DNA Sequencing

The sequencing of the Human Reference Genome, with Human Genome Project announced ten years ago, provided a roadmap that is the foundation for modern biomedical research. Reference Genome represents digital database founded by scientists which contains representative examples of species genomes. The need for sequencing has never been greater than it is today. Sequencing has found its applications within diverse research sectors including comparative genomics and evolution, forensics, epidemiology, and applied medicine for diagnostics and therapeutics. Arguably, the strongest rationale for ongoing sequencing is the question for identification and interpretation of human sequence variation as it relates to health and disease. The paper gives review of current DNA sequencing algorithms and techniques as well as next-generation of DNA sequencing. Since the DNA sequencing field is changing rapidly the information in this paper represent a snapshot of this particular moment

Exploring current practices and the potentials of Nanopore sequencing in metagenomics

For billions of years, microorganisms were the sole inhabitants of planet Earth. As major drivers behind many essential geochemical cycles such as the carbon cycle, microbial communities are integral to the continued support of life on Earth and are found in everywhere from the deep seas to our own bodies. In 1977, Frederick Sanger introduced a new method for determining the nucleotide sequences of DNA by chain-terminating inhibitors. This method later become known as Sanger sequencing and would go on to dominate the field for the next 30 or so years. In the mid-2000s, the development of high-throughput sequencing technology led to a revolution in microbial ecology. Often referred to as next-generation sequencing, these technologies were capable of generating tremendous amounts of data at much lower costs per sequenced base than traditional sequencing. This technology, however, rarely produces sequences above a few hundred bases in length, and thus genomes have to be reconstructed by piecing the small fragments back together like a jigsaw puzzle. As most genomes contain many repetitive regions of varying lengths, this reconstruction called assembly often cannot fully reconstruct the original genome due to the inability of short reads to resolve repeated sequences, and in response to this a third generation of sequencing is now on the rise, promising read lengths measured in kilobases and real-time output. Continued improvements in sequencing technologies has allowed researches to study the function and structure of microbial communities in great detail. Through metagenomics, a culture-independent technique that directly investigates the DNA isolated from an environmental sample, the study of hard to cultivate species from a host of high-interest niches is now possible.I milliarder av år var mikroorganismer de eneste beboerne på planeten. Som viktige drivere bak mange geokjemiske sykluser slik som karbonsyklusen, er mikrobielle samfunn helt essensielle for fortsatt liv på jorda og er å finne overalt, fra havdyp til våre egne kropper. I 1977 introduserte Frederick Sanger en ny metode for å sekvensere DNA ved hjelp av kjedeterminerende inhibitorer. Denne metoden ble senere kjent som Sanger sekvensering, og ville fortsette å dominere sitt felt i de neste 30 årene. På midten av 2000-tallet førte utviklingen av sekvenseringsteknologi med høy gjennomstrømning til en revolusjon i mikrobiell økologi. Disse teknologiene, ofte kalt neste generasjons sekvensering, var i stand til a generere enorme mengder med data til mye lavere pris per sekvenserte base en tradisjonelle metoder. Teknologien produserer sjelden sekvenser lenger enn et par hundre baser i lengde, og derfor måtte genomer rekonstrueres fra små biter som i et puslespill. Siden de fleste genomer inneholder repetitive sekvenser av varierende lengde, byr dette på problemer i monteringen («assembly») av genomer, siden de korte sekvensene ikke klarer å løse opp i disse. Som svar på dette er det nå en tredje generasjon av sekvensseringsteknologier som begynner å gjøre seg kjent, som lover lengre sekvenser, målt i kilobaser og sanntidsdata. Fortsatt utvikling av sekvenseringsteknologi har tillatt forskere å studere funksjon og struktur hos mikrobielle samfunn i detalj. Gjennom metagenomikk, en kultur-uavhengig metode som direkte undersøker DNA fra en miljøprøve har nå tidligere ukultiverbare arter fra en rekke nisjer av høy interesse blitt mulig.M-LU

Nanopores for detecting and sensing biological molecules

In spite of significant advances in the detection, separation and counting of single biological molecules (DNA, proteins, aminoacids, etc.) with solid-state nanopores, atomically-resolved scanning and detection of these molecules remains a significant challenge. In most nanopore-based DNA sequencing and single molecule detection techniques, ionic current blockade and blockade duration are the primary signatures associated with reading and scanning. Although these techniques are good enough for single molecule detection, they are not sophisticated enough to analyze and detect single DNA bases, fine structures, homologues and mutagenesis. Aside from the detection difficulties, low signal to noise ratio (SNR), fast speed of translocation, and lack of a cross-check signal are the biggest challenges of current nanopore technology. In this study, we explored different nanopore architectures and materials to find solutions to these current challenges. Using extensive atomistic simulations, we showed that a single layer molybdenum Disulfide (MoS2) nanopore is attractive pore for single base DNA detection with high SNR and multi-level conductance. We introduced and simulated MscL (Mechano-Sensitive Channel of Large Conductance) as an alternative to traditional biological nanopores (Alpha-Hemolysin, MspA) since it provides a flexible nanopore with adaptability to DNA base types. Induced tension in MscL is shown to be different and distinguishable for each DNA base type. The speed of DNA translocation is also decreased by one order of magnitude in MscL, providing a better detection resolution compared to its counterpart, e.g. MspA. Next, we explored DNA origami-graphene hybrid nanopore for DNA detection. We found that the dwell time of each base type in the hybrid pore is different and distinguishable compared to pristine graphene nanopore. The specific interaction (hydrogen bonds) between the complimentary bases at the edge of the pore and the translocating DNA bases give rise to distinguishable dwell time for each DNA. In addition to DNA sequencing studies, we also investigated the recognition of natively folded proteins using graphene nanopore. We specifically focused on the detection of Immunoglobin G subclasses since the separation and the detection of different subclasses of IgG is the signature of many diseases. These four subclasses differ only in their hinge regions and are 95% homologues. We showed that the one atom thick graphene is highly capable of distinguishing between the subclasses by using ionic current and water flux signals

