基于Nanopore测序技术的非洲猪瘟病毒全基因组测序方法建立

非洲猪瘟(ASF)是由非洲猪瘟病毒(ASFV)引起的一种高度传染性和致死性疫病,近年来给我国生猪产业的健康发展造成了沉重打击。ASFV庞大的基因组导致人们难以及时掌握流行毒株的全基因组序列。本研究旨在利用Nanopore三代测序技术建立一种简便可靠的ASFV全基因组测序方法。设计覆盖ASFV全基因组的31对引物并分为4个引物池对样本进行扩增,通过Nanopore测序技术对扩增产物进行测序,进一步优化相关生物信息学分析方法,最终成功建立了ASFV全基因组测序方法。应用该方法成功从某环境拭子样本中获取一株全长为189 416 bp的ASFV全基因组测序。经一代测序验证表明,在B646L、EP402R、E183L、MGF_360-12L、MGF_505-3R和I177L等关键基因及部分变异位点上,本方法结果与一代测序结果一致性100%;在全基因组水平上,本方法结果与二代测序结果一致性为99.94%。此外,在这项研究中,采用Nanopore测序技术发现了NP1450L基因与NP419L基因间区存在56 bp的重复序列插入(通过一代测序技术进行了验证),但是二代测序未能发现这一显著的变异特征。本研究成功建立了基于Nanopore技术的ASFV全基因组测序方法,该方法具有良好的简便性和可靠性,为当前ASF的防控和分子流行病学研究提供了一个重要手段。

Nanopore测序揭示m6A修饰铁死亡相关基因在中晚期头颈部鳞状细胞癌中的表达及临床意义

目的研究N6-甲基腺苷(m6A)修饰在中晚期头颈部鳞状细胞癌(HNSCC)的作用和基因相关通路,为头颈部HNSCC患者的个体化精准治疗提出新的观点及研究参考。方法选取3对临床中晚期HNSCC患者的癌及其癌旁组织(6个样本)进行测序,从癌和癌旁组织的转录组、m6A相关差异基因中筛选出两者共同的差异基因,通过基因本体(GO)分析、pathway分析及蛋白相互作用(PPI)分析,找出其中的枢纽(hub)基因,随后对网络中的基因进行基因集变异分析(GSVA),从而得到m6A修饰影响的基因及通路。结果从癌与癌旁组织的差异基因中筛选出96个转录组m6A相关的上调基因、159个m6A相关下调基因;信号通路分析发现PPAR通路、免疫相关通路下调,代谢通路上调;构建PPI后发现,CDK1呈现上调趋势,而DCN及ARG2均呈现下调趋势;GSVA分析发现PI3K/AKT信号通路在癌组织中上调;PPI中的hub基因共表达分析发现AGR2和CDK1在m6A调控下,与铁死亡相关基因联系很密切。结论m6A修饰与FOXA1、RPL8、DNC、CDK1、ApoE、POSTN、YWHAZ、MMP13等铁死亡相关基因紧密相关,m6A修饰可能通过铁死亡相关基因影响免疫、代谢相关通路,从而在中晚期HNSCC中起重要作用。

Shale gas transport in nanopores with mobile water films and water bridge

Gas flow properties in nanopores are significantly determined by the flow patterns. Slug flow pattern is a potential water–gas two phase flow pattern, in which gas molecules flow in form of gas slugs and water molecules separate gas slugs. Considering water slippage, a portion of water molecules accumulates at the wall with lower mobility, while the remaining water molecules take the shape of a water bridge. Adopting foam apparent viscosity model to represent slug rheological behavior, how water bridge disturbs on gas flow capacity is estimated. The results are compared with the water–gas two phase flow model that assumes annular flow pattern as well as the single gas flow model without the consideration of water. The comparison illustrates that gas molecular movement is significantly hindered by flow space reduction and loss of gas slippage. The impact from water phase of slug flow pattern is more significant than that of annular flow pattern on gas flow capacity. It is discovered that larger nanopores improve gas flow capacity while maintaining bulk water layer thickness and increasing water bridge thickness tend to reduce gas transport ability. A better understanding of the structure and transport of water and gas molecules is conducive to figure out the specific gas–water flow behavior and predict shale gas production.

