Infectious diseases are an enormous public health burden and a growing threat to human health worldwide.Emerging or classic recurrent pathogens,or pathogens with resistant traits,challenge our ability to diagnose and ...Infectious diseases are an enormous public health burden and a growing threat to human health worldwide.Emerging or classic recurrent pathogens,or pathogens with resistant traits,challenge our ability to diagnose and control infectious diseases.Nanopore sequencing technology has the potential to enhance our ability to diagnose,interrogate,and track infectious diseases due to the unrestricted read length and system portability.This review focuses on the application of nanopore sequencing technology in the clinical diagnosis of infectious diseases and includes the following:(i)a brief introduction to nanopore sequencing technology and Oxford Nanopore Technologies(ONT)sequencing platforms;(ii)strategies for nanopore-based sequencing technologies;and(iii)applications of nanopore sequencing technology in monitoring emerging pathogenic microorganisms,molecular detection of clinically relevant drug-resistance genes,and characterization of disease-related microbial communities.Finally,we discuss the current challenges,potential opportunities,and future outlook for applying nanopore sequencing technology in the diagnosis of infectious diseases.展开更多
Over the past decade,nanopore sequencing has experienced significant advancements and changes,transitioning from an initially emerging technology to a significant instrument in the field of genomic sequencing.However,...Over the past decade,nanopore sequencing has experienced significant advancements and changes,transitioning from an initially emerging technology to a significant instrument in the field of genomic sequencing.However,as advancements in next-generation sequencing technology persist,nanopore sequencing also improves.This paper reviews the developments,applications,and outlook on nanopore sequencing technology.Currently,nanopore sequencing supports both DNA and RNA sequencing,making it widely applicable in areas such as telomere-to-telomere(T2T)genome assembly,direct RNA sequencing(DRS),and metagenomics.The openness and versatility of nanopore sequencing have established it as a preferred option for an increasing number of research teams,signaling a transformative influence on life science research.As the nanopore sequencing technology advances,it provides a faster,more costeffective approach with extended read lengths,demonstrating the significant potential for complex genome assembly,pathogen detection,environmental monitoring,and human disease research,offering a fresh perspective in sequencing technologies.展开更多
Genome sequencing has shown strong capabilities in the initial stages of the COVID-19 pandemic such as pathogen identification and virus preliminary tracing.While the rapid acquisition of SARS-Co V-2 genome from clini...Genome sequencing has shown strong capabilities in the initial stages of the COVID-19 pandemic such as pathogen identification and virus preliminary tracing.While the rapid acquisition of SARS-Co V-2 genome from clinical specimens is limited by their low nucleic acid load and the complexity of the nucleic acid background.To address this issue,we modified and evaluated an approach by utilizing SARS-Co V-2-specific amplicon amplification and Oxford Nanopore Prometh ION platform.This workflow started with the throat swab of the COVID-19 patient,combined reverse transcript PCR,and multi-amplification in one-step to shorten the experiment time,then can quickly and steadily obtain high-quality SARS-Co V-2 genome within 24 h.A comprehensive evaluation of the method was conducted in 42 samples:the sequencing quality of the method was correlated well with the viral load of the samples;high-quality SARS-Co V-2 genome could be obtained stably in the samples with Ct value up to 39.14;data yielding for different Ct values were assessed and the recommended sequencing time was 8 h for samples with Ct value of less than 20;variation analysis indicated that the method can detect the existing and emerging genomic mutations as well;Illumina sequencing verified that ultra-deep sequencing can greatly improve the single read error rate of Nanopore sequencing,making it as low as 0.4/10,000 bp.In summary,high-quality SARS-Co V-2 genome can be acquired by utilizing the amplicon amplification and it is an effective method in accelerating the acquisition of genetic resources and tracking the genome diversity of SARSCo V-2.展开更多
BACKGROUND Infectious diseases are still one of the greatest threats to human health,and the etiology of 20%of cases of clinical fever is unknown;therefore,rapid identification of pathogens is highly important.Traditi...BACKGROUND Infectious diseases are still one of the greatest threats to human health,and the etiology of 20%of cases of clinical fever is unknown;therefore,rapid identification of pathogens is highly important.Traditional culture methods are only able to detect a limited number of pathogens and are time-consuming;serologic detection has window periods,false-positive and false-negative problems;and nucleic acid molecular detection methods can detect several known pathogens only once.Three-generation nanopore sequencing technology provides new options for identifying pathogens.CASE SUMMARY Case 1:The patient was admitted to the hospital with abdominal pain for three days and cessation of defecation for five days,accompanied by cough and sputum.Nanopore sequencing of the drainage fluid revealed the presence of orallike bacteria,leading to a clinical diagnosis of bronchopleural fistula.Cefoperazone sodium sulbactam treatment was effective.Case 2:The patient was admitted to the hospital with fever and headache,and CT revealed lung inflammation.Antibiotic treatment for Streptococcus pneumoniae,identified through nanopore sequencing of cerebrospinal fluid,was effective.Case 3:The patient was admitted to our hospital with intermittent fever and an enlarged neck mass that had persisted for more than six months.Despite antibacterial treatment,her symptoms worsened.The nanopore sequencing results indicate that voriconazole treatment is effective for Aspergillus brookii.The patient was diagnosed with mixed cell type classical Hodgkin's lymphoma with infection.CONCLUSION Three-generation nanopore sequencing technology allows for rapid and accurate detection of pathogens in human infectious diseases.展开更多
With the widespread presence of multidrug-resistant bacteria in medical environments,precise environmental monitoring and disinfection targeting drug-resistance genes have become increasingly important.Third-generatio...With the widespread presence of multidrug-resistant bacteria in medical environments,precise environmental monitoring and disinfection targeting drug-resistance genes have become increasingly important.Third-generation nanopore sequencing has significant application potential in genomics,clinical diagnosis,and other fields owing to its unique advantages of long read length,real-time sequencing,and portability.Research on nanopore sequencing has made significant progress;however,challenges remain in terms of technical principles,performance optimization,and the development of bioinformatics analysis tools.This paper aims to systematically review the latest advances in nanopore sequencing,analyze practical cases in monitoring antimicrobial resistance and environmental disinfection,and discuss current challenges faced and future development directions.By integrating relevant research findings,this review hopes to provide valuable references for researchers in related fields and promote the further development and application of nanopore sequencing.展开更多
