Application Of High-throughput Sequencing Research Articles

High-throughput sequencing for plant virology diagnostics and its potential in plant health certification

High-throughput sequencing (HTS) technologies have revolutionized plant virology through simultaneous detection of mixed viral infections. HTS advances have uncovered and improved understanding of virus biology, ecology, and evolution which is vital for viral disease management. Plant viruses continue to threaten global agricultural productivity and strict quarantine measures are essential to prevent the introduction and spread of virulent viruses around the world. The gradual decrease in HTS operational costs, including improved computational systems and automation through robotics, has facilitated the adoption of this tool for plant diagnostics, including its use in surveillance and quarantine programs. However, the speed of technology advancements and distinct HTS chemistries, laboratory procedures, data management, and bioinformatic analyses have proven challenging. In addition, the lack of viral species reference sequences, compared with the estimated number of distinct viral taxa, makes classification and identification of novel viruses difficult. There is a need for standardized HTS testing, especially within plant health programs. In this review, we consider the application of HTS in plant virology, explore the technical challenges faced and the opportunities for HTS in plant health certification. We propose standards for overcoming current barriers and for ensuring reliable and reproducible results. These efforts will impact global plant health by reducing the risk of introduction and the spread of damaging novel viruses.

Multi-omics dataset of bovine mammary epithelial cells stimulated by ten different essential amino acids

Application of high-throughput sequencing and screening help to detect the transcriptional and metabolic discrepancies in organs provided with various levels of nutrients. The influences of individual essential amino acid (EAA) administration on transcriptomic and metabolomic profilings of bovine mammary epithelial cells (BMECs) were systematically investigated. A RNA sequencing and liquid chromatography-tandem mass spectrometry generated a comprehensive comparison of transcriptomics, non-targeted metabolomics and targeted amino acids profilings of BMECs with individual EAA stimulation by turn. The sequencing data and raw LC-MS/MS data of samples were presented in the databases of Gene Expression Omnibus, MetaboLights and Figshare for efficient reuse, including exploring the divergences in metabolisms between different EAAs and screening valuable genes and metabolites regulating casein synthesis.

Application of High-Throughput Sequencing for Comprehensive Virome Profiling in Grapevines Shows Yellows in Iran.

A comprehensive study on the whole spectrum of viruses and viroids in five Iranian grapevine cultivars was carried out using sRNA libraries prepared from phloem tissue. A comparison of two approaches to virus detection from sRNAome data indicated a significant difference in the results and performance of the aligners in viral genome reconstruction. The results showed a complex virome in terms of viral composition, abundance, and richness. Thirteen viruses and viroids were identified in five Iranian grapevine cultivars, among which the grapevine red blotch virus and grapevine satellite virus were detected for the first time in Iranian vineyards. Grapevine leafroll-associated virus 1 (GLRaV1) and grapevine fanleaf virus (GFLV) were highly dominant in the virome. However, their frequency and abundance were somewhat different among grapevine cultivars. The results revealed a mixed infection of GLRaV1/grapevine yellow speckle viroid 1 (GYSVd1) and GFLV/GYSVd1 in grapevines that exhibited yellows and vein banding. We also propose a threshold of 14% of complete reconstruction as an appropriate threshold for detection of grapevine viruses that can be used as indicators for reliable grapevine virome profiling or in quarantine stations and certification programs.