Preliminary study on nanopores,nanofissures,and in situ accumulation of Gulong shale oil

The Qingshankou Formation shale oil in the Gulong Sag is an important oil and gas reservoir in the Daqing oilfield,with geological resources of 15.1 billion tons.The fabric of shale can reflect not only its genesis but also the nature of the reservoir space,its physical properties,oil content,and development value.Here,the characteristics of clay minerals in the Gulong shale oil reservoir were studied via electron microscopy,with the primary focus on the microfabrics and reservoir space;thereafter,the in situ accumulation was studied and discussed.Electron backscattering patterns revealed that nanometer pores and fissures were well developed in the Gulong shale oil reservoir.The nano pores were mostly 20-50 nm in diameter(median 20-30 nm),irregularly shaped,mostly,polygonal,and connected with nanofissures.The widths of nanofissures ranged mostly between 10-50 nm(median 20-30 nm);moreover,these fissures were mainly formed by F-F condensation of clay sheets(clay domains).The coagulation of clays was closely related to organic matter,especially algae.The clay colloids were negatively charged due to isocrystalline replacement;hence,metal cations were absorbed around the clay,forming a positive clay group.The positively charged clays subsequently adsorbed negatively charged humic acid(organic matter)and initially degraded algae to form an organic clay flocculant.When the organic clay flocculates reached the threshold for hydrocarbon generation and expulsion,the volume of organic matter decreased by 87%;thereafter,the generated and expelled hydrocarbon filled the nearby pores formed by this contraction.Moreover,the discharged hydrocarbon could not migrate due to capillary resistance(~12 MPa)of the nanopores;hence,the nanopores formed a unique continuous in situ reservoir within the Gulong shale oil.This study demonstrated that the Gulong shale oil reservoir is an actual clay-type shale reservoir with numerous nanopore and fissures.During coagulation,a large amount of organic matter(including layered algae)was absorbed by the clay,forming an organic clay condensate that could have provided the material foundation for hydrocarbon generation at a later stage.Thermal simulation experiments revealed that the volume of organic matter decreased sharply after hydrocarbon generation and expulsion.

A transient model integrating the nanoconfinement effect and pore structure characteristics of oil transport through nanopores

Understanding the integrated transport behavior of oil in shale nanopores is critical to efficient shale oil development. In this paper, based on the time-dependent Poiseuille flow momentum equation, we present a novel transient model to describe oil transport in unsteady and steady states. The model incorporates the effect of the critical shift density, apparent viscosity, slip length, and alkane property, as well as pore tortuosity and surface roughness. We evaluated our model through a comparison with other models, experiments, and molecular dynamics simulations. The results show that the development rates of the volume flows of C_(6)–C_(12) alkane confined in inorganic nanopores and C_(12) alkane confined in organic nanopores were faster than that of the corresponding bulk alkane. In addition, the critical drift density positively promoted the volume flow development rate in the unsteady state and negatively inhibited the mass flow rate in the steady state. This effect was clearest in pores with a smaller radius and lower-energy wall and in alkane with shorter chain lengths. Furthermore, both the nanoconfinement effect and pore structure determined whether the volume flow enhancement rate was greater than or less than 1. The rate increased or decreased with time and was controlled mainly by the nanoconfinement effect. Moreover, as the wall energy increased, the flow inhibition effect increased;as the carbon number of alkane increased, the flow promotion effect increased. The results indicate that the proposed model can accurately describe oil transport in shale nanopores.

Pressure-driven membrane inflation through nanopores on the cell wall

Walled cells,such as in plants and fungi,compose an important part of the model systems in biology.The cell wall primarily prevents the cell from over-expansion when exposed to water,and is a porous material distributed with nanosized pores on it.In this paper,we study the deformation of a membrane patch by an osmotic pressure through a nanopore on the cell wall.We find that there exists a critical pore size or a critical pressure beyond which the membrane cannot stand against the pressure and would inflate out through the pore and further expand.The critical pore size scales linearly with the membrane tension and quadratically with the spontaneous curvature.The critical pressure is inversely proportional to the pore radius.Our results also show that the fluid membrane expansion by pressure is mechanically different from the solid balloon expansion,and predict that the bending rigidity of the membrane in walled cells should be much larger than that of the mammalian cells so as to prevent membrane inflation through the pores on the cell wall.

