To Analyze the Sensitivity of RT-PCR Assays Employing S Gene Target Failure with Whole Genome Sequencing Data during Third Wave by SARS-CoV-2 Omicron Variant

Introduction: Omicron is a highly divergent variant of concern (VOCs) of a severe acute respiratory syndrome SARS-CoV-2. It carries a high number of mutations in its spike protein hence;it is more transmissible in the community by immune evasion mechanisms. Due to mutation within S gene, most Omicron variants have reported S gene target failure (SGTF) with some commercially available PCR kits. Such diagnostic features can be used as markers to screen Omicron. However, Whole Genome Sequencing (WGS) is the only gold standard approach to confirm novel microorganisms at genetically level as similar mutations can also be found in other variants that are circulating at low frequencies worldwide. This Retrospective study is aimed to assess RT-PCR sensitivity in the detection of S gene target failure in comparison with whole genome sequencing to detect variants of Omicron. Methods: We have analysed retrospective data of SARS-CoV-2 positive RT-PCR samples for S gene target failure (SGTF) with TaqPath COVID-19 RT-PCR Combo Kit (ThermoFisher) and combined with sequencing technologies to study the emerged pattern of SARS-CoV-2 variants during third wave at the tertiary care centre, Surat. Results: From the first day of December 2021 till the end of February 2022, a total of 321,803 diagnostic RT-PCR tests for SARS-CoV-2 were performed, of which 20,566 positive cases were reported at our tertiary care centre with an average cumulative positivity of 6.39% over a period of three months. In the month of December 21 samples characterized by the SGTF (70/129) were suggestive of being infected by the Omicron variant and identified as Omicron (B.1.1.529 lineage) when sequence. In the month of January, we analysed a subset of samples (n = 618) with SGTF (24%) and without SGTF (76%) with Ct values Conclusions: During the COVID-19 pandemic, it took almost more than 15 days to diagnose infection and identify pathogen by sequencing technology. In contrast to that molecular assay provided quick identification with the help of SGTF phenomenon within 5 hours of duration. This strategy helps scientists and health policymakers for the quick isolation and identification of clusters. That ultimately results in a decreased transmission of pathogen among the community.

Evaluation of A Single-reaction Method for Whole Genome Sequencing of Influenza A Virus using Next Generation Sequencing

Objective To evaluate a single-reaction genome amplification method, the multisegment reverse transcription-PCR （M-RTPCR）, for its sensitivity to full genome sequencing of influenza A virus, and the ability to differentiate mix-subtype virus, using the next generation sequencing （NGS） platform. Methods Virus genome copy was quantified and seria（iy diluted to different titers, followed by amplification with the M-RTPCR method and sequencing on the NGS platform. Furthermore, we manually mixed two subtype viruses to different titer rate and amplified the mixed virus with the M-RTPCR protocol, followed by whole genome sequencing on the NGS platform. We also used clinical samples to test the method performance. Results The M-RTPCR method obtained complete genome of testing virus at 125 copies/reaction and determined the virus subtype at titer of 25 copies/reaction. Moreover, the two subtypes in the mixed virus could be discriminated, even though these two virus copies differed by 200-fold using this amplification protocol. The sensitivity of this protocol we detected using virus RNA was also confirmed with clinical samples containing Iow-titer virus. Conclusion The M-RTPCR is a robust and sensitive amplification method for whole genome sequencing of influenza A virus using NGS platform.

Validation of the diagnostic potential of mtDNA copy number derived from whole genome sequencing

Diagnosis of mitochondrial DNA（mt DNA）disorders has traditionally been focused on the presence of point mutations and large deletions.However,deviations in mitochondrial abundance or mt DNA copy number can also be associated with many physiological and pathological conditions（Bai and Wong,2005）.

Whole genome sequencing and its applications in medical genetics

Fundamental improvement was made for genome sequencing since the next-generation sequencing （NGS） came out in the 2000s. The newer technologies make use of the power of massively-parallel short-read DNA sequencing, genome alignment and assembly methods to digitally and rapidly search the genomes on a revolutionary scale, which enable large-scale whole genome sequencing （WGS） accessible and practical for researchers. Nowadays, whole genome sequencing is more and more prevalent in detecting the genetics of diseases, studying causative relations with cancers, making genome-level comparative analysis, reconstruction of human population history, and giving clinical implications and instructions. In this review, we first give a typical pipeline of whole genome sequencing, including the lab template preparation, sequencing, genome assembling and quality control, variants calling and annotations. We compare the difference between whole genome and whole exome sequencing （WES）, and explore a wide range of applications of whole genome sequencing for both mendelian diseases and complex diseases in medical genetics. We highlight the impact of whole genome sequencing in cancer studies, regulatory variant analysis, predictive medicine and precision medicine, as well as discuss the challenges of the whole genome sequencing.

