High-throughput Sequencing Approach Research Articles

Single cell RNA-sequencing and RNA-tomography of the avian embryo extending body axis

Introduction: Vertebrate body axis formation initiates during gastrulation and continues within the tail bud at the posterior end of the embryo. Major structures in the trunk are paired somites, which generate the musculoskeletal system, the spinal cord—forming part of the central nervous system, and the notochord, with important patterning functions. The specification of these different cell lineages by key signalling pathways and transcription factors is essential, however, a global map of cell types and expressed genes in the avian trunk is missing.Methods: Here we use high-throughput sequencing approaches to generate a molecular map of the emerging trunk and tailbud in the chick embryo.Results and Discussion: Single cell RNA-sequencing (scRNA-seq) identifies discrete cell lineages including somites, neural tube, neural crest, lateral plate mesoderm, ectoderm, endothelial and blood progenitors. In addition, RNA-seq of sequential tissue sections (RNA-tomography) provides a spatially resolved, genome-wide expression dataset for the avian tailbud and emerging body, comparable to other model systems. Combining the single cell and RNA-tomography datasets, we identify spatially restricted genes, focusing on somites and early myoblasts. Thus, this high-resolution transcriptome map incorporating cell types in the embryonic trunk can expose molecular pathways involved in body axis development.

Unveiling Inter- and Intra-Patient Sequence Variability with a Multi-Sample Coronavirus Target Enrichment Approach

Amid the global challenges posed by the COVID-19 pandemic, unraveling the genomic intricacies of SARS-CoV-2 became crucial. This study explores viral evolution using an innovative high-throughput next-generation sequencing (NGS) approach. By taking advantage of nasal swab and mouthwash samples from patients who tested positive for COVID-19 across different geographical regions during sequential infection waves, our study applied a targeted enrichment protocol and pooling strategy to increase detection sensitivity. The approach was extremely efficient, yielding a large number of reads and mutations distributed across 10 distinct viral gene regions. Notably, the genes Envelope, Nucleocapsid, and Open Reading Frame 8 had the highest number of unique mutations per 1000 nucleotides, with both spike and Nucleocapsid genes showing evidence for positive selection. Focusing on the spike protein gene, crucial in virus replication and immunogenicity, our findings show a dynamic SARS-CoV-2 evolution, emphasizing the virus–host interplay. Moreover, the pooling strategy facilitated subtle sequence variability detection. Our findings painted a dynamic portrait of SARS-CoV-2 evolution, emphasizing the intricate interplay between the virus and its host populations and accentuating the importance of continuous genomic surveillance to understand viral dynamics. As SARS-CoV-2 continues to evolve, this approach proves to be a powerful, versatile, fast, and cost-efficient screening tool for unraveling emerging variants, fostering understanding of the virus’s genetic landscape.

Diversity and Functionality of Bacteria Associated with Different Tissues of Spider Heteropoda venatoria Revealed through Integration of High-Throughput Sequencing and Culturomics Approaches

Spiders host a diverse range of bacteria in their guts and other tissues, which have been found to play a significant role in their fitness. This study aimed to investigate the community diversity and functional characteristics of spider-associated bacteria in four tissues of Heteropoda venatoria using HTS of the 16S rRNA gene and culturomics technologies, as well as the functional verification of the isolated strains. The results of HTS showed that the spider-associated bacteria in different tissues belonged to 34 phyla, 72 classes, 170 orders, 277 families, and 458 genera. Bacillus was found to be the most abundant bacteria in the venom gland, silk gland, and ovary, while Stenotrophomonas, Acinetobacter, and Sphingomonas were dominant in the gut microbiota. Based on the amplicon sequencing results, 21 distinct cultivation conditions were developed using culturomics to isolate bacteria from the ovary, gut, venom gland, and silk gland. A total of 119 bacterial strains, representing 4 phyla and 25 genera, with Bacillus and Serratia as the dominant genera, were isolated. Five strains exhibited high efficiency in degrading pesticides in the in vitro experiments. Out of the 119 isolates, 28 exhibited antibacterial activity against at least one of the tested bacterial strains, including the pathogenic bacteria Staphylococcus aureus, Acinetobacter baumanii, and Enterococcus faecalis. The study also identified three strains, GL312, PL211, and PL316, which exhibited significant cytotoxicity against MGC-803. The crude extract from the fermentation broth of strain PL316 was found to effectively induce apoptosis in MGC-803 cells. Overall, this study offers a comprehensive understanding of the bacterial community structure associated with H. venatoria. It also provides valuable insights into discovering novel antitumor natural products for gastric cancer and xenobiotic-degrading bacteria of spiders.