Nanopore sequencing technology,an advanced third-generation sequencing technology,is a revolutionary sequencing method widely used in clinical diagnosis and genomic research because of its features such as real-time s...Nanopore sequencing technology,an advanced third-generation sequencing technology,is a revolutionary sequencing method widely used in clinical diagnosis and genomic research because of its features such as real-time sequencing,direct sequencing,long read length and portability.This paper outlines the basic principles and advantages of the technology,and briefly introduces its applications in clinical medicine such as diagnosis of diseases rare and genetic diseases,detection of infectious disease pathogens,public health emergency response,and cancer genomics screening.In genomics,nanopore sequencing is instrumental in genome assembly,structural variation detection,recovery of DNA from ancient organisms,and microbiological research.It enables direct sequencing and analysis of molecules,allowing for the identification of complex structural variations within the genome.This study finds that the technology also suffers from low accuracy,high cost associated with large data volumes,and significant requirements for data processing capabilities.These limitations can potentially be addressed through innovations such as improved nanopore materials and design,and integration with artificial intelligence.Finally,the latest innovations of the technology are analyzed,and the development trend and application prospects are outlooked.展开更多
This paper reports a case of Chlamydia psittaci pneumonia and pulmonary aspergillosis with concurrent liver injury.The patient was admitted to the hospital with coughing,expectoration,and fever for 5 days.Laboratory t...This paper reports a case of Chlamydia psittaci pneumonia and pulmonary aspergillosis with concurrent liver injury.The patient was admitted to the hospital with coughing,expectoration,and fever for 5 days.Laboratory tests upon admission revealed abnormalities in liver function.The patient was treated with broad-spectrum antibiotics and liver-protection therapy,but symptoms did not significantly improve.Further nanopore sequencing detected Chlamydia psittaci and Aspergillus flavus.The treatment plan was adjusted to include combined anti-infection and antifungal therapies.After treatment,the patient's symptoms improved significantly;their liver function returned to normal,and their condition improved.展开更多
AIM:To estimate if nanopore targeted sequencing(NTS)could identify pathogens causing postoperative endophthalmitis and further determine the feasibility of clinical application of NTS.METHODS:A total of 55 patients(55...AIM:To estimate if nanopore targeted sequencing(NTS)could identify pathogens causing postoperative endophthalmitis and further determine the feasibility of clinical application of NTS.METHODS:A total of 55 patients(55 eyes)with postoperative endophthalmitis were retrospectively included in this study with their medical records.Intraocular fluid samples were examined by NTS and microbial culture.All included patients had undergone examinations including measurement of best corrected visual acuity(BCVA)and intraocular pressure(IOP),slit-lamp biomicroscopy,and indirect ophthalmoscopy;additionally,they underwent B-ultrasound,anterior segment photography,and fundus photography if necessary.RESULTS:Among 55 patients with postoperative endophthalmitis,the age was 65.25±15.04y and there were 30 female(54.54%)patients.Forty-one(74.54%)vitreous humor samples and fourteen(25.45%)aqueous humor samples were sent for both NTS and microbial culture.NTS had a notable higher detection rate than microbial culture in detecting pathogens(90.91%vs 38.18%,χ^(2)=33.409,P<0.001).NTS exhibited high sensitivity of pathogen detection in both microbial culture positive and negative samples(100%and 85.29%,respectively).In 16 of 21(76.19%)patients who showed culture-positivity,their results corresponded with those of NTS.Moreover,in two patients(9.52%),NTS showed a better species resolution than microbial culture;in three patients(14.28%),NTS identified additional pathogens.As for fungus,the positive detection rate of NTS was significantly higher than that of microbial culture(20%vs 3.64%,χ^(2)=7.066,P=0.008).Also,NTS could detect multi-infection by bacteria and fungi than microbial culture(32.73%vs 0,χ^(2)=21.522,P<0.001).NTS could detect bacteria as well as fungi simultaneously within 48h in all patients.Meanwhile,NTS had a shorter detection time than microbial culture(1.13±0.34 vs 2.67±0.55d,Z=-9.218,P<0.001).After the NTS results were obtained,15 patients received additional intravitreal/intracameral anti-infection treatment.At follow-up,there was a statistically significant improvement in the visual acuity relative to the baseline(Z=−5.222,P<0.001).CONCLUSION:NTS can provide rapid identification and highly sensitive detection of pathogens among patients with postoperative endophthalmitis,which can guide anti-infection treatment and improve visual prognosis.展开更多
Objective Knowledge of an enterovirus genome sequence is very important in epidemiological investigation to identify transmission patterns and ascertain the extent of an outbreak. The MinION sequencer is increasingly ...Objective Knowledge of an enterovirus genome sequence is very important in epidemiological investigation to identify transmission patterns and ascertain the extent of an outbreak. The MinION sequencer is increasingly used to sequence various viral pathogens in many clinical situations because of its long reads, portability, real-time accessibility of sequenced data, and very low initial costs. However, information is lacking on MinION sequencing of enterovirus genomes. Methods In this proof-of-concept study using Enterovirus 71 （EV71） and Coxsackievirus A16 （CA16） strains as examples, we established an amplicon-based whole genome sequencing method using MinION. We explored the accuracy, minimum sequencing time, discrimination and high-throughput sequencing ability of MinION, and compared its performance with Sanger sequencing. Results Within the first minute （min） of sequencing, the accuracy of MinION was 98.5% for the single EV71 strain and 94.12%-97.33% for 10 genetically-related CA16 strains. In as little as 14 min, 99% identity was reached for the single EV71 strain, and in 17 min （on average）, 99% identity was achieved for 10 CA16 strains in a single run. Conclusion MinION is suitable for whole genome sequencing of enteroviruses with sufficient accuracy and fine discrimination and has the potential as a fast, reliable and convenient method for routine use.展开更多
DNA barcodes,short and unique DNA sequences,play a crucial role in sample identification when processing many samples simultaneously,which helps reduce experimental costs.Nevertheless,the low quality of long-read sequ...DNA barcodes,short and unique DNA sequences,play a crucial role in sample identification when processing many samples simultaneously,which helps reduce experimental costs.Nevertheless,the low quality of long-read sequencing makes it difficult to identify barcodes accurately,which poses significant challenges for the design of barcodes for large numbers of samples in a single sequencing run.Here,we present a comprehensive study of the generation of barcodes and develop a tool,PRO,that can be used for selecting optimal barcode sets and demultiplexing.We formulate the barcode design problem as a combinatorial problem and prove that finding the optimal largest barcode set in a given DNA sequence space in which all sequences have the same length is theoretically NP-complete.For practical applications,we developed the novel method PRO by introducing the probability divergence between two DNA sequences to expand the capacity of barcode kits while ensuring demultiplexing accuracy.Specifically,the maximum size of the barcode kits designed by PRO is 2,292,which keeps the length of barcodes the same as that of the official ones used by Oxford Nanopore Technologies(ONT).We validated the performance of PRO on a simulated nanopore dataset with high error rates.The demultiplexing accuracy of PRO reached 98.29%for a barcode kit of size 2,922,4.31%higher than that of Guppy,the official demultiplexing tool.When the size of the barcode kit generated by PRO is the same as the official size provided by ONT,both tools show superior and comparable demultiplexing accuracy.展开更多