Breed identification using breed‑informative SNPs and machine learning based on whole genome sequence data and SNP chip data

Background Breed identification is useful in a variety of biological contexts.Breed identification usually involves two stages,i.e.,detection of breed-informative SNPs and breed assignment.For both stages,there are several methods proposed.However,what is the optimal combination of these methods remain unclear.In this study,using the whole genome sequence data available for 13 cattle breeds from Run 8 of the 1,000 Bull Genomes Project,we compared the combinations of three methods(Delta,FST,and In)for breed-informative SNP detection and five machine learning methods(KNN,SVM,RF,NB,and ANN)for breed assignment with respect to different reference population sizes and difference numbers of most breed-informative SNPs.In addition,we evaluated the accuracy of breed identification using SNP chip data of different densities.Results We found that all combinations performed quite well with identification accuracies over 95%in all scenarios.However,there was no combination which performed the best and robust across all scenarios.We proposed to inte-grate the three breed-informative detection methods,named DFI,and integrate the three machine learning methods,KNN,SVM,and RF,named KSR.We found that the combination of these two integrated methods outperformed the other combinations with accuracies over 99%in most cases and was very robust in all scenarios.The accuracies from using SNP chip data were only slightly lower than that from using sequence data in most cases.Conclusions The current study showed that the combination of DFI and KSR was the optimal strategy.Using sequence data resulted in higher accuracies than using chip data in most cases.However,the differences were gener-ally small.In view of the cost of genotyping,using chip data is also a good option for breed identification.

Improving the accuracy of genomic prediction for meat quality traits using whole genome sequence data in pigs

Background Pork quality can directly affect customer purchase tendency and meat quality traits have become valu-able in modern pork production.However,genetic improvement has been slow due to high phenotyping costs.In this study,whole genome sequence(WGS)data was used to evaluate the prediction accuracy of genomic best linear unbiased prediction(GBLUP)for meat quality in large-scale crossbred commercial pigs.Results We produced WGS data(18,695,907 SNPs and 2,106,902 INDELs exceed quality control)from 1,469 sequenced Duroc×(Landrace×Yorkshire)pigs and developed a reference panel for meat quality including meat color score,marbling score,L*(lightness),a*(redness),and b*(yellowness)of genomic prediction.The prediction accuracy was defined as the Pearson correlation coefficient between adjusted phenotypes and genomic estimated breeding values in the validation population.Using different marker density panels derived from WGS data,accuracy differed substantially among meat quality traits,varied from 0.08 to 0.47.Results showed that MultiBLUP outperform GBLUP and yielded accuracy increases ranging from 17.39%to 75%.We optimized the marker density and found medium-and high-density marker panels are beneficial for the estimation of heritability for meat quality.Moreover,we conducted genotype imputation from 50K chip to WGS level in the same population and found average concord-ance rate to exceed 95%and r^(2)=0.81.Conclusions Overall,estimation of heritability for meat quality traits can benefit from the use of WGS data.This study showed the superiority of using WGS data to genetically improve pork quality in genomic prediction.

Genome‑wide DNA methylation and transcriptome analyses reveal the key gene for wool type variation in sheep