Molecular insight into the oil displacement mechanism of CO_(2) flooding in the nanopores of shale oil reservoir

With the increasing demand for petroleum,shale oil with considerable reserves has become an important part of global oil resources.The shale oil reservoir has a large number of nanopores and a complicated mineral composition,and the effect of nanopore confinement and pore type usually makes the effective development of shale oil challenging.For a shale oil reservoir,CO_(2) flooding can effectively reduce the oil viscosity and improve the reservoir properties,which can thus improve the recovery performance.In this study,the method of non-equilibrium molecular dynamics(NEMD)simulation is used to simulate the CO_(2) flooding process in the nanoscale pores of shale oil reservoir.The performance difference between the organic kerogen slit nanopore and four types of inorganic nanopores is discussed.Thus,the effects of nanopore type and displacement velocity on the nanoscale displacement behavior of CO_(2) are analyzed.Results indicate that the CO_(2) flooding process of different inorganic pores is different.In comparison,the displacement efficiency of light oil components is higher,and the transport distance is longer.The intermolecular interaction can significantly affect the CO_(2) displacement behavior in nanopores.The CO_(2) displacement efficiency is shown as montmorillonite,feldspar>quartz>calcite>kerogen.On the other hand,it is found that a lower displacement velocity can benefit the miscibility process between alkane and CO_(2),which is conducive to the overall displacement process of CO_(2).The displacement efficiency can significantly decrease with the increase in displacement velocity.But once the displacement velocity is very high,the strong driving force can promote the alkane to move forward,and the displacement efficiency will recover slightly.This study further reveals the microscopic oil displacement mechanism of CO_(2) in shale nanopores,which is of great significance for the effective development of shale oil reservoirs by using the method of CO_(2) injection.

Surveillance of emerging SARS-CoV-2 variants by nanopore technology-based genome sequencing

Objective:To surveill emerging variants by nanopore technology-based genome sequencing in different COVID-19 waves in Sri Lanka and to examine the association with the sample characteristics,and vaccination status.Methods:The study analyzed 207 RNA positive swab samples received to sequence laboratory during different waves.The N gene cut-off threshold of less than 30 was considered as the major inclusion criteria.Viral RNA was extracted,and elutes were subjected to nanopore sequencing.All the sequencing data were uploaded in the publicly accessible database,GISAID.Results:The Omicron,Delta and Alpha variants accounted for 58%,22%and 4%of the variants throughout the period.Less than 1%were Kappa variant and 16%of the study samples remained unassigned.Omicron variant was circulated among all age groups and in all the provinces.Ct value and variants assigned percentage was 100%in Ct values of 10-15 while only 45%assigned Ct value over 25.Conclusions:The present study examined the emergence,prevalence,and distribution of SARS-CoV-2 variants locally and has shown that nanopore technology-based genome sequencing enables whole genome sequencing in a low resource setting country.

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.

宏基因组Nanopore测序在儿童呼吸道病原微生物快速检测中的应用价值

目的探索宏基因组Nanopore(三代)测序在儿童呼吸道病原微生物快速检测中的应用价值。方法对收集的1例重症肺炎患儿的肺泡灌洗液标本,传统方法检测为腺病毒抗原阳性,肺炎支原体和EB病毒PCR检测阳性,患儿经治疗痊愈出院。以此肺泡灌洗液标本提取DNA后,分别行宏基因组三代和二代测序。建立微生物参考基因组数据库,建立三代测序数据实时分析流程,分别将三代和二代测序数据和参考数据库比对,检测并鉴定病原微生物。结果三代、二代测序和传统方法检测从收到样本至检出腺病毒用时分别为3.2 h、55.5 h和48 h,检测费用人民币4200、3600和1790元。三代测序在开始后的12 min即检出1093条腺病毒序列,测序完成后,共得到4920000条序列,检出腺病毒99284条序列,占总序列数的2.02%;检出肺炎支原体和EB病毒序列各1条。二代测序检出的微生物组成和三代类似。结论宏基因组三代测序能实现对所有潜在儿童呼吸道病原微生物的快速实时检测。