Identifying archived insect bulk samples using DNA metabarcoding: A case study using the long‐term Rothamsted Insect Survey

AbstractInsect populations are declining in many parts of the world, but a lack of long‐term monitoring data is impeding our ability to understand and mitigate the causes of insect biodiversity loss. Whilst high‐throughput sequencing (HTS) approaches, such as DNA metabarcoding, have the potential to revolutionize insect biomonitoring through rapid scalable identification, it is unclear to what extent HTS can be applied to long‐term stored insect samples. Archived insect samples could inform forecasting and provide valuable information regarding past changes to biodiversity. Here, we assess the efficacy of DNA metabarcoding to identify archived samples from the longest passive monitoring scheme in the United Kingdom: the Rothamsted Insect Survey (RIS). With a focus on aphids as the target taxa of a national network of suction‐traps, we analyze a 16‐year time‐series of stored samples (2003–2018) using DNA metabarcoding from one of the RIS suction traps as an exemplar. We achieved this by using a non‐destructive DNA extraction protocol, ensuring the integrity of archival samples for further studies. We compared the identities of aphids determined by both metabarcoding (as inferred amplicon sequence variants [ASVs]) and morphological identification and found that metabarcoding detected most genera with varying success (mean > 76%). When comparing the two methods objectively (i.e., including taxa not detected morphologically), however, congruence decreased (51%). We show that minimum sequence copy thresholds can minimize metabarcoding false positives, but at the expense of introducing false negatives, highlighting the need for careful data curation. Detectability of taxa identified morphologically and similarity between the two methods did not significantly vary over time, demonstrating the viability of metabarcoding for screening archival samples. We discuss the advantages and challenges of metabarcoding for insect biomonitoring, particularly from archival samples, including improvements to sample handling, processing, and archiving. We highlight the wider potential of HTS approaches for stored samples from insect monitoring schemes, unlocking the immense potential of global historical time series.

Microbial mechanisms for improved soil phosphorus mobilization in monoculture conifer plantations by mixing with broadleaved trees

Replanting broadleaved trees in monoculture conifer plantations has been shown to improve the ecological environment. However, not much is known about the distribution properties of soil phosphate-mobilizing bacteria (PMB) under different mixed plantings or how PMB affects biometabolism-driven phosphorus (P) bioavailability. The phoD and pqqC genes serve as molecular markers of PMB because they regulate the mobilization of organic (Po) and inorganic (Pi) P. Differences in soil bioavailable P concentration, phoD- and pqqC-harboring PMB communities, and their main regulators were analyzed using biologically-based P (BBP) and high-throughput sequencing approaches after combining coniferous trees (Pinus massoniana) and five individual broadleaved trees (Bretschneidera sinensis, Michelia maudiae, Cercidiphyllum japonicum, Manglietia conifera, and Camellia oleifera). The findings revealed that the contents of litter P, soil organic carbon (SOC), available Pi (CaCl2–P), and labile Po (Enzyme-P) were significantly higher in conifer-broadleaf mixed plantations than those in the monospecific Pinus massoniana plantations (PM), especially in the mixed stands with the introduction of Cercidiphyllum japonicum, Michelia maudiae, and Camellia oleifera. Conifer-broadleaf mixing had little effect on the abundance of phoD and pqqC genes but significantly altered species composition within the communities. Conifer-broadleaf mixing improved soil microbial habitat mainly by increasing the pH, increasing carbon source availability and nutrient content, decreasing exchangeable Fe3+ and Al3+ content, and decreasing the activation degrees of Fe and Al oxides in acidic soils. A small group of taxa (phoD: Bradyrhizobium, Tardiphaga, Nitratireductor, Mesorhizobium, Herbaspirillum, and Ralstonia; pqqC: Burkholderia, Variovorax, Bradyrhizobium, and Leptothrix) played a key role in the synthesis of P-related enzymes (e.g., alkaline phosphomonoesterase, ALP) and in lowering the levels of mineral-occluded (HCl–P) and chelated (Citrate-P) Pi. Overall, our findings highlight that mixing conifers and broadleaves could change the PMB communities that produce ALP and dissolve Pi to make P more bioavailable.