The high molecular diversity in Noctiluca scintillans is dominated by intra-genomic variations revealed by single cell high-throughput sequencing of 18S rDNA V4

The application of high-throughput sequencing (HTS) technologies has revolutionized research on phytoplankton biodiversity by generating an unprecedented amount of molecular data in marine ecosystem surveys. However, high-level of molecular diversity uncovered in HTS-based metabarcoding analyses may lead to overinterpretation of phytoplankton diversity due to excessive intra-genomic variations (IGVs). The aims in this study are to explore the nature of phytoplankton molecular diversity and to test the hypothesis. We carried out single-cell metabarcoding analysis of 18S rDNA V4 sequences obtained in single Noctiluca scintillans cells isolated from various sites in coastal waters of China. Results showed that each single N. scintillans cell harbored a high level of IGVs with about 100 amplicon sequence variants (ASVs). The large numbers of non-dominant ASVs identified in N. scintillans cells, which might correspond to the larger numbers of ASVs annotated as N. scintillans and showed similar temporal dynamics in metabarcoding analyses, could inflate the inter-species diversity or intra-species genetic diversity. In addition, there were large numbers of additional ASVs that were not annotated as N. scintillans. These non-N. scintillans ASVs might represent diverse preys for N. scintillans, consistent with previous reports that N. scintillans may act as chance predator of a broad-spectrum preys. This single-cell study has unambiguously demonstrated that the existence of high levels of IGVs in N. scintillans and most likely many other phytoplankton species, demonstrating that the majority of the molecular diversity revealed in metabarcoding analysis, which were generally interpreted as the sum of inter-species diversity and intra-species diversity, actually included high levels of IGVs and should be interpreted with caution.

Klinische Anwendung der Hochdurchsatz-Sequenzierung (next-generation sequencing, NGS) bei der Diagnostik des Usher-Syndroms

Purpose. The aim of this publication is to describe the clinical application of high-throughput sequencing (next-generation sequencing, NGS) and its advantages compared with Sanger sequencing using Usher syndrome as an example. Material and Methods. Genetic diagnostics is subject to rapid progress. The identification of disease-causing genetic alter- ations enables increasingly accurate diagnosis of hereditary diseases, making it possible to give affected individuals more precise information about the nature of the disease (course, treatment options, prognosis). Genetic diagnostics of patients and their relatives enables not only to determine the diagnosis of a disease, but its recurrence risk in the family. Results. Usher syndrome is an autosomal recessively inherited disorder, characterized by sensorineural hearing impairment and vision loss (due to retinitis pigmentosa). Several genes are known to be associated with 3 clinical subtypes of Usher syndrome. Conclusion. Advances in genetic diagnostics, including high-throughput sequencing (next-generation sequencing, NGS), today enable uncomplicated, quick and reliable diagno- sis of genetic eye diseases such as Usher syndrome. Through their own diagnosis (or the diagnosis of their child), patients and families can be informed about the prognosis and use meaningful tools early on. Family members of an affected person can be tested for carriership. Keywords Usher syndrome, next-generation sequencing, genetic testing, genetic counseling, genetic heterogeneity

Analysis of Xylem Cells by Nucleus-Based Transcriptomics and Chromatin Profiling.

Nuclei contain essential information for cell states, including chromatin and RNA profiles - features which are nowadays accessible using high-throughput sequencing applications. Here, we describe analytical pipelines including nucleus isolation from differentiated xylem tissues by fluorescence-activated nucleus sorting (FANS), as well as subsequent SMART-seq2-based transcriptome profiling and assay for transposase-accessible chromatin (ATAC)-seq-based chromatin analysis. Combined with tissue-specific expression of nuclear fluorescent reporters, these pipelines allow obtaining tissue-specific data on gene expression and on chromatin structure and are applicable for a large spectrum of cell types, tissues, and organs. Considering, however, the extreme degree of differentiation found in xylem cells with programmed cell death happening during vessel element formation and their role as a long-term depository for atmospheric CO2 in the form of wood, xylem cells represent intriguing and relevant objects for large-scale profilings of their cellular signatures.

Similarities and differences in the microbial structure of surface soils of different vegetation types.