Background Wool fibers are valuable materials for textile industry.Typical wool fibers are divided into medullated and non-medullated types,with the former generated from primary wool follicles and the latter by either primary or secondary wool follicles.The medullated wool is a common wool type in the ancestors of fine wool sheep before breeding.The fine wool sheep have a non-medullated coat.However,the critical period determining the type of wool follicles is the embryonic stage,which limits the phenotypic observation and variant contrast,making both selection and studies of wool type variation fairly difficult.Results During the breeding of a modern fine(MF)wool sheep population with multiple-ovulation and embryo transfer technique,we serendipitously discovered lambs with ancestral-like coarse(ALC)wool.Whole-genome rese-quencing confirmed ALC wool lambs as a variant type from the MF wool population.We mapped the significantly associated methylation locus on chromosome 4 by using whole genome bisulfite sequencing signals,and in turn identified the SOSTDC1 gene as exons hypermethylated in ALC wool lambs compare to their half/full sibling MF wool lambs.Transcriptome sequencing found that SOSTDC1 was expressed dozens of times more in ALC wool lamb skin than that of MF and was at the top of all differentially expressed genes.An analogy with the transcriptome of coarse/fine wool breeds revealed that differentially expressed genes and enriched pathways at postnatal lamb stage in ALC/MF were highly similar to those at the embryonic stage in the former.Further experiments validated that the SOSTDC1 gene was specifically highly expressed in the nucleus of the dermal papilla of primary wool follicles.Conclusion In this study,we conducted genome-wide differential methylation site association analysis on differen-tial wool type trait,and located the only CpG locus that strongly associated with primary wool follicle development.Combined with transcriptome analysis,SOSTDC1 was identified as the only gene at this locus that was specifically overexpressed in the primary wool follicle stem cells of ALC wool lamb skin.The discovery of this key gene and its epigenetic regulation contributes to understanding the domestication and breeding of fine wool sheep.

The evolution of cancer genomic medicine in Japan and the role of the National Cancer Center Japan

The journey to implement cancer genomic medicine(CGM)in oncology practice began in the 1980s,which is considered the dawn of genetic and genomic cancer research.At the time,a variety of activating oncogenic alterations and their functional significance were unveiled in cancer cells,which led to the development of molecular targeted therapies in the 2000s and beyond.Although CGM is still a relatively new discipline and it is difficult to predict to what extent CGM will benefit the diverse pool of cancer patients,the National Cancer Center(NCC)of Japan has already contributed considerably to CGM advancement for the conquest of cancer.Looking back at these past achievements of the NCC,we predict that the future of CGM will involve the following:1)A biobank of paired cancerous and non-cancerous tissues and cells from various cancer types and stages will be developed.The quantity and quality of these samples will be compatible with omics analyses.All biobank samples will be linked to longitudinal clinical information.2)New technologies,such as whole-genome sequencing and artificial intelligence,will be introduced and new bioresources for functional and pharmacologic analyses(e.g.,a patient-derived xenograft library)will be systematically deployed.3)Fast and bidirectional translational research(bench-to-bedside and bedside-to-bench)performed by basic researchers and clinical investigators,preferably working alongside each other at the same institution,will be implemented;4)Close collaborations between academia,industry,regulatory bodies,and funding agencies will be established.5)There will be an investment in the other branch of CGM,personalized preventive medicine,based on the individual's genetic predisposition to cancer.

Identification of novel genes associated with atherosclerosis in Bama miniature pig

Background:Atherosclerosis is a chronic cardiovascular disease of great concern.However,it is difficult to establish a direct connection between conventional small animal models and clinical practice.The pig's genome,physiology,and anatomy reflect human biology better than other laboratory animals,which is crucial for studying the pathogenesis of atherosclerosis.Methods:We used whole-genome sequencing data from nine Bama minipigs to perform a genome-wide linkage analysis,and further used bioinformatic tools to filter and identify underlying candidate genes.Candidate gene function prediction was performed using the online prediction tool STRING 12.0.Immunohistochemistry and immunofluorescence were used to detect the expression of proteins encoded by candidate genes.Results:We mapped differential single nucleotide polymorphisms(SNPs)to genes and obtained a total of 102 differential genes,then we used GO and KEGG pathway enrichment analysis to identify four candidate genes,including SLA-1,SLA-2,SLA-3,and TAP2.nsSNPs cause changes in the primary and tertiary structures of SLA-I and TAP2 proteins,the primary structures of these two proteins have undergone amino acid changes,and the tertiary structures also show slight changes.In addition,immunohistochemistry and immunofluorescence results showed that the expression changes of TAP2 protein in coronary arteries showed a trend of increasing from the middle layer to the inner layer.Conclusions:We have identified SLA-I and TAP2 as potential susceptibility genes of atherosclerosis,highlighting the importance of antigen processing and immune response in atherogenesis.