Deciphering the microbial communities of alkaline hot spring in Panamik, Ladakh, India using a high-throughput sequencing approach.

Due to their distinctive physicochemical characteristics, hot springs are extremely important. The whole genome metagenomic sequencing technology can be utilized to analyze the diverse microbial community that thrives in this habitat due to the particular selection pressure that prevails there. The current investigation emphasizes on culture-independent metagenomic study of the Panamik hot spring and its nearby areas from Ladakh, India. Based on different diversity indices, sequence analysis of the soil reservoir showed higher species richness and diversity in comparison to water and sediment samples. The mineral content and various physicochemical pameters like temperature, pH had an impact on the composition of the microbial community of the geothermal springs. The phyla Proteobacteria, Cyanobacteria, Bacteroidetes, Actinobacter, Firmicutes, and Verrucomicrobia in bacterial domain dominate the thermos-alkaline spring at Panamik in different concentrations. Economically significant microbes from the genera Actinobacter, Thermosynechoccus, Candidatus Solibacter, Chthoniobacter, Synechoccus, Pseudomonas and Sphingomonas, were prevalent in hot spring. In the archaeal domain, the most dominant phylum and genera were Euryarchaeota and Thermococcus in all the samples. Further, the most abundant species were Methanosarcina barkeri, Nitrospumilus maritimus and Methanosarcina acetivorans. The present study which only examined one of the several thermal springs present in the Himalayan geothermal area, should be regarded as a preliminary investigation of the microbiota that live in the hot springs on these remote areas. These findings suggest that further investigations should be undertaken to characterize the ecosystems of the Panamik hot spring, which serve as a repository for unidentified microbial lineages.

Towards Understanding and Identification of Human Viral Co-Infections.

Viral co-infections, in which a host is infected with multiple viruses simultaneously, are common in the human population. Human viral co-infections can lead to complex interactions between the viruses and the host immune system, affecting the clinical outcome and posing challenges for treatment. Understanding the types, mechanisms, impacts, and identification methods of human viral co-infections is crucial for the prevention and control of viral diseases. In this review, we first introduce the significance of studying human viral co-infections and summarize the current research progress and gaps in this field. We then classify human viral co-infections into four types based on the pathogenic properties and species of the viruses involved. Next, we discuss the molecular mechanisms of viral co-infections, focusing on virus-virus interactions, host immune responses, and clinical manifestations. We also summarize the experimental and computational methods for the identification of viral co-infections, emphasizing the latest advances in high-throughput sequencing and bioinformatics approaches. Finally, we highlight the challenges and future directions in human viral co-infection research, aiming to provide new insights and strategies for the prevention, control, diagnosis, and treatment of viral diseases. This review provides a comprehensive overview of the current knowledge and future perspectives on human viral co-infections and underscores the need for interdisciplinary collaboration to address this complex and important topic.