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...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.展开更多
The Human Genome Project opened an era of(epi)genomic research,and also provided a platform for the development of new sequencing technologies.During and after the project,several sequencing technologies continue to d...The Human Genome Project opened an era of(epi)genomic research,and also provided a platform for the development of new sequencing technologies.During and after the project,several sequencing technologies continue to dominate nucleic acid sequencing markets.Currently,Illumina(short-read),PacBio(long-read),and Oxford Nanopore(longread)are the most popular sequencing technologies.Unlike PacBio or the popular short-read sequencers before it,which,as examples of the second or so-called Next-Generation Sequencing platforms,need to synthesize when sequencing,nanopore technology directly sequences native DNA and RNA molecules.Nanopore sequencing,therefore,avoids converting mRNA into cDNA molecules,which not only allows for the sequencing of extremely long native DNA and full-length RNA molecules but also document modifications that have been made to those native DNA or RNA bases.In this review on direct DNA sequencing and direct RNA sequencing using Oxford Nanopore technology,we focus on their development and application achievements,discussing their challenges and future perspective.We also address the problems researchers may encounter applying these approaches in their research topics,and how to resolve them.展开更多
To achieve the enormous potential of gene-editing technology in clinical therapies,one needs to evaluate both the on-target efficiency and unintended editing consequences comprehensively.However,there is a lack of a p...To achieve the enormous potential of gene-editing technology in clinical therapies,one needs to evaluate both the on-target efficiency and unintended editing consequences comprehensively.However,there is a lack of a pipelined,large-scale,and economical workflow for detecting genome editing outcomes,in particular insertion or deletion of a large fragment.Here,we describe an approach for efficient and accurate detection of multiple genetic changes after CRISPR/Cas9 editing by pooled nanopore sequencing of barcoded long-range PCR products.Recognizing the high error rates of Oxford nanopore sequencing,we developed a novel pipeline to capture the barcoded sequences by grepping reads of nanopore amplicon sequencing(GREPore-seq).GREPore-seq can assess nonhomologous end-joining(NHEJ)-mediated double-stranded oligodeoxynucleotide(dsODN)insertions with comparable accuracy to Illumina next-generation sequencing(NGS).GREPore-seq also reveals a full spectrum of homology-directed repair(HDR)-mediated large gene knock-in,correlating well with the fluorescence-activated cell sorting(FACS)analysis results.Of note,we discovered low-level fragmented and full-length plasmid backbone insertion at the CRISPR cutting site.Therefore,we have established a practical workflow to evaluate various genetic changes,including quantifying insertions of short dsODNs,knock-ins of long pieces,plasmid insertions,and large fragment deletions after CRISPR/Cas9-mediated editing.GREPore-seq is freely available at GitHub(https://github.com/lisiang/GREPore-seq)and the National Genomics Data Center(NGDC)BioCode(https://ngdc.cncb.ac.cn/biocode/tools/BT007293).展开更多
Transposable element insertions(TEIs) are an important source of genomic innovation by contributing to plant adaptation, speciation, and the production of new varieties. The often large,complex plant genomes make iden...Transposable element insertions(TEIs) are an important source of genomic innovation by contributing to plant adaptation, speciation, and the production of new varieties. The often large,complex plant genomes make identifying TEIs from short reads difficult and expensive. Moreover, rare somatic insertions that refect mobilome dynamics are difficult to track using short reads.To address these challenges, we combined Cas9-targeted Nanopore sequencing(CANS) with the novel pipeline Nano Cas TE to trace both genetically inherited and somatic TEIs in plants. We performed CANS of the EVADé(EVD) retrotransposon in wild-type Arabidopsis thaliana and rapidly obtained up to 40× sequence coverage.Analysis of hemizygous T-DNA insertion sites and genetically inherited insertions of the EVD transposon in the ddm1(decrease in DNA methylation1) genome uncovered the crucial role of DNA methylation in shaping EVD insertion preference.We also investigated somatic transposition events of the ONSEN transposon family, finding that genes that are downregulated during heat stress are preferentially targeted by ONSENs. Finally, we detected hypomethylation of novel somatic insertions for two ONSENs. CANS and Nano Cas TE are effective tools for detecting TEIs and exploring mobilome organization in plants in response to stress and in different genetic backgrounds, as well as screening T-DNA insertion mutants and transgenic plants.展开更多
Nanopore sequencing harnesses changes in ionic current as nucleotides traverse a nanopore,enabling real-time decoding of DNA/RNA sequences.The instruments for the dynamic behavior of substances in the nanopore on the ...Nanopore sequencing harnesses changes in ionic current as nucleotides traverse a nanopore,enabling real-time decoding of DNA/RNA sequences.The instruments for the dynamic behavior of substances in the nanopore on the molecular scale are still very limited experimentally.This study employs all-atom molecular dynamics(MD)simulations to explore the impact of charge densities on graphene nanopore in the translocation of single-stranded DNA(ssDNA).We find that the magnitude of graphene’s charge,rather than the charge disparity between ssDNA and graphene,significantly influences ssDNA adsorption and translocation speed.Specifically,high negative charge densities on graphene nanopores are shown to substantially slow down ssDNA translocation,highlighting the importance of hydrodynamic effects and electrostatic repulsions.This indicates translocation is crucial for achieving distinct ionic current blockades,which plays a central role for DNA sequencing accuracy.Our findings suggest that negatively charged graphene nanopores hold considerable potential for optimizing DNA sequencing,marking a critical advancement in this field.展开更多
The DNA sequencing technology has achieved a leapfrog development in recent years. As a new generation of the DNA sequencing technology, nanopore sequenc- ing has shown a broad application prospect and attracted vast ...The DNA sequencing technology has achieved a leapfrog development in recent years. As a new generation of the DNA sequencing technology, nanopore sequenc- ing has shown a broad application prospect and attracted vast research interests since it was proposed. In the present study, the dynamics of the electric-driven translocation of a homopolymer through a nanopore is investigated by the dissipative particle dynam- ics （DPD）, in which the homopolymer is modeled as a worm-like chain （WLC）. The DPD simulations show that the polymer chain undergoes conformation changes during the translocation process. The different structures of the polymer in the translocation process, i.e., single-file, double folded, and partially folded, and the induced current block- ades are analyzed. It is found that the current blockades have different magnitudes due to the polymer molecules traversing the pore with different folding conformations. The nanoscale vortices caused by the concentration polarization layers （CPLs） in the vicinity of the sheet are also studied. The results indicate that the translocation of the polymer has the effect of eliminating the vortices in the polyelectrolyte solution. These findings are expected to provide the theoretical guide for improving the nanopore sequencing tech- nique.展开更多