A Combination of N2 and CO2 Adsorption to Characterize Nanopore Structure of Organic-Rich Lower Silurian Shale in the Upper Yangtze Platform, South China： Implications for Shale Gas Sorption Capacity

The pores in shales are mainly of nanometer-scale, and their pore size distribution is very important for the preservation and exploitation of shale gas. This study focused on the organic-rich Lower Silurian black shale from four wells in the Upper Yangtze Platform, and their TOC, mineralogical composition and pore characterization were investigated. Low pressure N2 and CO2 adsorption were conducted at 77.35 K and 273.15 K, respectively, and the pore structures were characterized by modified Brunauer-Emmett-Teller （BET）, Dubinin-Radushkevich （DR）, t-plot, Barrett- Joyner-Halenda （BJH） and density functional theory （DFT） methods and then the relationship between pore structure and shale gas sorption capacity was discussed. The results indicate that （1） The Lower Silurian shale has high TOC content of 0.92%~96%, high quartz content of 30.6%-69.5%, and high clays content of 24.1%-51.2%. The total specific surface area varies from 7.56 m^2/g to 25.86 m^2/g. Both the total specific surface area and quartz content are positively associated with the TOC content. （2） Shale samples with higher TOC content have more micropores, which results in more complex nanopore structure. Micropore volumes/surface areas and non-micropore surface areas all increase with the increasing TOC content. （3） A combination of N2 and CO2 adsorption provides the most suitable detection range （~0.3-60 nm） and has high reliability and accuracy for nanopore structure characterization. （4） The TOC content is the key factor to control the gas sorption capacity of the Lower Silurian shale in the Upper Yangtze Platform.

An analytical model for water-oil two-phase flow in inorganic nanopores in shale oil reservoirs

The existence of water phase occupies oil flow area and impacts the confined oil flow behavior at the solid substrate in inorganic nanopores of shale oil reservoirs,resulting in a completely different flow pattern when compared with the single oil phase flow.This study proposes an analytical model to describe the water-oil two-phase flow.In this model,water slippage at the solid substrate is considered while oil slip is introduced to calculate the oil movement at the solid-oil boundary in dry conditions.It is proven that the oil flow profiles of both the two-phase model and single-phase model show parabolic shapes,but the oil flow capacity drops when water takes up the flow space and the impact of water is more significant when the pore dimension is smaller than 30 nm.Also,the oil flow velocity at a pore center is found to drop linearly given a larger water saturation in wet conditions.The effects of surface wettability and oil properties on water-oil flow are also discussed.Compared with the existing singlephase models,this model describes oil flow pattern in the wet condition with the incorporation of the influence of nanopore properties,which better predicts the oil transport in actual reservoir conditions.Water-oil relative permeability curves are also obtained to improve oil yield.

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.

Manipulation and optimization of electron transport by nanopore array targets

The transport of sub-picosecond laser-driven fast electrons in nanopore array targets is studied.Attributed to the generation of micro-structured magnetic fields,most fast electron beams are proven to be effectively guided and restricted during the propagation.Different transport patterns of fast electrons in the targets are observed in experiments and reproduced by particle-in-cell simulations,representing two components:initially collimated low-energy electrons in the center and high-energy scattering electrons turning into surrounding annular beams.The critical energy for confined electrons is deduced theoretically.The electron guidance and confinement by the nano-structured targets offer a technological approach to manipulate and optimize the fast electron transport by properly modulating pulse parameters and target design,showing great potential in many applications including ion acceleration,microfocus x-ray sources and inertial confinement fusion.

A biosensor based on graphene nanoribbon with nanopores:a first-principles devices-design

A biosensor device, built from graphene nanoribbons （GNRs） with nanopores, was designed and studied by first- principles quantum transport simulation. We have demonstrated the intrinsic transport properties of the device and the effect of different nucleobases on device properties when they are located in the nanopores of GNRs. It was found that the device＇s current changes remarkably with the species of nucleobases, which originates from their different chemical compositions and coupling strengths with GNRs. In addition, our first-principles results clearly reveal that the distinguished ability of a device＇s current depends on the position of the pore to some extent. These results may present a new way to read off the nucleobases sequence of a single-stranded DNA （ssDNA） molecule by such GNRs-based device with designed nanopores

A glass nanopore electrode for single molecule detection

We have developed a simple method for fabricating robust and low noise glass nanopore electrodes with pore size 10±5 nm to detect single molecules.β-Cyclodextrin was used as model compound for characterization.In 1.0 mol/L NaCl solution,the molecules generated current pulses of 2-5 pA with noise level less than 0.8 pA.A slide mode and a plug mode were suggested for the way ofβ-cyclodextrin single molecule moving into the glass nanopores.