Abstract 403: APOBEC3 enzymes induce chromosomal translocations in solid cancer

Abstract Introduction: Chromosomal translocations are common oncogenic drivers in human cancers, and an increasing number of translocations are being considered as crucial diagnostic and prognostic markers in clinics. In B-cell lymphoma, a number of chromosomal translocations are directed by the off-target activity of the activation-induced cytidine deaminase (AID; a B-cell specific apolipoprotein B mRNA-editing enzyme, catalytic polypeptode-like (APOBEC) family enzyme). However, in solid tumors, no such enzymes capable of directing chromosomal translocations have been identified. Given recent findings on APOBEC-mediated mutagenesis in solid cancers, APOBECs may be involved in the formation of chromosomal translocations in solid tumors, like AID does in B cells. In human, the APOBEC3 (A3) family consists of seven members (A3A-A3H) and is associated with C <t or="" c<g="" substitutions.="" recent="" evidence="" shows="" that="" a3a="" might="" be="" involved="" in="" chromosomal="" instability="" leading="" to="" drug="" resistance="" lung="" cancer.="" however,="" it="" has="" not="" been="" explored="" whether="" a3s="" generate="" dna="" dsbs,="" eventually="" translocations.="" this="" study,="" we="" investigated="" the="" role="" of="" and="" their="" mechanisms="" translocations="" solid="" cancer.Methods: We applied high-throughput genome-wide translocation sequencing (HTGTS) approach to map genome-wide chromosomal translocations induced by human A3s in mouse fibroblast and human epithelial cells. We correlated patterns of translocations induced by A3s with several other cellular features including transcription, DNA accessibility, DNA replication timing, and mutational signatures. We validated the contribution of A3 to the generation of oncogenic translocations in lung cancer. Results: Ectopic expression of A3s significantly increased chromosomal translocations in mouse fibroblasts and human epithelial cells. A3s and AID preferentially induced translocations in different parts of the genome, but some of the translocation hotspots were shared among them. Hierarchical clustering of hotspots showed that A3-mediated translocations were distinguished from AID-mediated translocations. Notably, the junction distribution of breakpoint clusters showed three distinct translocation patterns based on the locations of breakpoint clusters: (1) AID-preferred; (2) A3-preferred; and (3) AID/A3-mixed. AID-mediated translocations were heavily enriched near the transcription start site, whereas A3-mediated translocations were dispersed across the gene body and transcription end site. Furthermore, DNA replication timing and genomic architectures were linked to the formation of A3-mediated chromosomal translocations in solid cancer. Finally, A3 contributed to the formation of oncogenic rearrangements in lung cancer. Conclusions: A3 enzymes induced chromosomal translocations with distinct patterns compared to AID and facilitated chromosomal translocation formation in solid cancers. Citation Format: Taek-Chin Cheong, Qi Wang, Ahram Jang, Elif Karaca-Atabay, Roberto Chiarle. APOBEC3 enzymes induce chromosomal translocations in solid cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 403.

Implementation of a high-throughput whole genome sequencing approach with the goal of maximizing efficiency and cost effectiveness to improve public health.

Public Health Laboratories that implement whole genome sequencing (WGS) technologies may struggle to find the balance between sample volume and cost effectiveness. We present a method that allows for sequencing of a variety of bacterial isolates in a cost-effective manner. This report provides specific strategies to implement high-volume WGS, including an innovative, low-cost solution utilizing a novel quarter volume sequencing library preparation. The methods described support the use of high-throughput DNA extraction and WGS within budgetary constraints, strengthening public health responses to outbreaks and disease surveillance.

Soil Microbiomes and their Arsenic Functional Genes in Chronically High-Arsenic Contaminated Soils.

Microbial arsenic transformations play essential roles in controlling pollution and ameliorating risk. This study combined high-throughput sequencing and PCR-based approaches targeting both the 16S rRNA and arsenic functional genes to investigate the temporal and spatial dynamics of the soil microbiomes impacted by high arsenic contamination (9.13 to 911.88mg/kg) and to investigate the diversity and abundance of arsenic functional genes in soils influenced by an arsenic gradient. The results showed that the soil microbiomes were relatively consistent and mainly composed of Actinobacteria (uncultured Gaiellales and an unknown_67 - 14 bacterium), Proteobacteria, Firmicutes (particularly, Bacillus), Chloroflexi, and Acidobacteria (unknown_Subgroup_6). Although a range of arsenic functional genes (e.g., arsM, arsC, arrA, and aioA) were identified by shotgun metagenomics, only the arsM gene was detected by the PCR-based method. The relative abundance of the arsM gene accounted for 0.20%-1.57% of the total microbial abundance. Combining all analyses, arsenic methylation mediated by the arsM gene was proposed to be a key process involved in the arsenic biogeochemical cycle and mitigation of arsenic toxicity. This study advances our knowledge about arsenic mechanisms over the long-term in highly contaminated soils.