Endophthalmitis is a serious ophthalmic disease characterized by changes in the eye's posterior segment,such as hypopyon and intraocular inflammation,vitritis being a hallmark.Infection-caused endophthalmitis can ...Endophthalmitis is a serious ophthalmic disease characterized by changes in the eye's posterior segment,such as hypopyon and intraocular inflammation,vitritis being a hallmark.Infection-caused endophthalmitis can lead to irreversible vision loss,accompanied by eye pain or eye distention,and in the most severe cases the removal of the eyeball.Microorganisms such as bacteria,fungi,viruses,and parasites typically account for the disease and the entry pathways of the microbial can be divided into either endogenous or exogenous approaches,according to the origin of the etiological agents.Exogenous endophthalmitis can be derived from various occasions(such as postoperative complications or trauma)while endogenous endophthalmitis results from the bloodstream which carries pathogens to the eye.This review aims to summarize the application of new technology in pathogen identification of endophthalmitis so as to prevent the disease and better guide clinical diagnosis and treatment.展开更多
The efficacy of DNA sequencing,particularly long reads nanopore sequencing,is critically dependent on the amount and quality of the input DNA.However,extracting high concentrations of DNA from low biomass samples,espe...The efficacy of DNA sequencing,particularly long reads nanopore sequencing,is critically dependent on the amount and quality of the input DNA.However,extracting high concentrations of DNA from low biomass samples,especially from solid matrices,presents significant challenges,this limitation not only substantially hampers the scope of environmental microbiology studies but also makes enhancing DNA yield indispensable in many instances.Therefore,in this study,we systematically evaluated the impact of four different DNA enrichment methods on both amplicon and metagenomic community analyses of solid-phase,low-biomass samples:permafrost soil and biofilm of sand filter.These methods include multiple displacement amplification(MDA),centrifugal filtration(CF),freeze vacuum drying at(FVD)as well as vacuum centrifugal at 35,45,and 60°C(namely VC35,VC45,VC60).Our results indicate that FVD was the most effective for increasing DNA concentration,while VC methods best preserved DNA fragment length.In contrast,the widely used MDA and CF methods exhibited biases,preferentially enriching low-GC content sequences,which affected both assembly and annotation outcomes.Metagenomic assembly from MDA and CF samples was suboptimal,with fewer contigs and no middle quality MAGs recovered compared to other methods.Community composition analysis revealed significant shifts across all enrichment methods,with Sphingomonas and Sphingorhabdus genera could be obviously enriched.These findings highlight the necessity and importance of carefully selecting DNA enrichment methods to ensure reliable metagenomic investigation of low-biomass environmental samples.展开更多
Background:Infective endocarditis(IE)and myocarditis are serious heart diseases that can lead to life-threatening complications.These illnesses can have infectious origins,including viral,bacterial,or fungal pathogens...Background:Infective endocarditis(IE)and myocarditis are serious heart diseases that can lead to life-threatening complications.These illnesses can have infectious origins,including viral,bacterial,or fungal pathogens.Traditional detection methods,such as culture-based methods,have limited ability to detect causative pathogens because of antibiotic use and the difficulty in cultivating intracellular and fastidious bacteria as well as viruses.In clinical settings,rapid diagnostics for pathogen identification are essential for timely treatment and appropriate antimicrobial therapy.Methods:We successfully developed a method based on nanopore targeted sequencing(NTS)with pathogen-specific panels for testing myocarditis and IE.As part of this pilot study,a sample-to-results protocol was developed with an optimized in-house pipeline and bioinformatics analysis solution.Results:The performance of NTS met our expectations for sensitivity,specificity,and turnaround time.The pathogen-specific panel testing was accomplished in a 10-h turnaround time,achieving a detection limit of 20 copies/test for the IE target panel and 10 copies/test for the myocarditis target panel.NTS achieved a clinical performance of 85.0% sensitivity and 96.3% specificity compared with culture testing methods,using 74 clinical specimens from patients(53 male,21 female)associated with IE.Conclusions:The rapid turnaround time of NTS is advantageous for managing acute infections,such as IE and myocarditis.NTS is a powerful tool for rapidly diagnosing infections in IE and myocarditis with significant potential for broader clinical applications.展开更多
The pursuit of complete telomere-to-telomere(T2T)genome assembly in plants,challenged by genomic complexity,has been advanced by Oxford Nanopore Technologies(ONT),which offers ultra-long,realtime sequencing.Despite it...The pursuit of complete telomere-to-telomere(T2T)genome assembly in plants,challenged by genomic complexity,has been advanced by Oxford Nanopore Technologies(ONT),which offers ultra-long,realtime sequencing.Despite its promise,sequencing length and gap filling remain significant challenges.This study optimized DNA extraction and library preparation,achieving DNA lengths exceeding 485 kb;average N50 read lengths of 80.57 kb,reaching up to 440 kb;and maximum reads of 5.83 Mb.Importantly,we demonstrated that combining ultra-long sequencing and adaptive sampling can effectively fill gaps during assembly,evidenced by successfully filling the remaining gaps of a near-complete Arabidopsis genome assembly and resolving the sequence of an unknown telomeric region in watermelon genome.Collectively,our strategies improve the feasibility of complete T2T genomic assemblies across various plant species,enhancing genome-based research in diverse fields.展开更多
基金supported by CAMS Innovation Fund for Medical Sciences (CIFMS)[2021-I2M-1-038]
文摘Infectious diseases are an enormous public health burden and a growing threat to human health worldwide.Emerging or classic recurrent pathogens,or pathogens with resistant traits,challenge our ability to diagnose and control infectious diseases.Nanopore sequencing technology has the potential to enhance our ability to diagnose,interrogate,and track infectious diseases due to the unrestricted read length and system portability.This review focuses on the application of nanopore sequencing technology in the clinical diagnosis of infectious diseases and includes the following:(i)a brief introduction to nanopore sequencing technology and Oxford Nanopore Technologies(ONT)sequencing platforms;(ii)strategies for nanopore-based sequencing technologies;and(iii)applications of nanopore sequencing technology in monitoring emerging pathogenic microorganisms,molecular detection of clinically relevant drug-resistance genes,and characterization of disease-related microbial communities.Finally,we discuss the current challenges,potential opportunities,and future outlook for applying nanopore sequencing technology in the diagnosis of infectious diseases.