Isolation and Characterization of SARS-CoV-2 in Kenya

The discovery of Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) in Wuhan, Hubei province, China, in December 2019 raised global health warnings. Quickly, in 2020, the virus crossed borders and infected individuals across the world, evolving into the COVID-19 pandemic. Notably, early signs of the virus’s existence were observed in various countries before the initial outbreak in Wuhan. As of 12th of April, the respiratory disease had infected over 762 million people worldwide, with over 6.8 million deaths recorded. This has led scientists to focus their efforts on understanding the virus to develop effective means to diagnose, treat, prevent, and control this pandemic. One of the areas of focus is the isolation of this virus, which plays a crucial role in understanding the viral dynamics in the laboratory. In this study, we report the isolation and detection of locally circulating SARS-CoV-2 in Kenya. The isolates were cultured on Vero Cercopithecus cell line (CCL-81) cells, RNA extraction was conducted from the supernatants, and reverse transcriptase-polymerase chain reaction (RT-PCR). Genome sequencing was done to profile the strains phylogenetically and identify novel and previously reported mutations. Vero CCL-81 cells were able to support the growth of SARS-CoV-2 in vitro, and mutations were detected from the two isolates sequenced (001 and 002). Genome sequencing revealed the circulation of two isolates that share a close relationship with the Benin isolate with the D614G common mutation identified along the S protein. These virus isolates will be expanded and made available to the Kenya Ministry of Health and other research institutions to advance SARS-CoV-2 research in Kenya and the region.

Epidemiology and Clonal Spread Evidence of Carbapenem-Resistant Organisms in the Center of Care and Protection of Orphaned Children, Vietnam

Objective: To determine the prevalence of colonization and transmission of carbapenem-resistant Gram-negative organisms in order to develop of an effective infection prevention program. Design: Cross-sectional study with carbapenem-resistant organisms (CRO) colonization detection from the fecal specimens of 20 Health Care Workers (HCWs) and 67 residents and 175 random environment specimens from September 2022 to September 2023. Setting: A Care and Protection Centre of Orphaned Children in South of HCM City. Participants: It included 20 HCWs, 67 residents, and 175 randomly collected environmental specimens. Method: Rectal and environmental swabs were collected from 20 HCWs, 67 residents (most of them were children), and 175 environmental specimens. MELAB Chromogenic CARBA agar plates, Card NID, and NMIC-500 CPO of the BD Phoenix TM Automated Microbiology System and whole genome sequencing (WGS) were the tests to screen, confirm CROs, respectively and determine CRO colonization and transmission between HCWs, residents, and the environment. Result: We detected 36 CRO isolates, including 6, 11 and 19 CROs found in 6 HCWs, 10 residents and 19 environments. The prevalence of detectable CRO was 30% (6/20) in HCWs, 14.92% (10/67) in residents, and 10.86% (19/175) in environmental swabs in our study. WGS demonstrated CRO colonization and transmission with the clonal spread of E. coli and A. nosocomialis, among HCWs and residents (children). Conclusion: Significant CRO colonization and transmission was evident in HCWs, residents, and the center environment. Cleaning and disinfection of the environment and performing regular hand hygiene are priorities to reduce the risk of CRO colonization and transmission.

A Roadmap for Whitefly Genomics Research:Lessons from Previous Insect Genome Projects

Due to evolving molecular and informatics technologies,modern genome sequencing projects have more different characteristics than what most biologists have become accustomed to during the capillary-based sequencing era.In this paper,we explore the characteristics that made past insect genome projects successful and place them in the context of next-generation sequencing.By taking into account the intricacies of whitefly biology and the community,we present a roadmap for whitefly-omics,which focuses on the formation of an international consortium,deployment of informatic platforms and realistic generation of reference sequence data.

Impact of next-generation sequencing on molecular diagnosis of inherited non-syndromic hearing loss

Hearing loss is one of the most common birth defects,with inherited genetic defects play an important role,contributing to about 60%of deafness occurring in infants.However,hearing impairment is genetically heterogeneous,with both common and rare forms occurring due to mutations in estimated 500 genes.Due to the large number and presumably low mutation frequencies of those genes,it would be highly expensive and time-consuming to address this issue by conventional gene-by-gene Sanger sequencing.Next-generation sequencing is a revolutionary technology that allows the simultaneous screening of mutations in a large number of genes.It is cost effective compared to classical strategies of linkage analysis and direct sequencing when the number or size of genes is large,and thus has become a highly efficient strategy for identifying novel causative genes and mutations involved in heritable disease.In this review, we describe major NGS methodologies currently used for genetic disorders and highlight applications of these technologies in studies of molecular diagnosis and the discovery of genes implicated in non-syndromic hearing loss.