Utilising eDNA Methods and Interactive Data Dashboards for Managing Sustainable Drinking Water

Generating rapid, easy-to-interpret community data for drinking reservoirs as a means of tackling water quality management is of increasing demand within the water industry. Taste and odour (T&O) is one of many increasing concerns to water companies worldwide, incurring huge costs as customer complaints accumulate and additional treatment and resource management are required. However, there remains a two-fold issue in addressing T&O management: firstly, predicting the initial onset of a T&O event relies on a highly complex understanding of environmental considerations and their interaction with T&O-related taxa, and secondly, there remains a lag between the notification of a T&O event and the resolution of the issue by reservoir management staff. This is partly due to slow, low-resolution methods of detecting and reliably identifying problem taxa in samples. These methods are unable to provide information on the huge plethora of taxa related to T&O metabolite production and often cannot provide data in a timely enough manner for an opportune management response. This means the water industry is often forced to use a reactive, rather than proactive, approach to water quality monitoring. Here, we present methods for implementing a high-throughput sequencing approach to monitoring drinking reservoirs for water quality and improving the sustainability of water supplies, as well as methods for presenting these data on easy-to-interpret data dashboards that can be updated rapidly as new data are generated. Our methods and dashboarding approaches are currently being trialled and tested within the UK water industry, and so here, we show anonymised examples of those data presentations. We propose that these methods can greatly aid reservoir management teams in their approach to T&O monitoring and can be used to implore more sustainable management pipelines, safeguarding future water sources.

Analysis of Culturable Bacterial Diversity of Pangong Tso Lake via a 16S rRNA Tag Sequencing Approach.

The Pangong Tso lake is a high-altitude freshwater habitat wherein the resident microbes experience unique selective pressures, i.e., high radiation, low nutrient content, desiccation, and temperature extremes. Our study attempts to analyze the diversity of culturable bacteria by applying a high-throughput amplicon sequencing approach based on long read technology to determine the spectrum of bacterial diversity supported by axenic media. The phyla Pseudomonadota, Bacteriodetes, and Actinomycetota were retrieved as the predominant taxa in both water and sediment samples. The genera Hydrogenophaga and Rheinheimera, Pseudomonas, Loktanella, Marinomonas, and Flavobacterium were abundantly present in the sediment and water samples, respectively. Low nutrient conditions supported the growth of taxa within the phyla Bacteriodetes, Actinomycetota, and Cyanobacteria and were biased towards the selection of Pseudomonas, Hydrogenophaga, Bacillus, and Enterococcus spp. Our study recommends that media formulations can be finalized after analyzing culturable diversity through a high-throughput sequencing effort to retrieve maximum species diversity targeting novel/relevant taxa.

Analysis of HBV integration in mitochondrial DNA of HepAD38 cells by the high-throughput HBV integration sequencing and RNASeq approaches

Analysis of HBV integration in mitochondrial DNA of HepAD38 cells by the high-throughput HBV integration sequencing and RNASeq approaches

Analysis of immunoglobulin/T-cell receptor repertoires by high-throughput RNA sequencing reveals a continuous dynamic of positive clonal selection in follicular lymphoma.

Follicular lymphoma (FL) course is highly variable, making its clinical management challenging. In this incurable and recurring pathology, the interval between relapses tends to decrease while aggressiveness increases, sometimes resulting in the transformation to higher-grade lymphoma. These evolutions are particularly difficult to anticipate, resulting from complex clonal evolutions where multiple subclones compete and thrive due to their capacity to proliferate and resist therapies. Here, to apprehend further these processes, we used a high-throughput RNA sequencing approach to address simultaneously the B-cell immunoglobulin repertoires and T-cell immunoglobulin repertoires repertoires of lymphoma cells and their lymphoid microenvironment in a large cohort of 131 FL1/2-3A patients. Our data confirm the existence of a high degree of intra-clonal heterogeneity in this pathology, resulting from ongoing somatic hyper-mutation and class switch recombination. Through the evaluation of the Simpson ecological-diversity index, we show that the contribution of the cancerous cells increases during the course of the disease to the detriment of the reactive compartment, a phenomenon accompanied by a concomitant decrease in the diversity of the tumoral population. Clonal evolution in FL thus contrasts with many tumors, where clonal heterogeneity steadily increases over time and participates in treatment evasion. In this pathology, the selection of lymphoma subclones with proliferative advantages progressively outweighs clonal diversification, ultimately leading in extreme cases to transformation to high-grade lymphoma resulting from the rapid emergence of homogeneous subpopulations.

Genome characterization of influenza A and B viruses in New South Wales, Australia, in 2019: A retrospective study using high-throughput whole genome sequencing.