基金financially supported by the Natural Science Foundation of China(32470055 and U23A20148)the China Postdoctoral Science Foundation(2024M753580)the Agricultural Science and Technology Innovation Program(CAAS-ZDRW202308)。
文摘Over the past decade,nanopore sequencing has experienced significant advancements and changes,transitioning from an initially emerging technology to a significant instrument in the field of genomic sequencing.However,as advancements in next-generation sequencing technology persist,nanopore sequencing also improves.This paper reviews the developments,applications,and outlook on nanopore sequencing technology.Currently,nanopore sequencing supports both DNA and RNA sequencing,making it widely applicable in areas such as telomere-to-telomere(T2T)genome assembly,direct RNA sequencing(DRS),and metagenomics.The openness and versatility of nanopore sequencing have established it as a preferred option for an increasing number of research teams,signaling a transformative influence on life science research.As the nanopore sequencing technology advances,it provides a faster,more costeffective approach with extended read lengths,demonstrating the significant potential for complex genome assembly,pathogen detection,environmental monitoring,and human disease research,offering a fresh perspective in sequencing technologies.
基金supported by grants from the Foundation for National Mega Project on Major Infectious Disease Prevention(grant number 2017ZX10103005-005)National Key Research and Development Program of China(2020YFC0845800 and 2020YFC0845600)the National Natural Science Foundation of China(31970548 and 91631110)。
文摘Genome sequencing has shown strong capabilities in the initial stages of the COVID-19 pandemic such as pathogen identification and virus preliminary tracing.While the rapid acquisition of SARS-Co V-2 genome from clinical specimens is limited by their low nucleic acid load and the complexity of the nucleic acid background.To address this issue,we modified and evaluated an approach by utilizing SARS-Co V-2-specific amplicon amplification and Oxford Nanopore Prometh ION platform.This workflow started with the throat swab of the COVID-19 patient,combined reverse transcript PCR,and multi-amplification in one-step to shorten the experiment time,then can quickly and steadily obtain high-quality SARS-Co V-2 genome within 24 h.A comprehensive evaluation of the method was conducted in 42 samples:the sequencing quality of the method was correlated well with the viral load of the samples;high-quality SARS-Co V-2 genome could be obtained stably in the samples with Ct value up to 39.14;data yielding for different Ct values were assessed and the recommended sequencing time was 8 h for samples with Ct value of less than 20;variation analysis indicated that the method can detect the existing and emerging genomic mutations as well;Illumina sequencing verified that ultra-deep sequencing can greatly improve the single read error rate of Nanopore sequencing,making it as low as 0.4/10,000 bp.In summary,high-quality SARS-Co V-2 genome can be acquired by utilizing the amplicon amplification and it is an effective method in accelerating the acquisition of genetic resources and tracking the genome diversity of SARSCo V-2.
基金Supported by Research and Development Funding for Medical and Health Institutions,No.2021YL007.
文摘BACKGROUND Infectious diseases are still one of the greatest threats to human health,and the etiology of 20%of cases of clinical fever is unknown;therefore,rapid identification of pathogens is highly important.Traditional culture methods are only able to detect a limited number of pathogens and are time-consuming;serologic detection has window periods,false-positive and false-negative problems;and nucleic acid molecular detection methods can detect several known pathogens only once.Three-generation nanopore sequencing technology provides new options for identifying pathogens.CASE SUMMARY Case 1:The patient was admitted to the hospital with abdominal pain for three days and cessation of defecation for five days,accompanied by cough and sputum.Nanopore sequencing of the drainage fluid revealed the presence of orallike bacteria,leading to a clinical diagnosis of bronchopleural fistula.Cefoperazone sodium sulbactam treatment was effective.Case 2:The patient was admitted to the hospital with fever and headache,and CT revealed lung inflammation.Antibiotic treatment for Streptococcus pneumoniae,identified through nanopore sequencing of cerebrospinal fluid,was effective.Case 3:The patient was admitted to our hospital with intermittent fever and an enlarged neck mass that had persisted for more than six months.Despite antibacterial treatment,her symptoms worsened.The nanopore sequencing results indicate that voriconazole treatment is effective for Aspergillus brookii.The patient was diagnosed with mixed cell type classical Hodgkin's lymphoma with infection.CONCLUSION Three-generation nanopore sequencing technology allows for rapid and accurate detection of pathogens in human infectious diseases.
文摘With the widespread presence of multidrug-resistant bacteria in medical environments,precise environmental monitoring and disinfection targeting drug-resistance genes have become increasingly important.Third-generation nanopore sequencing has significant application potential in genomics,clinical diagnosis,and other fields owing to its unique advantages of long read length,real-time sequencing,and portability.Research on nanopore sequencing has made significant progress;however,challenges remain in terms of technical principles,performance optimization,and the development of bioinformatics analysis tools.This paper aims to systematically review the latest advances in nanopore sequencing,analyze practical cases in monitoring antimicrobial resistance and environmental disinfection,and discuss current challenges faced and future development directions.By integrating relevant research findings,this review hopes to provide valuable references for researchers in related fields and promote the further development and application of nanopore sequencing.
