Diagnosis and treatment of refractory infectious diseases using nanopore sequencing technology:Three case reports

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.

Application of Nanopore Sequencing Technology in the Clinical Diagnosis of Infectious Diseases

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.

Nanopore sequencing:flourishing in its teenage years

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.

Generating barcodes for nanopore sequencing data with PRO

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.

Rapid and Accurate Sequencing of Enterovirus Genomes Using MinION Nanopore Sequencer

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.

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.

Rapid Acquisition of High-Quality SARS-CoV-2 Genome via Amplicon-Oxford Nanopore Sequencing

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.

Applications and potentials of nanopore sequencing in the(epi)genome and(epi)transcriptome era

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.

GREPore-seq: A Robust Workflow to Detect Changes After Gene Editing Through Long-range PCR and Nanopore Sequencing

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).

Cas9-targeted Nanopore sequencing rapidly elucidates the transposition preferences and DNA methylation profiles of mobile elements in plants

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.

Polymer translocation through nanopore under external electric field:dissipative particle dynamics study

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.

Nanopore techniques as a potent tool in the diagnosis and treatment of endophthalmitis:a literature review

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.

Nanopore ultra-long sequencing and adaptive sampling spur plant complete telomere-totelomere genome assembly

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.

DNA sequencing using nanopores and kinetic proofreading

We discuss the feasibility of using a nanopore sandwich device to implement the principle of kinetic proofreading to discriminate incorrect hybridizing oligonucleotides on a target DNA or RNA.We propose a method of sequencing DNA or RNA using this approach.The design parameters for such a DNA sequencer are estimated from the Hopfield・Ninio theory of kinetic proofreading and Schrodinger's first-passage-time distribution function.

Translocation of ssDNA through Charged Graphene Nanopores:Effect of the Charge Density

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.

Nanopore-based Fourth-generation DNA Sequencing Technology

Nanopore-based sequencers, as the fourth-generation DNA sequencing technology, have the potential to quickly and reliably sequence the entire human genome for less than $1000, and possibly for even less than $100. The single-molecule techniques used by this technology allow us to further study the interaction between DNA and protein, as well as between protein and protein.Nanopore analysis opens a new door to molecular biology investigation at the single-molecule scale.In this article, we have reviewed academic achievements in nanopore technology from the past as well as the latest advances, including both biological and solid-state nanopores, and discussed their recent and potential applications.

Regulations of m^(6)A methylation on tomato fruit chilling injury

Tomato fruit are sensitive to chilling injury(CI)during cold storage.Several factors have been discovered to be involved in chilling injury of tomato fruit.Plant hormones play an important regulatory role,however,the relationship between chilling injury and N6-methyladenosine(m^(6)A)methylation of transcripts in plant hormone pathways has not been reported yet.In order to clarify the complex regulatory mechanism of m^(6)A methylation on chilling injury in tomato fruit,Nanopore direct RNA sequencing was employed.A large number of enzymes and transcription factors were found to be involved in the regulation process of fruit chilling injury,which were associated with plant hormone,such as 1-aminocyclopropane 1-carboxylate synthase(ACS),aspartate aminotransferase(AST),auxin response factor(ARF2),ethylene response factor 2(ERF2),gibberellin 20-oxidase-3(GA20ox)and jasmonic acid(JA).By conjoint analysis of the differential expression transcripts related to chilling injury andm^(6)Amethylation differential expression transcripts 41 differential expression transcripts were identified involved in chilling injury including 1-aminocyclopropane-1-carboxylate oxidase(ACO)and pectinesterase(PE)were down-regulated and heat shock cognate 70 kD protein 2(cpHSC70),HSP70-binding protein(HspBP)and salicylic acid-binding protein 2(SABP2)were up-regulated.Our results will provide a deeper understanding for chilling injury regulatory mechanism and post-harvest cold storage of tomato fruit.

Recent advances of DNA sequencing via nanopore-based technologies

This review briefly summarizes recent progress in nanopore DNA sequencing from the beginning of 2012 to July 2014. Although partial successes have been achieved in different types of nanopores, biological pores such as α- hemolysin and MspA afford most promising results.

Nanopore DNA sequencing:Are we there yet?

Nanopores have been studied as a unique DNA sequencing technology that can quickly read long stretched DNA sequences. A DNA molecule could pass through a nanopore in a speed of microsecond per base and even faster. With this speed, a human genome can potentially be sequenced by one nanopore in 〈1 h. In contrast to next- generation DNA sequencing （NGS）, the nanopore sequencing is enzyme free without need of sample amplification due to its single-molecule nature. The nanopore sequencing has been envisioned as a new generation of DNA sequencing technology in the post-NGS era. This progress focuses on status quo of the nanopore DNA sequencing and discusses the opportunities and challenges in this rapidly growing field.

Graphene nanopores toward DNA sequencing： a review of experimental aspects

Nanopores for DNA sequencing have drawn much attention due to their potentials to achieve amplification-free, low-cost, and high-throughput analysis of nuclei acids. The material configuration and fabrication of the nanopore has become one important consideration in the nanopore based DNA sequencing research. Among various materials, the newly emerged graphene has brought more opportunities to the development of sequencing technology because of its unique structures and properties. This review mainly focuses on the experimental aspects of graphene nanopore research including the nanopore fabrication methods and processes. Meanwhile, the challenges in the present graphene nanopore research including hydrophobicity, translocation velocity and noise are also addressed and discussed.