounting for about 39.17%. Ks and syntenic depth analyses revealed that the polyploidization history of three platycodonoid clade species involved only the γ-WGT event. Karyotype evolutionary analysis identified an ancestral karyotype with 9 protochromosomes for the three platycodonoid clade species. Moreover, non-WGD genes, particularly those arising from tandem duplications, were found to contribute significantly to gene family expansion.DiscussionThese findings provide essential insights into the genetic diversity and evolutionary biology of C. pilosula, aiding its conservation and sustainable use.

Enhancing SARS-CoV-2 Lineage Surveillance through the Integration of a Simple and Direct qPCR-Based Protocol Adaptation with Established Machine Learning Algorithms.

Emerging and evolving Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) lineages, adapted to changing epidemiological conditions, present unprecedented challenges to global public health systems. Here, we introduce an adapted analytical approach that complements genomic sequencing, applying a cost-effective quantitative polymerase chain reaction (qPCR)-based assay. Viral RNA samples from SARS-CoV-2 positive cases detected by diagnostic laboratories or public health network units in Ceará, Brazil, were tracked for genomic surveillance and analyzed by using paired-end sequencing combined with integrative genomic analysis. Validation of a key structural variation was conducted with gel electrophoresis for the presence of a specific open reading frame 7a(ORF7a) gene deletion within the "BE.9" lineages tracked. The analytical innovation of our method is the optimization of a simple intercalating dye-based qPCR assay through repositioning primers from the ARTIC v4.1 amplicon panel to detect large molecular patterns. This assay distinguishes between "BE.9" and "non-BE.9" lineages, particularly BQ.1, without the need for expensive probes or sequencing. The protocol was validated against lineage predictions from next-generation sequencing (NGS) using 525 paired samples, achieving 93.3% sensitivity, 95.1% specificity, and 92.4% agreement, as measured by Cohen's Kappa coefficient. Machine learning (ML) models were trained using the melting curves from intercalating dye-based qPCR of 1724 samples, enabling highly accurate lineage assignment. Among them, the support vector machine (SVM) model had the best performance and after fine-tuning showed ∼96.52% (333/345) accuracy in comparison to the test data set. Our integrated approach provides an adapted analytical method that is both cost-effective and scalable, suitable for rapid assessment of emerging variants, especially in resource-limited settings. In this work, the protocol is applied to improve the monitoring of SARS-CoV-2 sublineages but can be extended to track any key molecular signature, including large insertions and deletions (indels) commonly observed in pathogenic agent subtypes. By offering a complement to traditional sequencing methods and utilizing easily trainable machine learning algorithms, our methodology contributes to enhanced molecular surveillance strategies and supports global efforts in pandemic control.

Distinct molecular responses of mangrove plants to hypoxia and reoxygenation stresses contribute to their resilience in coastal wetland environment

Mangroves adapt to periodical submergence and constitute resilient ecosystems in coastal environments. The question is whether they can sustain long submergence stress when sea level rises as a consequence of climate change. To address this, seedlings of two representative mangrove species that acclimate to low to mid tide (Avicennia marina) and mid to high tide (Kandelia obovata) conditions were treated with continual submergence for 7 days as extended hypoxia, or semi-diurnal cyclic submergence and reoxygenation for 7 days. At specific time points, leaves were collected to construct RNA-Sequencing libraries for gene expression analysis. Through the lens of transcriptome, the initial response of A. marina to submergence was mild but more dramatic after prolong immersion. However, the initial response of K. obovata was drastic and reduced in latitude later, suggesting distinct species-specific responses. After adapting to diurnal cycles, both species minimized transcriptome fluctuations similarly. Metabolically, during initial response, sucrose and starch were converted into glucose for fermentation to increase glycolytic flux, coupled with regeneration of NAD+. The energy amelioration was accompanied by longer term phytohormone regulations where ethylene signal transduction pathway was enhanced, but abscisic acid biosynthesis was reduced. Notably, gibberellic acids biosynthesis increased in A. marina but decreased in K. obovata as a unique feature. Genomic level analysis indicated that only about 30 % of the conserved plant submergence responsive genes were expressed during submergence in these mangroves. The function of an ethylene responsive gene was validated in transgenic Arabidopsis. This research elucidates distinct molecular mechanisms and metabolic pathways that empower A. marina and K. obovata to endure prolonged submergence and hypoxia. By highlighting their unique adaptive strategies in response to rising sea levels, these findings enhance our understanding of mangrove resilience and provide insights for the conservation and management of these essential coastal ecosystems in the face of climate change.