Soil microbial community diversity serves as a highly sensitive indicator for assessing the response of terrestrial ecosystems to various changes, and it holds significant ecological relevance in terms of indicating ecological alterations. At the global scale, vegetation type acts as a major driving force behind the diversity of soil microbial communities, encompassing both bacterial and fungal components. Modifications in vegetation type not only induce transformations in the visual appearance of land, but also influence the soil ecosystem's material cycle and energy flow, resulting in substantial impacts on the composition and performance of soil microbes. In order to examine the disparities in the structure and diversity of soil microbial communities across distinct vegetation types, we opted to utilize sample plots representing four specific vegetation types. These included a woodland with the dominant tree species Drypetes perreticulata, a woodland with the dominant tree species Horsfieldia hainanensis, a Zea mays farmland and a Citrus reticulata fields. Through the application of high-throughput sequencing, the 16S V3_V4 region of soil bacteria and the ITS region of fungi were sequenced in this experiment. Subsequently, a comparative analysis was conducted to explore and assess the structure and dissimilarities of soil bacterial and fungal communities of the four vegetation types were analyzed comparatively. Our findings indicated that woodland soil exhibit a higher richness of microbial diversity compared to farmland soils. There were significant differences between woodland and farmland soil microbial community composition. However, all four dominant phyla of soil fungi were Ascomycota across the four vegetation types, but the bacterial dominant phyla were different in the two-farmland soil microbial communities with the highest similarity. Furthermore, we established a significant correlation between the nutrient content of different vegetation types and the relative abundance of soil microorganisms at both phyla and genus levels. This experiment serves as a crucial step towards unraveling the intricate relationships between plants, soil microbes, and soil, as well as understanding the underlying driving mechanism.

Combined application of high-throughput sequencing and LC-MS-based lipidomics in the evaluation of microorganisms and lipidomics of restructured ham of different salted substitution

The optimization of processed meats through salt replacement using KCl and k-lactate may reduce the risk of chronic diseases through reduction in dietary sodium. The objective of this study was to investigate the changes and relationships between microbial and lipid metabolism during the fermentation of restructured duck ham with different salt substitutions. Lactobacillus and Staphylococcus were found to be the dominant bacterial species in the 30 % KCl + 70 % NaCl (w/w) and 25 % k-lactate + 75 % NaCl (w/w). The LefSe analysis showed that different biomarkers were present in different ham groups, and the PLS-DA showed that triglycerides (GL) and glycerophospholipids (GP) were the two classes with the highest abundance. Besides, the KEGG pathway analysis revealed that glycerophospholipid metabolism and triglyceride metabolism were also the main metabolic pathways. According to the correlation study, Staphylococcus, Halomonas, and Lactobacillus were mostly linked to the important metabolic pathways in restructured ham. Our findings serve as a foundation for quality assurance and product enhancement for low-salt restructured ham.

Ancient DNA is preserved in fish fossils from tropical lake sediments.

Tropical freshwater lakes are well known for their high biodiversity, and particularly the East African Great Lakes are renowned for their adaptive radiation of cichlid fishes. While comparative phylogenetic analyses of extant species flocks have revealed patterns and processes of their diversification, little is known about evolutionary trajectories within lineages, the impacts of environmental drivers, or the scope and nature of now-extinct diversity. Time-structured palaeodata from geologically young fossil records, such as fossil counts and particularly ancient DNA (aDNA) data, would help fill this large knowledge gap. High ambient temperatures can be detrimental to the preservation of DNA, but refined methodology now allows data generation even from very poorly preserved samples. Here, we show for the first time that fish fossils from tropical lake sediments yield endogenous aDNA. Despite generally low endogenous content and high sample dropout, the application of high-throughput sequencing and, in some cases, sequence capture allowed taxonomic assignment and phylogenetic placement of 17% of analysed fish fossils to family or tribe level, including remains which are up to 2700 years old or weigh less than 1mg. The relationship between aDNA degradation and the thermal age of samples is similar to that described for terrestrial samples from cold environments when adjusted for elevated temperature. Success rates and aDNA preservation differed between the investigated lakes Chala, Kivu and Victoria, possibly caused by differences in bottom water oxygenation. Our study demonstrates that the sediment records of tropical lakes can preserve genetic information on rapidly diversifying fish taxa over time scales of millennia.