Genetic Analysis of the Entire Genome of a A/duck/Shanghai/Y20/2006 (H4N6) Avian Influenza Virus

[ Objective] This paper aimed to investigate the origin, characteristics and molecular evolution of duck derived H4N6 subtype avian influ- enza virus （DK/SH/Y20/06） and enrich the epidemiologic data of the waterfowl origin AIV. [Method] The entire genome of DK/SH/Y20/06 was amplified and subjected to genome sequencing. The molecular software was used for sequence analysis and phylogenetic tree construction of DK/ SH/Y20/06 with some other reference sequences in GenBank. [Result] The results indicated that the amino acid sequence adjacent to HA cleav- age site was PEKASR ↓ GLF, which was the typical characteristics of the LPAIV. The phylogenetic analysis indicated that the HA gene of the isolate was derived from the Eurasian lineage in the eastern hemisphere. The NA gene was at the same branch with A/rnallard/Yan chen/2005（ H4N6）, sharing 98.3% sequence identity. The PB2, PB1, NP and PA gene of this isolate had genetically close relationships with H6 subtype AIV which is epidemic in China at present. The M gene fell into the same branch with A/environment/Korea/CSM05/2004（ H3N1 ）. The NS segment had the highest similarity with A/wild duck/Korea/YS44/2004（H1N2）. The eight genes were not at the same branch and shared a low similarity with other H4N6 subtype avian influenza viruses isolated in North America. [Condusion] These data showed that DK/SH/Y20/06（H4N6） was possibly a re- combinant virus derived from H4N6 subtype, H6N2, H6N5, H3N1 and H1 N2 subtype AIV by complex gene recombination in duck.

Antimicrobial resistance and molecular typing of Salmonella in the chicken production chain in Hubei Province, China

Salmonella is a significant foodborne zoonotic pathogen that endangers both human and animal health.The goal of this research is to gain a preliminary understanding of Salmonella contamination and antimicrobial resistance in the chicken production chain in Hubei Province,China.1149 animal and environmental samples were collected from chicken farms,slaughterhouses,and retail markets in six cities across Hubei Province in China from 2019 to 2020,yielding Salmonella isolation rates of 4.68%(28/598),12.21%(47/385),and 9.64%(16/166),respectively.Seventeen distinct serotypes were detected among 53 non-clonal Salmonella strains,of which Meleagridis(26.42%,14/53)was the dominant serotype.Almost half of the strains(49.06%,26/53)were multi-drug resistant(MDR).Whole-genome sequencing(WGS)showed that 10 resistance genes(tetA,bla_(TEM),parC,qnrs1,floR,aac(6'-ly,aph(6)-ld,aph(3")-b,aac(6')-laa and sul2)and 7 categories of virulence genes were present in all three links in 22 non-clonal dominant serotype strains.It was shown that Salmonella in the chicken production chain in Hubei Province had a high resist-ance rate to Tetracycline(TET,73.58%),Ofloxacin(OFL,69.81%),Florfenicol(FFC,60.38%)and Ampicillin(AMP,39.62%)which was consistent with the widespread use of these drugs in the husbandry industry in China.Salmonella ST types determined by MLST and serotypes determined by WGS had a one-to-one correlation.Minimum spanning tree analysis revealed that there was cross contamination of Salmonella in farms and slaughterhouses,slaughterhouses and markets,animal samples and environmental samples.This work provides useful information for the prevention and control of contamination and antimicrobial resistance of Salmonella in the chicken production chain,as well as demonstrating the dependable role of WGS in Salmonella molecular typing.

A Campylobacteriosis Outbreak Caused by One Asymptomatic Food Handler Carrier

In August 2021,three students with diarrhea from the same school visited a local hospital in the S district of Beijing.An epidemic investigation showed that there were more students with diarrhea in the same school and they had one meal together.Campylobacter jejuni was isolated from both patients with diarrhea and asymptomatic food handlers;however,the latter also carried Campylobacter coli.Phylogenomic analysis showed that there was a campylobacteriosis outbreak among the students,and the asymptomatic food handler may have been the source of the infection.Routine inspection and surveillance for Campylobacter is needed for the food producing staff,particularly those cooking in the cafeteria in schools or other public food services.