Insight into the effects of electrochemical factors on host-guest interaction induced signature events in a biological nanopore

The signature events caused by host-guest interactions in the nanopore system can be used as a novel and characteristic signal in quantitative detection and analysis of various molecules.However,the effect of several electrochemical factors on the host-guest interactions in nanopore still remains unknown.Here,we systematically studied host-guest interactions,especially oscillation of DNA-azide adamantane@cucurbit[6]inα-Hemolysin nanopore under varying pH,concentration of electrolytes and counterions(Li+,Na+,K+).Our results indicate correlations between the change of pH and the duration of the oscillation signal.In addition,the asymmetric electrolyte concentration and the charge of the counterions affects the frequency of signature events in oscillation signals,and even the integrity of the protein nanopore.This study provides insight into the design of a future biosensing platform based on signature oscillation signals of the host-guest interaction within a nanopore.

Noise in nanopore sensors: Sources, models, reduction, and benchmarking

Label-free nanopore sensors have emerged as a new generation technology of DNA sequencing and have been widely used for single molecule analysis.Since the firstα-hemolysin biological nanopore,various types of nanopores made of different materials have been under extensive development.Noise represents a common challenge among all types of nanopore sensors.The nanopore noise can be decomposed into four components in the frequency domain(1/f noise,white noise,dielectric noise,and amplifier noise).In this work,we reviewed and summarized the physicalmodels,origins,and reduction methods for each of these noise components.For the first time,we quantitatively benchmarked the root mean square(RMS)noise levels for different types of nanopores,demonstrating a clear material-dependent RMS noise.We anticipate this review article will enhance the understanding of nanopore sensor noises and provide an informative tutorial for developing future nanopore sensors with a high signal-to-noise ratio.

Achieving a sub-10 nm nanopore array in silicon by metal-assisted chemical etching and machine learning

Solid-state nanopores with controllable pore size and morphology have huge application potential.However,it has been very challenging to process sub-10 nm silicon nanopore arrays with high efficiency and high quality at low cost.In this study,a method combining metal-assisted chemical etching and machine learning is proposed to fabricate sub-10 nm nanopore arrays on silicon wafers with various dopant types and concentrations.Through a SVM algorithm,the relationship between the nanopore structures and the fabrication conditions,including the etching solution,etching time,dopant type,and concentration,was modeled and experimentally verified.Based on this,a processing parameter window for generating regular nanopore arrays on silicon wafers with variable doping types and concentrations was obtained.The proposed machine-learning-assisted etching method will provide a feasible and economical way to process high-quality silicon nanopores,nanostructures,and devices.

Effect of confinement on the three-phase equilibrium of water-oil-CO_(2)mixtures in nanopores

Accurate characterization of fluid phase behavior is an important aspect of CO_(2) enhanced shale oil recovery.So far,however,there has been little discussion about the nanopore confinement effect,including adsorption and capillarity on the phase equilibrium of water-oil-CO_(2) mixtures.In this study,an improved three-phase flash algorithm is proposed for calculating the phase behavior of water-oil-gas mixture on the basis of an extended Young-Laplace equation and a newly developed fugacity calculation model.The fugacity model can consider the effect of water-oil-gas adsorption on phase equilibrium.A water-Bakken oil-CO_(2) mixture is utilized to verify the accuracy of the flash algorithm and investigate the confinement effect.Results show that the confinement effect promotes the transfer of all components in the vapor phase to other phases,while the transfer of water,CO_(2),and lighter hydrocarbons is more significant.This leads to a large decrease,a large increase,and a small increase in the mole fraction of the vapor,oleic,and aqueous phases,respectively.When the confinement effect is considered,the density difference of vaporoleic phases decreases,and the interfacial tension of vapor-oleic phases decreases;however,the density difference of vapor-aqueous phases increases,the interfacial tension of vapor-aqueous phases still decreases.