During the 2019 severe influenza season, New South Wales (NSW) experienced the highest number of cases in Australia. This study retrospectively investigated the genetic characteristics of influenza viruses circulating in NSW in 2019 and identified genetic markers related to antiviral resistance and potential virulence. The complete genomes of influenza A and B viruses were amplified using reverse transcription-polymerase chain reaction (PCR) and sequenced with an Illumina MiSeq platform. When comparing the sequencing data with the vaccine strains and reference sequences, the phylogenetic analysis revealed that most NSW A/H3N2 viruses (n= 68; 94%) belonged to 3C.2a1b and a minority (n= 4; 6%) belonged to 3C.3a. These viruses all diverged from the vaccine strain A/Switzerland/8060/2017. All A/H1N1pdm09 viruses (n= 20) showed genetic dissimilarity from vaccine strain A/Michigan/45/2015, with subclades 6B.1A.5 and 6B.1A.2 identified. All B/Victoria-lineage viruses (n= 21) aligned with clade V1A.3, presenting triple amino acid deletions at positions 162-164 in the hemagglutinin protein, significantly diverging from the vaccine strain B/Colorado/06/2017. Multiple amino acid substitutions were also found in the internal proteins of influenza viruses, some of which have been previously reported in hospitalized influenza patients in Thailand. Notably, the oseltamivir-resistant marker H275Y was present in one immunocompromised patient infected with A/H1N1pdm09 and the resistance-related mutation I222V was detected in another A/H3N2-infected patient. Considering antigenic drift and the constant evolution of circulating A and B strains, we believe continuous monitoring of influenza viruses in NSW via the high-throughput sequencing approach provides timely and pivotal information for both public health surveillance and clinical treatment.

Deep RNA sequencing of muscle tissue reveals absence of viral signatures in dermatomyositis.

Objective: To explore a possible connection between active viral infections and manifestation of dermatomyositis (DM). Methods: Skeletal muscle biopsies were analyzed from patients diagnosed with juvenile (n=10) and adult (n=12) DM. Adult DM patients harbored autoantibodies against either TIF-1γ (n=7) or MDA5 (n=5). Additionally, we investigated skeletal muscle biopsies from non-diseased controls (NDC, n=5). We used an unbiased high-throughput RNA sequencing (HTS) approach to detect viral sequences. To further increase sequencing depth, a host depletion approach was applied. Results: In this observational study, no relevant viral sequences were detected either by native sequencing or after host depletion. The absence of detectable viral sequences makes an active viral infection of the muscle tissue unlikely to be the cause of DM in our cohorts. Discussion: Type I interferons (IFN) play a major role in the pathogenesis of both juvenile and adult DM. The IFN response is remarkably conserved between DM subtypes classified by specific autoantibodies. Certain acute viral infections are accompanied by a prominent type I IFN response involving similar downstream mechanisms as in DM. Aiming to elucidate the pathogenesis of DM in skeletal muscle tissue, we used deep RNA sequencing and a host depletion approach to detect possible causative viruses.

Exploring N6-methyladenosine (m6A) modification in tree species: opportunities and challenges.

N 6-methyladenosine (m6A) in eukaryotes is the most common and widespread internal modification in mRNA. The modification regulates mRNA stability, translation efficiency, and splicing, thereby fine-tuning gene regulation. In plants, m6A is dynamic and critical for various growth stages, embryonic development, morphogenesis, flowering, stress response, crop yield, and biomass. Although recent high-throughput sequencing approaches have enabled the rapid identification of m6A modification sites, the site-specific mechanism of this modification remains unclear in trees. In this review, we discuss the functional significance of m6A in trees under different stress conditions and discuss recent advancements in the quantification of m6A. Quantitative and functional insights into the dynamic aspect of m6A modification could assist researchers in engineering tree crops for better productivity and resistance to various stress conditions.