Using high-throughput sequencing to investigate summer truffle consumption by chipmunks in relation to retention forestry

Intensive management of forests for wood modifies forest biodiversity, affecting the composition of plant and animal communities as well as microorganisms such as bacteria and fungi. Ectomycorrhizal fungi (EMF) form mutualistic relationships with trees, but there is evidence that the abundance and diversity of EMF declines under intensive forest management. The dispersal of some EMF, including that of truffles, depends on small mammals consuming their fruiting bodies and dispersing spores through feces. To better understand provisioning of this ecosystem service within intensively managed forests, we applied high-throughput, DNA amplicon sequencing to scat collected from Townsend’s chipmunks (Neotamias townsendii) inhabiting retention patches in recently clearcut-conifer stands. Across two summers, we collected 165 chipmunk scat samples from 43 clearcut-conifer stands in the Pacific Northwest (USA). We identified 81 truffle species representing 16 families, which included many rare and uncommon taxa. Retention patch sizes varied from 9 to 222 trees (0.002–0.83 ha) but we did not detect an effect of retention patch size on the richness of truffles in chipmunk scat samples. However, truffle richness was slightly higher in scat samples collected from chipmunks in retention patches compared to individuals sampled in adjacent clearcuts. Furthermore, the abundance of certain truffle species in chipmunk scat varied in relation to retention patch size and location (riparian or upland), suggesting retention forestry practices may influence truffle community composition. Throughout the sampling season, we detected a 44% decline in truffle species richness and a significant reduction in abundance (i.e., sequence reads) for > 50% of common truffle taxa. Our application of high-throughput sequencing of scat effectively captured variation in truffle species consumption by chipmunks. Our results confirm that common small mammals play an important role in the dispersal of EMF across recently logged forests (<8 years since harvest) and indicate that retaining relatively small (∼10 tree) green-tree patches may promote truffle dispersal by providing small mammal habitat.

Application of high-throughput sequencing to analyze the diet of black croaker (Atrobucca nibe) in southern coastal waters of Zhejiang

This study investigated the feeding habits of black croaker (Atrobucca nibe) to fully understand its role in the food web in southern coastal waters of Zhejiang, China. In total, 225 black croakers were collected in a bottom trawl survey, of which 195 were used for the stomach contents analysis and 30 for high-throughput sequencing (HTS). Both approaches showed that the main prey was shrimp, with Alpheus japonicus being the most predominant. In addition, black croaker also fed on other benthic species, suggesting that it was a demersal predator. Feeding intensity also varied with black croaker body length, as did prey preference, from small-sized species to larger species with increases in fish size; in addition, prey species were more diverse in the large-bodied prey group. Canonical correspondence analysis indicated that the feeding habit of black croaker was most significantly correlated with temperature and dissolved oxygen. HTS was shown to be a feasible high precision semi-quantitative analytical approach, although it must be combined with morphological methods to obtain more complete feeding data; thus, it could provide an effective scientific basis for feeding ecology studies of marine organisms.

Phylogenetic analysis of Shewanella spp. isolated from fish.

Different species of Shewanella spp. widely inhabit freshwater and marine environments. Some of them are opportunistic fish pathogens. The application of high-throughput sequencing enabled the characterization and taxonomic reclassification of many Shewanella spp. species. Still, some strains collected from fish need to be better recognized. The aim of the present study was to classify and determine the phylogenetic relationships of Shewanella spp. collected from fish. The complete genomes of 94 strains of Shewanella spp. from different fish species were sequenced using Illumina platform (MiSeq). The 16S rRNA gene, genomic features and whole-genome relationships of those bacteria were comprehensively analysed in comparison to reference strains. Whole-genome analysis showed that the tested Shewanella spp. strains were clustered into six groups similar to reference strains of S. xiamenensis, S. oneidensis, S. glacialipiscicola, S. hafniensis, S. baltica and S. oncorhynchi. Our study indicates that the whole-genome sequence analysis enabled taxonomic classification and assessment of the diversity of the Shewanella spp. strains, as opposed to recently the gold standard method of 16S rRNA amplicon sequencing. The high genetic diversity and low similarity to the reference genome of S. oneidensis indicate that the group of strains may be a subspecies or even new species. Furthermore, we showed that the most frequent Shewanella spp. species occurring in freshwater fish in our study is the recently described species S. oncorhynchi.