文摘Nanopore sequencing technology,an advanced third-generation sequencing technology,is a revolutionary sequencing method widely used in clinical diagnosis and genomic research because of its features such as real-time sequencing,direct sequencing,long read length and portability.This paper outlines the basic principles and advantages of the technology,and briefly introduces its applications in clinical medicine such as diagnosis of diseases rare and genetic diseases,detection of infectious disease pathogens,public health emergency response,and cancer genomics screening.In genomics,nanopore sequencing is instrumental in genome assembly,structural variation detection,recovery of DNA from ancient organisms,and microbiological research.It enables direct sequencing and analysis of molecules,allowing for the identification of complex structural variations within the genome.This study finds that the technology also suffers from low accuracy,high cost associated with large data volumes,and significant requirements for data processing capabilities.These limitations can potentially be addressed through innovations such as improved nanopore materials and design,and integration with artificial intelligence.Finally,the latest innovations of the technology are analyzed,and the development trend and application prospects are outlooked.
基金supported by the Guangxi Zhuang Autonomous Region Health Commission Self-Financed Scientific Research Project(Grant No.Z-A20231211)the Nanning Scientific Research and Technology Development Plan Project(Grant No.20233069).
文摘This paper reports a case of Chlamydia psittaci pneumonia and pulmonary aspergillosis with concurrent liver injury.The patient was admitted to the hospital with coughing,expectoration,and fever for 5 days.Laboratory tests upon admission revealed abnormalities in liver function.The patient was treated with broad-spectrum antibiotics and liver-protection therapy,but symptoms did not significantly improve.Further nanopore sequencing detected Chlamydia psittaci and Aspergillus flavus.The treatment plan was adjusted to include combined anti-infection and antifungal therapies.After treatment,the patient's symptoms improved significantly;their liver function returned to normal,and their condition improved.
基金Supported by Open Project of Key Laboratory of Hubei Province(No.2023KFZZ026).
文摘AIM:To estimate if nanopore targeted sequencing(NTS)could identify pathogens causing postoperative endophthalmitis and further determine the feasibility of clinical application of NTS.METHODS:A total of 55 patients(55 eyes)with postoperative endophthalmitis were retrospectively included in this study with their medical records.Intraocular fluid samples were examined by NTS and microbial culture.All included patients had undergone examinations including measurement of best corrected visual acuity(BCVA)and intraocular pressure(IOP),slit-lamp biomicroscopy,and indirect ophthalmoscopy;additionally,they underwent B-ultrasound,anterior segment photography,and fundus photography if necessary.RESULTS:Among 55 patients with postoperative endophthalmitis,the age was 65.25±15.04y and there were 30 female(54.54%)patients.Forty-one(74.54%)vitreous humor samples and fourteen(25.45%)aqueous humor samples were sent for both NTS and microbial culture.NTS had a notable higher detection rate than microbial culture in detecting pathogens(90.91%vs 38.18%,χ^(2)=33.409,P<0.001).NTS exhibited high sensitivity of pathogen detection in both microbial culture positive and negative samples(100%and 85.29%,respectively).In 16 of 21(76.19%)patients who showed culture-positivity,their results corresponded with those of NTS.Moreover,in two patients(9.52%),NTS showed a better species resolution than microbial culture;in three patients(14.28%),NTS identified additional pathogens.As for fungus,the positive detection rate of NTS was significantly higher than that of microbial culture(20%vs 3.64%,χ^(2)=7.066,P=0.008).Also,NTS could detect multi-infection by bacteria and fungi than microbial culture(32.73%vs 0,χ^(2)=21.522,P<0.001).NTS could detect bacteria as well as fungi simultaneously within 48h in all patients.Meanwhile,NTS had a shorter detection time than microbial culture(1.13±0.34 vs 2.67±0.55d,Z=-9.218,P<0.001).After the NTS results were obtained,15 patients received additional intravitreal/intracameral anti-infection treatment.At follow-up,there was a statistically significant improvement in the visual acuity relative to the baseline(Z=−5.222,P<0.001).CONCLUSION:NTS can provide rapid identification and highly sensitive detection of pathogens among patients with postoperative endophthalmitis,which can guide anti-infection treatment and improve visual prognosis.
基金supported by the National key research and development plan(2016TFC1202700,2016YFC1200900)Beijing Municipal Science&Technology Commission project(grant numbers D151100002115003)Guangzhou Municipal Science&Technology Commission project(grant numbers 2015B2150820)
文摘Objective Knowledge of an enterovirus genome sequence is very important in epidemiological investigation to identify transmission patterns and ascertain the extent of an outbreak. The MinION sequencer is increasingly used to sequence various viral pathogens in many clinical situations because of its long reads, portability, real-time accessibility of sequenced data, and very low initial costs. However, information is lacking on MinION sequencing of enterovirus genomes. Methods In this proof-of-concept study using Enterovirus 71 （EV71） and Coxsackievirus A16 （CA16） strains as examples, we established an amplicon-based whole genome sequencing method using MinION. We explored the accuracy, minimum sequencing time, discrimination and high-throughput sequencing ability of MinION, and compared its performance with Sanger sequencing. Results Within the first minute （min） of sequencing, the accuracy of MinION was 98.5% for the single EV71 strain and 94.12%-97.33% for 10 genetically-related CA16 strains. In as little as 14 min, 99% identity was reached for the single EV71 strain, and in 17 min （on average）, 99% identity was achieved for 10 CA16 strains in a single run. Conclusion MinION is suitable for whole genome sequencing of enteroviruses with sufficient accuracy and fine discrimination and has the potential as a fast, reliable and convenient method for routine use.
文摘DNA barcodes,short and unique DNA sequences,play a crucial role in sample identification when processing many samples simultaneously,which helps reduce experimental costs.Nevertheless,the low quality of long-read sequencing makes it difficult to identify barcodes accurately,which poses significant challenges for the design of barcodes for large numbers of samples in a single sequencing run.Here,we present a comprehensive study of the generation of barcodes and develop a tool,PRO,that can be used for selecting optimal barcode sets and demultiplexing.We formulate the barcode design problem as a combinatorial problem and prove that finding the optimal largest barcode set in a given DNA sequence space in which all sequences have the same length is theoretically NP-complete.For practical applications,we developed the novel method PRO by introducing the probability divergence between two DNA sequences to expand the capacity of barcode kits while ensuring demultiplexing accuracy.Specifically,the maximum size of the barcode kits designed by PRO is 2,292,which keeps the length of barcodes the same as that of the official ones used by Oxford Nanopore Technologies(ONT).We validated the performance of PRO on a simulated nanopore dataset with high error rates.The demultiplexing accuracy of PRO reached 98.29%for a barcode kit of size 2,922,4.31%higher than that of Guppy,the official demultiplexing tool.When the size of the barcode kit generated by PRO is the same as the official size provided by ONT,both tools show superior and comparable demultiplexing accuracy.