Spike-Seq: An amplicon-based high-throughput sequencing approach for the sensitive detection and characterization of SARS-CoV-2 genetic variations in environmental samples

Ever since the outbreak of COVID-19 disease in Wuhan, China, different variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been identified. Wastewater-based epidemiology (WBE), an approach that has been successfully applied in numerous case studies worldwide, offers a cost-effective and rapid way for monitoring trends of SARS-Cov-2 in the community level without selection bias. Despite being a gold-standard procedure, WBE is a challenging approach due to the sample instability and the moderate efficiency of SARS-CoV-2 concentration in wastewater. In the present study, we introduce Spike-Seq, a custom amplicon-based approach for the S gene sequencing of SARS-CoV-2 in wastewater samples, which enables not only the accurate identification of the existing Spike-related genetic markers, but also the estimation of their frequency in the investigated samples. The implementation of Spike-Seq involves the combination of nested PCR-based assays that efficiently amplify the entire nucleotide sequence of the S gene and next-generation sequencing, which enables the variant detection and the estimation of their frequency. In the framework of the current work, Spike-Seq was performed to investigate the mutational profile of SARS-CoV-2 in samples from the Wastewater Treatment Plant (WWTP) of Athens, Greece, which originated from multiple timepoints, ranging from March 2021 until July 2022. Our findings demonstrate that Spike-Seq efficiently detected major genetic markers of B.1.1.7 (Alpha), B.1.617.2 (Delta) as well as B.1.1.529 (Omicron) variants in wastewater samples and provided their frequency levels, showing similar variant distributions with the published clinical data from the National Public Health organization. The presented approach can prove to be a useful tool for the detection of SARS-CoV-2 in challenging wastewater samples and the identification of the existing genetic variants of S gene.

Awakening soil microbial utilization of nitrate by carbon regulation to lower nitrogen pollution

Soil microbial immobilization of nitrate (NO3−-N, INO3) and its role in mitigating NO3−-N pollution in agricultural ecosystems globally is often neglected because of the tenet that microbes preferentially use ammonium over NO3−-N. Soil INO3 driven by heterotrophic microorganisms is often carbon (C)-limited, and may therefore be stimulated by C sources with high C/nitrogen (N) ratios. Here, using 15NO3−-N labelling coupled with acetylene inhibition, quantitative PCR and Illumina high-throughput sequencing approaches, we demonstrated that INO3 was stimulated from zero to a substantial level by crop residue amendment without any environmental risk; the degree of stimulation depending on residue quality and soil type. The stimulation was predicted well by C mineralization of the added residue. Soil with a lower initial nutrient status had reduced INO3 efficiency due to stronger C constraint and lower return on energy investment. Fast-growing bacterial r-strategists were identified as the key driver of INO3. We estimate that, globally, residue amendment could cause a total NO3−-N loss reduction of about 33.6 Tg N yr−1 through INO3 stimulation. We suggest that soil INO3 driven by addition of exogenous C is an overlooked process that can help curb NO3−-N accumulation in soil and its subsequent release to the environment, the “nitrate time bomb”.

Revealing microbial patterns in the rhizosphere of pecan trees asymptomatic and symptomatic for Texas root rot using a high-throughput sequencing approach

The rhizosphere harbors diverse microbial communities that play crucial roles in nutrient cycling and plant health. Understanding the composition and dynamics of these communities is essential for optimizing agricultural productivity and disease management. In this study, bacterial and fungal communities in the rhizosphere of pecan trees (Carya illinoinensis) asymptomatic and symptomatic for Texas root rot were characterized using high-throughput sequencing of the 16S rRNA gene and ITS region. The results indicated that Proteobacteria and Actinobacteria were the predominant bacterial phyla in both conditions. The predominant fungal phyla comprised Basidiomycota and Ascomycota in symptomatic conditions and Ascomycota and Mortierellomycota in asymptomatic ones. At the genus level, Streptomyces and Pseudomonas were the most abundant bacterial genera; among fungi, Scleroderma and Psathyrella were predominant in symptomatic samples and Mortierella in asymptomatic samples. α-diversity analysis showed significant differences in the fungal community. Specific bacterial and fungal biomarkers for asymptomatic and symptomatic conditions were identified, highlighting their importance in the pecan tree rhizosphere. A total of six bacterial and seven fungal taxa were detected in symptomatic samples, while four bacterial and 16 fungal taxa were detected in asymptomatic ones. These included genera known for their roles in soil health, such as Pseudomonas, Kaistobacter, Streptomyces, Psathyrella, Scleroderma, and Mortierella. Co-occurrence network analysis revealed higher complexity and interaction levels among microbial communities in asymptomatic samples, suggesting a healthier network supporting microbial communities. This study provides comprehensive and valuable insights into microbiome dynamics and microbial resources for future studies aimed at improving agricultural production through the study of rhizosphere microorganisms.