The human gut phageome: composition, development, and alterations in disease.

The human gastrointestinal tract is colonized by a large number of microorganisms, including bacteria, archaea, viruses, and eukaryotes. The bacterial community has been widely confirmed to have a significant impact on human health, while viruses, particularly phages, have received less attention. Phages are viruses that specifically infect bacteria. They are abundant in the biosphere and exist in a symbiotic relationship with their host bacteria. Although the application of high-throughput sequencing and bioinformatics technology has greatly improved our understanding of the genomic diversity, taxonomic composition, and spatio-temporal dynamics of the human gut phageome, there is still a large portion of sequencing data that is uncharacterized. Preliminary studies have predicted that the phages play a crucial role in driving microbial ecology and evolution. Prior to exploring the function of phages, it is necessary to address the obstacles that hinder establishing a comprehensive sequencing database with sufficient biological properties and understanding the impact of phage-bacteria interactions on human health. In this study, we provide an overview of the human gut phageome, including its composition, structure, and development. We also explore the various factors that may influence the phageome based on current research, including age, diet, ethnicity, and geographical location. Additionally, we summarize the relationship between the phageome and human diseases, such as IBD, IBS, obesity, diabetes, and metabolic syndrome.

Application and significance of circRNA and high throughput sequencing in different metastatic pathways of gastric cancer

In this study, we will explore the expression profile of circular RNA (cRNA) in gastric cancer tissues and plasma in order to evaluate its potential as a molecular marker for the detection of gastric cancer. Additionally, we will research the expression profile of cRNA in normal gastric tissue. Reverse transcription drop digital polymerase chain reaction (RT ddPCR), circRNA expression profiling, the prediction of circRNA-microRNA interactions, the design of a method for RT ddPCR, as well as the analysis of clinicopathological factors and survival outcomes are all included in the recommended approach. The results of the research indicate that some circRNAs have a distinct expression pattern in the plasma and tissues of patients with gastric cancer. In addition, a quantitative RT ddPCR approach was created, and the identification of certain circRNAs that are connected with clinicopathological factors and prognosis was accomplished. The results of this study provide significant new insights into the ability of circRNAs to function not only as diagnostic markers but also as independent prognostic factors for gastric cancer. In addition, a dye-based RT ddPCR method is shown to be an effective and economical technique for the detection of plasma circRNA.

Relic DNA effects on the estimates of bacterial community composition and taxa dynamics in soil

DNA-based analyses have become routine methods in soil microbial research, for their high throughput and resolution in characterizing microbial communities. Yet, concerns arise regarding the interference of relic DNA in estimates of viable bacterial community composition and individual taxa dynamics in soils that recovered from post-gamma irradiation. In this study, different soil samples with varying bacterial diversity but similar soil properties were randomly selected. We split each sample into two parts: one part was treated with propidium monoazide (PMA) before DNA extraction, PMA can bind to relic DNA and inhibit PCR amplification by chemical modification; DNA of the other part was extracted following the same process but without PMA pretreatment. Then, soil bacterial abundance was quantified by quantitative polymerase chain reaction, and bacterial community structure was examined by Illumina metabarcoding sequencing of 16S rRNA gene. The results showed that the higher bacterial richness and evenness were estimated when relic DNA was present. The variation trends of bacterial abundance, alpha diversity, and beta diversity remained the same, as reflected by the significant correlations between PMA-treated and -untreated samples (P < 0.05). Moreover, as the mean abundance increased, the reproducibility of detecting individual taxa dynamics between relic DNA present and absent treatments increased. These findings provide empirical evidence that a more even distribution of species abundance derived from relic DNA would result in the overestimation of richness in the total DNA pools and also have crucial implications for guiding proper application of high-throughput sequencing to estimate bacterial community diversity and taxonomic population dynamic. KEY POINTS: • Relic DNA effects on the bacterial community in sterilized soils were assessed. • More even species abundance distribution in relic DNA overestimates true richness. • The reproducibility of individual taxa dynamics increased with their abundance.