Role of hepatitis B virus DNA integration in human hepatocarcinogenesis

Liver cancer ranks sixth in cancer incidence, and is the third leading cause of cancer-related deaths worldwide. Hepatocellular carcinoma (HCC) is the most common type of liver cancer, which arises from hepatocytes and accounts for approximately 70%-85% of cases. Hepatitis B virus (HBV) frequently causes liver inflammation, hepatic damage and subsequent cirrhosis. Integrated viral DNA is found in 85%-90% of HBV-related HCCs. Its presence in tumors from non-cirrhotic livers of children or young adults further supports the role of viral DNA integration in hepatocarcinogenesis. Integration of subgenomic HBV DNA fragments into different locations within the host DNA is a significant feature of chronic HBV infection. Integration has two potential consequences: (1) the host genome becomes altered (“cis” effect); and (2) the HBV genome becomes altered (“trans” effect). The cis effect includes insertional mutagenesis, which can potentially disrupt host gene function or alter host gene regulation. Tumor progression is frequently associated with rearrangement and partial gain or loss of both viral and host sequences. However, the role of integrated HBV DNA in hepatocarcinogenesis remains controversial. Modern technology has provided a new paradigm to further our understanding of disease mechanisms. This review summarizes the role of HBV DNA integration in human carcinogenesis.

Whole genome analyses reveal significant convergence in obsessive-compulsive disorder between humans and dogs

Obsessive-compulsive disorder(OCD)represents a heterogeneous collection of diseases with diverse levels of phenotypic,genetic,and etiologic variability,making it difficult to identify the underlying genetic and biological mechanisms in humans.Domestic dogs exhibit several OCD-like behaviors.Using continuous circling as a representative phenotype for OCD,we screened two independent dog breeds,the Belgian Malinois and Kunming Dog and subsequently sequenced ten circling dogs and ten unaffected dogs for each breed.Using population differentiation analyses,we identified 11 candidate genes in the extreme tail of the differentiated regions between cases and controls.These genes overlap significantly with genes identified in a genome wide association study(GWAS)of human OCD,indicating strong convergence between humans and dogs.Through gene expressional analysis and functional exploration,we found that two candidate OCD risk genes,PPP2R2B and ADAMTSL3,affected the density and morphology of dendritic spines.Therefore,changes in dendritic spine may underlie some common biological and physiological pathways shared between humans and dogs.Our study revealed an unprecedented level of convergence in OCD shared between humans and dogs,and highlighted the importance of using domestic dogs as a model species for many human diseases including OCD.

Coinheritance of OLFM2 and SIX6 variants in a Chinese family with juvenile-onset primary open-angle glaucoma: A case report

BACKGROUND Juvenile-onset primary open-angle glaucoma(JOAG),characterized by severe elevation of intraocular pressure and optic neuropathy prior to the age of 40,is a rare subtype of primary open-angle glaucoma.Several genetic mutations have been associated with JOAG.CASE SUMMARY The proband patient was a young male,diagnosed with primary open-angle glaucoma at the age of 27.The patient and his unaffected parents who have been excluded from classic genetic mutations for primary open-angle glaucoma were included to explore for other possible genetic variants through whole genome sequencing and bioinformatics analysis.In this trio,we found two heterozygous variants inherited from the parents in the proband:c.281G>A,p.Arg94His in OLFM2 and c.177C>G,p.Ile59Met in SIX6.Both genetic mutations are predicted through bioinformatics analysis to replace evolutionary conserved amino acids,therefore rendering a pathogenic effect on proteins.In contrast,very low frequencies for these genetic mutations were recorded in most common control databases.CONCLUSION This is the first report on coinherited mutations of OLFM2 and SIX6 in a JOAG family,which shows the complexity of JOAG inheritance.Large-scale clinical screening and molecular functional investigations on these coinherited mutations are imperative to improve our understanding of the development of JOAG.

Application of Biological Nanopore Sequencing Technology in the Detection of Microorganisms

Environmental pollution and the spread of pathogenic microorganisms pose a significant threat to the health of humans and the planet.Thus,understanding and detecting microorganisms is crucial for maintaining a healthy living environment.Nanopore sequencing is a single-molecule detection method developed in the 1990s that has revolutionized various research fields.It offers several advantages over traditional sequencing methods,including low cost,label-free,time-saving detection speed,long sequencing reading,real-time monitoring,convenient carrying,and other significant advantages.In this review,we summarize the technical principles and characteristics of nanopore sequencing and discuss its applications in amplicon sequencing,metagenome sequencing,and whole-genome sequencing of environmental microorganisms,as well as its in situ application under some special circumstances.We also analyze the advantages and challenges of nanopore sequencing in microbiology research.Overall,nanopore sequencing has the potential to greatly enhance the detection and understanding of microorganisms in environmental research,but further developments are needed to overcome the current challenges.