High-throughput Sequencing Approach Research Articles

Characterization of Six Ampeloviruses Infecting Pineapple in Reunion Island Using a Combination of High-Throughput Sequencing Approaches

Characterization of Six Ampeloviruses Infecting Pineapple in Reunion Island Using a Combination of High-Throughput Sequencing Approaches

A new geminialphasatellite associated with wheat dwarf virus identified in winter barley in France.

Using a high-throughput sequencing (HTS) approach, we report the discovery of a new alphasatellite identified in a winter barley plant collected in France in 2022 that was also infected by wheat dwarf virus (WDV). The presence of the satellite and of WDV was confirmed by several independent PCR assays, and the complete genome sequence was determined. The circular satellite genome is 1424 nt long and shows typical hallmarks of members of the subfamily Geminialphasatellitinae, including a replication-associated hairpin with a CAGTATTAC sequence and a Rep-encoding open reading frame (ORF). It also possesses a second ORF, embedded in a different frame within the Rep ORF, which is also observed in clecrusatellites and a few other members of the family Alphasatellitidae. Pairwise sequence comparisons and phylogenetic analysis showed that this satellite represents a novel species. Its closest relatives are in the genus Colecusatellite, but it likely represents a new genus given its divergence from other genera of the subfamily Geminialphasatellitinae. Given that WDV was the only virus observed in coinfection with the satellite, the name "wheat dwarf virus-associated alphasatellite" is proposed for this novel agent.

Genomic profiling of a skin adnexal carcinomas cohort using a comprehensive high throughput sequencing approach.

In this study, we have conducted a comprehensive molecular characterization of a cohort of 77 SAC and compared our findings to those of other skin carcinomas. We highlighted genomic profiles bringing insights regarding classification, tumorigenesis processes and therapeutic options.

Effects of polypropylene micro(nano)plastics on soil bacterial and fungal community assembly in saline-alkaline wetlands

Microplastic pollution is a major environmental threat, especially to terrestrial ecosystems. To better understand the effects of microplastics on soil microbiota, the influence of micro- to nano-scale polypropylene plastics was investigated on microbial community diversity, functionality, co-occurrence, assembly, and their interaction with soil-plant using high-throughput sequencing approaches and multivariate analyses. The results showed that polypropylene micro/nano-plastics mainly reduced bacterial diversity, not fungal, and that plastic size had a stronger effect than concentration on the assembly of microbial communities. Nano-plastics decreased the complexity and connectivity of both bacterial and fungal networks compared to micro-plastics. Moreover, bacteria were more sensitive and deterministic to polypropylene micro/nano-plastic stress than fungi, as shown by their different growth rates, guanine-cytosine content, and cell structure. Interestingly, the dominant ecological process for bacteria shifted from stochastic drift to deterministic selection with polypropylene micro/nano-plastic exposure. Furthermore, nano-plastics directly or indirectly disrupted the interactions within intra-microbes and between soil-bacteria-plant by altering soil nutrients and stoichiometry (C:N:P) or plant diversity. Collectively, the results indicate that polypropylene nano-plastics pose more ecological risks to soil microbes and their plant-soil interactions. This study sheds light on the potential ecological consequences of polypropylene micro/nano-plastic pollution in terrestrial ecosystems.

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.

Plants against cancer: towards green Taxol production through pathway discovery and metabolic engineering

AbstractThe diversity of plant natural products presents a rich resource for accelerating drug discovery and addressing pressing human health issues. However, the challenges in accessing and cultivating source species, as well as metabolite structural complexity, and general low abundance present considerable hurdles in developing plant-derived therapeutics. Advances in high-throughput sequencing, genome assembly, gene synthesis, analytical technologies, and synthetic biology approaches, now enable us to efficiently identify and engineer enzymes and metabolic pathways for producing natural and new-to-nature therapeutics and drug candidates. This review highlights challenges and progress in plant natural product discovery and engineering by example of recent breakthroughs in identifying the missing enzymes involved in the biosynthesis of the anti-cancer agent Taxol®. These enzyme resources offer new avenues for the bio-manufacture and semi-synthesis of an old blockbuster drug.

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.