Identification and validation of the reference genes in the echiuran worm Urechis unicinctus based on transcriptome data

BackgroundReal-time quantitative PCR (RT-qPCR) is a crucial and widely used method for gene expression analysis. Selecting suitable reference genes is extremely important for the accuracy of RT-qPCR results. Commonly used reference genes are not always stable in various organisms or under different environmental conditions. With the increasing application of high-throughput sequencing, transcriptome analysis has become an effective method for identifying novel stable reference genes.ResultsIn this study, we identified candidate reference genes based on transcriptome data covering embryos and larvae of early development, normal adult tissues, and the hindgut under sulfide stress using the coefficient of variation (CV) method in the echiuran Urechis unicinctus, resulting in 6834 (15.82%), 7110 (16.85%) and 13880 (35.87%) candidate reference genes, respectively. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses revealed that the candidate reference genes were significantly enriched in cellular metabolic process, protein metabolic process and ribosome in early development and normal adult tissues as well as in cellular localization and endocytosis in the hindgut under sulfide stress. Subsequently, ten genes including five new candidate reference genes and five commonly used reference genes, were validated by RT-qPCR. The expression stability of the ten genes was analyzed using four methods (geNorm, NormFinder, BestKeeper, and ∆Ct). The comprehensive results indicated that the new candidate reference genes were more stable than most commonly used reference genes. The commonly used ACTB was the most unstable gene. The candidate reference genes STX12, EHMT1, and LYAG were the most stable genes in early development, normal adult tissues, and hindgut under sulfide stress, respectively. The log2(TPM) of the transcriptome data was significantly negatively correlated with the Ct values of RT-qPCR (Ct = − 0.5405 log2(TPM) + 34.51), which made it possible to estimate the Ct value before RT-qPCR using transcriptome data.ConclusionOur study is the first to select reference genes for RT-qPCR from transcriptome data in Echiura and provides important information for future gene expression studies in U. unicinctus.

Evaluating the Reproducibility of Amplicon Sequencing Data Derived from Deep-Sea Cold Seep Sediment-Associated Microbiota.

Benefiting from the rapid developments and wide applications of high-throughput sequencing, great advancements have been made in investigating microbiota, which are highly diverse and play key roles in both element cycling and the energy flow of ecosystems. There have been inherent limitations of amplicon sequencing that could introduce uncertainty and raise concerns about the accuracy and reproducibility of this technology. However, studies focusing on the reproducibility of amplicon sequencing are limited, especially in characterizing microbial communities in deep-sea sediments. To evaluate reproducibility, 118 deep-sea sediment samples were used for 16S rRNA gene sequencing in technical replicates (repeated measurements of the same sample) that demonstrate the variability of amplicon sequencing. The average occurrence-based overlaps were 35.98% and 27.02% between two and among three technical replicates, respectively, whereas their abundance-based overlaps reached 84.88% and 83.16%, respectively. Although variations of alpha and beta diversity indices were found between/among technical replicates, alpha diversity indices were similar across samples, and the average beta diversity indices were much smaller for technical replicates than among samples. Moreover, clustering methods (i.e., operational taxonomic units [OTUs] and amplicon sequence variants [ASVs]) were shown to have little impact on the alpha and beta diversity patterns of microbial communities. Taken together, although there are variations between/among technical replicates, amplicon sequencing is still a powerful tool with which to reveal diversity patterns of microbiota in deep-sea sediments. IMPORTANCE The reproducibility of amplicon sequencing is vital for whether the diversities of microbial communities could be accurately estimated. Thus, reproducibility influences the drawing of sound ecological conclusions. Nevertheless, few studies have focused on the reproducibility of microbial communities that are characterized by amplicon sequencing, and studies focusing on microbiota in deep-sea sediments have been especially lacking. In this study, we evaluated the reproducibility of amplicon sequencing targeting microbiota in deep-sea sediments of cold seep. Our results revealed that there were variations between/among technical replicates and that amplicon sequencing was still a powerful tool with which to characterize the diversities of microbial communities in deep-sea sediments. This study provides valuable guidelines for the reproducibility evaluation of future work in experimental design and interpretation.