基金grateful to the financial support from the National Natural Science Foundation of China(Nos.22025407,21974144)Institute of Chemistry,Chinese Academy of Sciences。
文摘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.
基金supported by the Key-Areas Research and Development Program of Guangdong Province(2020B020220004)the Youth Innovation Promotion Association,Chinese Academy of Sciences(2017399)+2 种基金the Science and Technology Program of Guangzhou(202002030097)the Hong Kong Research Grants Council Area of Excellence Scheme(AoE/M-403/16),the ECS(27204518)TRS of the HKSAR government(T21-705/20-N).
文摘The Human Genome Project opened an era of(epi)genomic research,and also provided a platform for the development of new sequencing technologies.During and after the project,several sequencing technologies continue to dominate nucleic acid sequencing markets.Currently,Illumina(short-read),PacBio(long-read),and Oxford Nanopore(longread)are the most popular sequencing technologies.Unlike PacBio or the popular short-read sequencers before it,which,as examples of the second or so-called Next-Generation Sequencing platforms,need to synthesize when sequencing,nanopore technology directly sequences native DNA and RNA molecules.Nanopore sequencing,therefore,avoids converting mRNA into cDNA molecules,which not only allows for the sequencing of extremely long native DNA and full-length RNA molecules but also document modifications that have been made to those native DNA or RNA bases.In this review on direct DNA sequencing and direct RNA sequencing using Oxford Nanopore technology,we focus on their development and application achievements,discussing their challenges and future perspective.We also address the problems researchers may encounter applying these approaches in their research topics,and how to resolve them.
基金supported by the National Key R&D Program of China(Grant Nos.2016YFA0100600,2019YFA0110800,and 2019YFA0110204)the National Natural Science Foundation of China(Grant Nos.81890990,81730006,81770198,81870149,and 82070115)the Chinese Academy of Medical Sciences(CAMS)Innovation Fund for Medical Sciences(CIFMS)(Grant Nos.2019-I2M-1-006 and 2021-I2M-1-041).
文摘To achieve the enormous potential of gene-editing technology in clinical therapies,one needs to evaluate both the on-target efficiency and unintended editing consequences comprehensively.However,there is a lack of a pipelined,large-scale,and economical workflow for detecting genome editing outcomes,in particular insertion or deletion of a large fragment.Here,we describe an approach for efficient and accurate detection of multiple genetic changes after CRISPR/Cas9 editing by pooled nanopore sequencing of barcoded long-range PCR products.Recognizing the high error rates of Oxford nanopore sequencing,we developed a novel pipeline to capture the barcoded sequences by grepping reads of nanopore amplicon sequencing(GREPore-seq).GREPore-seq can assess nonhomologous end-joining(NHEJ)-mediated double-stranded oligodeoxynucleotide(dsODN)insertions with comparable accuracy to Illumina next-generation sequencing(NGS).GREPore-seq also reveals a full spectrum of homology-directed repair(HDR)-mediated large gene knock-in,correlating well with the fluorescence-activated cell sorting(FACS)analysis results.Of note,we discovered low-level fragmented and full-length plasmid backbone insertion at the CRISPR cutting site.Therefore,we have established a practical workflow to evaluate various genetic changes,including quantifying insertions of short dsODNs,knock-ins of long pieces,plasmid insertions,and large fragment deletions after CRISPR/Cas9-mediated editing.GREPore-seq is freely available at GitHub(https://github.com/lisiang/GREPore-seq)and the National Genomics Data Center(NGDC)BioCode(https://ngdc.cncb.ac.cn/biocode/tools/BT007293).
基金supported by the Russian Science Foundation (grant no. 20-74-10055RNA-seq and mobilome analyses of plants after heat stress)+1 种基金Kurchatov Genomic Center of All-Russia Research Institute of Agricultural Biotechnology (agreement no. 075-15-20191667CANS development)。
文摘Transposable element insertions(TEIs) are an important source of genomic innovation by contributing to plant adaptation, speciation, and the production of new varieties. The often large,complex plant genomes make identifying TEIs from short reads difficult and expensive. Moreover, rare somatic insertions that refect mobilome dynamics are difficult to track using short reads.To address these challenges, we combined Cas9-targeted Nanopore sequencing(CANS) with the novel pipeline Nano Cas TE to trace both genetically inherited and somatic TEIs in plants. We performed CANS of the EVADé(EVD) retrotransposon in wild-type Arabidopsis thaliana and rapidly obtained up to 40× sequence coverage.Analysis of hemizygous T-DNA insertion sites and genetically inherited insertions of the EVD transposon in the ddm1(decrease in DNA methylation1) genome uncovered the crucial role of DNA methylation in shaping EVD insertion preference.We also investigated somatic transposition events of the ONSEN transposon family, finding that genes that are downregulated during heat stress are preferentially targeted by ONSENs. Finally, we detected hypomethylation of novel somatic insertions for two ONSENs. CANS and Nano Cas TE are effective tools for detecting TEIs and exploring mobilome organization in plants in response to stress and in different genetic backgrounds, as well as screening T-DNA insertion mutants and transgenic plants.
基金This work was financially supported by the National Natural Science Foundation of China(Nos.22373025 and 22227804).
文摘Nanopore sequencing harnesses changes in ionic current as nucleotides traverse a nanopore,enabling real-time decoding of DNA/RNA sequences.The instruments for the dynamic behavior of substances in the nanopore on the molecular scale are still very limited experimentally.This study employs all-atom molecular dynamics(MD)simulations to explore the impact of charge densities on graphene nanopore in the translocation of single-stranded DNA(ssDNA).We find that the magnitude of graphene’s charge,rather than the charge disparity between ssDNA and graphene,significantly influences ssDNA adsorption and translocation speed.Specifically,high negative charge densities on graphene nanopores are shown to substantially slow down ssDNA translocation,highlighting the importance of hydrodynamic effects and electrostatic repulsions.This indicates translocation is crucial for achieving distinct ionic current blockades,which plays a central role for DNA sequencing accuracy.Our findings suggest that negatively charged graphene nanopores hold considerable potential for optimizing DNA sequencing,marking a critical advancement in this field.