Genomic Insights into Drug Abuse: The Role of DNA and Protein-Coding Regions in Addiction and Genetic Mutation

Abstract This paper explores the genetic and epigenetic impacts of drug abuse, focusing on how chronic drug use can induce mutations in DNA, particularly within protein-coding regions, and alter gene expression through mechanisms such as DNA methylation. The study highlights the significant role of these genetic changes in the brain's reward pathways, which contribute to the development and persistence of addiction. Through a comprehensive literature review, the paper identifies key genetic factors associated with addiction susceptibility and the long-term effects of drug-induced epigenetic modifications. A proposed research framework is presented, emphasizing the need for longitudinal studies to track epigenetic changes over time, targeted genomic analysis to identify critical mutations, and the integration of multi-omics approaches to provide a holistic understanding of drug abuse's impact on the genome. The paper also addresses ethical considerations, including the importance of protecting genetic privacy and preventing stigmatization based on genetic findings. The study concludes that advancing our understanding of the genetic and epigenetic mechanisms underlying addiction can lead to more effective, personalized treatment strategies and inform public health policies aimed at mitigating the effects of drug abuse.

Comparative Genomic Analysis Reveals Distinct Virulence and Resistance Mechanisms in 21 Bacterial Fish Pathogens

The rising bacterial infections threaten world aquaculture and wild fish populations, making it imperative to increase the understanding of the mechanisms of pathogenic virulence and resistance. This study applies comparative genomic analysis to 21 bacterial fish pathogens, using whole-genome sequences from public genomic resources and sophisticated bioinformatics tools for screening of virulence factors, mobile genetic elements, antibiotic resistance genes, anti-phage defense mechanisms and secretory systems. We have seen that the different pathogens depict a wide range of variability regarding virulence and resistance potential, which may be attributed to species-specific adaptation. Notably, Streptococcus agalactiae and Mycobacterium salmoniphilum were found to possess high offensive and defensive virulence potential, but at different regulative controls. We also found diverse secretion systems and intricate mechanisms for antibiotic resistance, which have provided very important insights into how pathogens adapt to their environments. By categorizing functional genes and finding anti-phage systems, our analysis has revealed new insights into the complex interactions among bacterial virulence, resistance, and host defense mechanisms. These findings not only shed new light on the bacterial pathogenesis process in aquaculture but also provide the bases for focused, therapeutically-based strategies and genomic surveillance programs able to improve disease management and sustainability in aquaculture environments.

Comparative genomic analysis and characterization of novel high-quality draft genomes from the coal metagenome.

Coal, a sedimentary rock harbours a complex microbial community that plays a significant role in its formation and characteristics. However, coal metagenome sequencing and studies were less, limiting our understanding of this complex ecosystem. This study aimed to reconstruct high-quality metagenome-assembled genomes (MAGs) from the coal sample collected in the Neyveli mine to explore the unrevealed diversity of the coal microbiome. Using Illumina sequencing, we obtained high-quality raw reads in FASTQ format. Subsequently, de novo assembly and binning with metaWRAP software facilitated the reconstruction of coal MAGs. Quality assessment using CheckM identified 10 High-Quality MAGs (HQ MAGs), 7 medium-quality MAGs (MQ MAGs), and 6 low-quality MAGs (LQ MAGs). Further analysis using GTDB-Tk revealed four HQ MAGs as known species like Dermacoccus abyssi, Sphingomonas aquatilis, Acinetobacter baumannii, and Burkholderia cenocepacia. The remaining six HQ MAGs were classified as Comamonas, Arthrobacter, Noviherbaspirillum, Acidovorax, Oxalicibacterium, and Bordetella and designated as novel genomes by the validation of digital DNA-DNA hybridization (dDDH). Phylogenetic analysis and further pangenome analysis across the phylogenetic groups revealed a similar pattern with a high proportion of cloud genes. We further analysed the functional potential of these MAGs and closely related genomes using COG. The comparative functional genomics revealed that novel genomes are highly versatile, potentially reflecting adaptations to the coal environment. BlastKOALA was used to conduct a detailed analysis of the metabolic pathways associated with the MAGs. This study highlights the comparative genomic analysis of novel coal genomes with their closely related genomes to understand the evolutionary relationships and functional properties.