Amplicon-Based High-Throughput Sequencing Method for Genotypic Characterization of Norovirus in Oysters.

Norovirus is a highly diverse RNA virus often implicated in foodborne outbreaks, particularly those associated with shellfish. Shellfish are filter feeders, and when harvested in bays exposed to wastewater overflow or storm overflows, they can harbor various pathogens, including human-pathogenic viruses. The application of Sanger or amplicon-based high-throughput sequencing (HTS) technologies to identify human pathogens in shellfish faces two main challenges: (i) distinguishing multiple genotypes/variants in a single sample and (ii) low concentrations of norovirus RNA. Here, we assessed the performance of a novel norovirus capsid amplicon HTS method. We generated a panel of spiked oysters containing various norovirus concentrations with different genotypic compositions. Several DNA polymerases and reverse transcriptases (RTs) were compared, and performance was evaluated based on (i) the number of reads passing quality filters per sample, (ii) the number of correct genotypes identified, and (iii) the sequence identity of outputs compared to Sanger-derived sequences. A combination of the reverse transcriptase LunaScript and the DNA polymerase AmpliTaq Gold provided the best results. The method was then employed, and compared with Sanger sequencing, to characterize norovirus populations in naturally contaminated oysters. IMPORTANCE While foodborne outbreaks account for approximately 14% of norovirus cases (L. Verhoef, J. Hewitt, L. Barclay, S. Ahmed, R. Lake, A. J. Hall, B. Lopman, A. Kroneman, H. Vennema, J. Vinjé, and M. Koopmans, Emerg Infect Dis 21:592-599, 2015), we do not have standardized high-throughput sequencing methods for genotypic characterization in foodstuffs. Here, we present an optimized amplicon high-throughput sequencing method for the genotypic characterization of norovirus in oysters. This method can accurately detect and characterize norovirus at concentrations found in oysters grown in production areas impacted by human wastewater discharges. It will permit the investigation of norovirus genetic diversity in complex matrices and contribute to ongoing surveillance of norovirus in the environment.

The Principles and Applications of High-Throughput Sequencing Technologies

The advancement in high-throughput sequencing (HTS) technology has revolutionized the field of biology, including genomics, epigenomics, transcriptomics, and metagenomics. This technology has become a crucial tool in many areas of research, allowing scientists to generate vast amounts of genetic data at a much faster pace than traditional methods. With this increased speed and scale of data generation, researchers can now address critical questions and gain new insights into the inner workings of living organisms, as well as the underlying causes of various diseases. Although the first HTS technology have been introduced about two decades ago, it can still be challenging for those new to the field to understand and use effectively. This review aims to provide a comprehensive overview of commonly used HTS technologies these days and their applications in terms of genome sequencing, transcriptome, DNA methylation, DNA-protein interaction, chromatin accessibility, three-dimensional genome organization, and microbiome.

Plastisphere microbiome: Methodology, diversity, and functionality.

Broad topics of the plastisphere in various environments are reviewed, including its methodologies, diversity, functionality, and outlook.