基金supported by the National Natural Science Foundation of China(Nos.11272197 and11372175)the Innovation Program of Shanghai Municipality Education Commission,China(No.14ZZ095)
文摘The DNA sequencing technology has achieved a leapfrog development in recent years. As a new generation of the DNA sequencing technology, nanopore sequenc- ing has shown a broad application prospect and attracted vast research interests since it was proposed. In the present study, the dynamics of the electric-driven translocation of a homopolymer through a nanopore is investigated by the dissipative particle dynam- ics （DPD）, in which the homopolymer is modeled as a worm-like chain （WLC）. The DPD simulations show that the polymer chain undergoes conformation changes during the translocation process. The different structures of the polymer in the translocation process, i.e., single-file, double folded, and partially folded, and the induced current block- ades are analyzed. It is found that the current blockades have different magnitudes due to the polymer molecules traversing the pore with different folding conformations. The nanoscale vortices caused by the concentration polarization layers （CPLs） in the vicinity of the sheet are also studied. The results indicate that the translocation of the polymer has the effect of eliminating the vortices in the polyelectrolyte solution. These findings are expected to provide the theoretical guide for improving the nanopore sequencing tech- nique.
文摘Endophthalmitis is a serious ophthalmic disease characterized by changes in the eye's posterior segment,such as hypopyon and intraocular inflammation,vitritis being a hallmark.Infection-caused endophthalmitis can lead to irreversible vision loss,accompanied by eye pain or eye distention,and in the most severe cases the removal of the eyeball.Microorganisms such as bacteria,fungi,viruses,and parasites typically account for the disease and the entry pathways of the microbial can be divided into either endogenous or exogenous approaches,according to the origin of the etiological agents.Exogenous endophthalmitis can be derived from various occasions(such as postoperative complications or trauma)while endogenous endophthalmitis results from the bloodstream which carries pathogens to the eye.This review aims to summarize the application of new technology in pathogen identification of endophthalmitis so as to prevent the disease and better guide clinical diagnosis and treatment.
基金supported by the National Natural Science Foundation of China (No. 42277103)the Shenzhen Science and Technology Innovation Committee, China (Nos. JCYJ20240813095659001 and KCXFZ20240903094205008)+1 种基金the Guangdong Provincial Key Laboratory of Soil and Groundwater Pollution Control, China (No. 2023B1212060002)the Guangdong Provincial Key Laboratory of Environmental Health and Land Resource, China (No. 2020B121201014) and the high level special funds, China (Nos. G03050K001 and G030290001)。
文摘The efficacy of DNA sequencing,particularly long reads nanopore sequencing,is critically dependent on the amount and quality of the input DNA.However,extracting high concentrations of DNA from low biomass samples,especially from solid matrices,presents significant challenges,this limitation not only substantially hampers the scope of environmental microbiology studies but also makes enhancing DNA yield indispensable in many instances.Therefore,in this study,we systematically evaluated the impact of four different DNA enrichment methods on both amplicon and metagenomic community analyses of solid-phase,low-biomass samples:permafrost soil and biofilm of sand filter.These methods include multiple displacement amplification(MDA),centrifugal filtration(CF),freeze vacuum drying at(FVD)as well as vacuum centrifugal at 35,45,and 60°C(namely VC35,VC45,VC60).Our results indicate that FVD was the most effective for increasing DNA concentration,while VC methods best preserved DNA fragment length.In contrast,the widely used MDA and CF methods exhibited biases,preferentially enriching low-GC content sequences,which affected both assembly and annotation outcomes.Metagenomic assembly from MDA and CF samples was suboptimal,with fewer contigs and no middle quality MAGs recovered compared to other methods.Community composition analysis revealed significant shifts across all enrichment methods,with Sphingomonas and Sphingorhabdus genera could be obviously enriched.These findings highlight the necessity and importance of carefully selecting DNA enrichment methods to ensure reliable metagenomic investigation of low-biomass environmental samples.
基金funded by the National Key R&D Program of China(grant number:2018YFF01012105).
文摘Background:Infective endocarditis(IE)and myocarditis are serious heart diseases that can lead to life-threatening complications.These illnesses can have infectious origins,including viral,bacterial,or fungal pathogens.Traditional detection methods,such as culture-based methods,have limited ability to detect causative pathogens because of antibiotic use and the difficulty in cultivating intracellular and fastidious bacteria as well as viruses.In clinical settings,rapid diagnostics for pathogen identification are essential for timely treatment and appropriate antimicrobial therapy.Methods:We successfully developed a method based on nanopore targeted sequencing(NTS)with pathogen-specific panels for testing myocarditis and IE.As part of this pilot study,a sample-to-results protocol was developed with an optimized in-house pipeline and bioinformatics analysis solution.Results:The performance of NTS met our expectations for sensitivity,specificity,and turnaround time.The pathogen-specific panel testing was accomplished in a 10-h turnaround time,achieving a detection limit of 20 copies/test for the IE target panel and 10 copies/test for the myocarditis target panel.NTS achieved a clinical performance of 85.0% sensitivity and 96.3% specificity compared with culture testing methods,using 74 clinical specimens from patients(53 male,21 female)associated with IE.Conclusions:The rapid turnaround time of NTS is advantageous for managing acute infections,such as IE and myocarditis.NTS is a powerful tool for rapidly diagnosing infections in IE and myocarditis with significant potential for broader clinical applications.
基金supported by the Key R&D Program of Shandong Province,China(grant no.ZR202211070163)the National Natural Science Foundation of China(grant nos.32170574 and 32200249)+1 种基金the Natural Science Foundation of Shandong Province(grant nos.ZR2023QC026 and ZR2023QC106)the Young Taishan Scholars Program and Yuandu Scholars Program.
文摘The pursuit of complete telomere-to-telomere(T2T)genome assembly in plants,challenged by genomic complexity,has been advanced by Oxford Nanopore Technologies(ONT),which offers ultra-long,realtime sequencing.Despite its promise,sequencing length and gap filling remain significant challenges.This study optimized DNA extraction and library preparation,achieving DNA lengths exceeding 485 kb;average N50 read lengths of 80.57 kb,reaching up to 440 kb;and maximum reads of 5.83 Mb.Importantly,we demonstrated that combining ultra-long sequencing and adaptive sampling can effectively fill gaps during assembly,evidenced by successfully filling the remaining gaps of a near-complete Arabidopsis genome assembly and resolving the sequence of an unknown telomeric region in watermelon genome.Collectively,our strategies improve the feasibility of complete T2T genomic assemblies across various plant species,enhancing genome-based research in diverse fields.