Ensemble-Based Coarse-Grained Molecular Dynamics Simulations of Multifunctional DNA Nanopores

Transmembrane pores are highly specialised nano-devices, with intrinsic specificity and gate-keeping properties that can be exploited in the field of nanobiotechnology. Recently, DNA-origami inspired transmembrane pores with tailorable surface chemistry and programmable dimensions have been rationally designed in an effort to overcome the limitations of protein-based membrane pores such as their fixed lumen size and limited structural repertoire. Ongoing experimental research into the potential applications of triethylene glycol-cholesterol DNA nanopores (DNPs) has been fruitful, with a particular emphasis on drug delivery and biosensing. In this thesis, I describe an ensemble-based coarse-grained MD protocol devised to probe the interactions between bilayer lipids and DNPs, and to determine the effect of membrane encapsulation and salt concentration on the dynamics, structure and conductance of these nanopores. Furthermore, I aim to elucidate the mechanisms by which DNPs mediate translocation of small molecules across lipid bilayers, and the energetics associated with these mechanisms with constant-velocity steered MD and umbrella sampling simulations. I have found that the DNP has no distinct lumen in bulk solution, where it adopts a bloated, amorphous structure with strained and constricted termini regardless of the salt conditions, with significant kinking and fraying of helices. However, salt conditions have a profound effect on the structure of a DNP as it spans a planar lipid bilayer, where it assumes a barrel-like structure with a defined lumen. Sites of constriction in the lumen of the membrane-spanning DNP present a significant barrier to translocation of fluorophores bearing dense negative charges

Metallic Nanopores for Single Molecule Biosensing

This thesis describes a novel approach to the fabrication and characterisation of metallic nanopores and their application for the detection of single DNA molecules. Metallic nanopores with apparent diameters below 20 nm are produced using electrochemical deposition and real-time ionic current feedback. Beginning with large nanopores (diameter 100-200 nm) milled into gold silicon nitride membranes using a focused ion beam, platinum metal is electrodeposited onto the gold surface, thus reducing the effective pore diameter. By simultaneously observing the ion current feedback, the shrinking of the nanopore can be monitored and terminated at any pre-defined value of the pore conductance in a precisely controlled and reproducible way. The ion transport properties of the metallic nanopore system are investigated by characterising the pore conductance at varying potentials across the nanopore and concentrations of electrolyte. The results are compared to conventional bare silicon nitride nanopore systems. Chemical modification at the nanopore surface is also studied using thiolisation to reduce the capacitive charging effects observed with metallic nanopores. Further to this, impedance measurements are carried out to study the resistive behaviour exhibited in these systems. An equivalent circuit model is proposed to validate the results obtained from the experimental studies. To evaluate the suitability of these nanopores for applications in single-molecule biosensing, translocation experiments using λ-DNA are performed. DNA molecules are electrokinetically driven through the nanopore under an applied electric field, hence as the DNA translocates through the pore, current blockade events are detected. Each event is the result of a single molecular interaction of DNA with the nanopore and is characterised by its dwell time and amplitude. Characterisation studies and noise analysis towards the applicability of metallic nanopores as single molecule detectors are also studied and compared to current bare silicon nitride pore systems

Genomic analyses of the immune system

Project 1. Genetic variation in key immune system components Genes underpinning the diversity and plasticity of the human adaptive immune system, such as the HLA and immunoglobulins, are known for their complex structures and polymorphism. The emergence of long-read sequencing technologies has revolutionised genomics research, in particular the characterisation of segmental duplications and structural variation. Here, using long-read sequencing and additional genomics data from a healthy donor identified as HV31, I built two iterations of de novo personal genome assemblies for HV31 as a foundation to study the genetic variation of the immune system. I analysed complex structural variants found in genomic regions encoding key immune system components, and validated them against sequencing data. I also evaluated long-read sequencing accuracy and developed a tool for genomic data visualisation. Collectively, these efforts demonstrate the applications of personal genome assemblies in studying the immune system. Project 2. Effects of low-dose IL-2 immunotherapy in T and NK cells Low-dose interleukin-2 (IL-2) immunotherapy is a promising treatment for type 1 diabetes (T1D). IL-2 supresses autoimmune reactions by increasing the number of regulatory T cells (Tregs). To better understand the mechanism of action of low- dose IL-2 immunotherapy, I analysed single-cell multiomics data of T and NK cells collected from T1D patients before and after low-dose IL-2 treatment. I confirmed that low-dose IL-2 selectively expanded thymic-derived FOXP3+ HELIOS+ regulatory T cells and CD56br NK cells, and showed that the treatment reduced the frequency of IL-21-producing CD4+ T cells. In addition, I identified a long-lived gene expression signature induced by IL-2, which featured the upregulation of CISH and downregulation of AREG. Notably, I found that the signature remained detectable one month after the treatment. Further analyses of publicly available COVID-19 cohort data revealed that SARS-CoV-2 infection induced opposite changes that persisted for several months after recovery. These findings suggested potential mechanisms of long COVID and longer-term benefits of IL-2 immunotherapy

Theoretical and computational studies of the correlated ionic motion in narrow ion channels

An ion channel is a protein with a hole down its middle embedded into the cytoplasmic membrane of a living biological cells. Ion channels facilitate ionic transport across the membrane, thus bridging the intra- and extra-cellular compartments. Properly functioning channels contribute to the healthy state of an organism, making them one of the main targets for pharmaceutical applications. The description and prediction of a channel’s performance --conductivity, selectivity, blocking etc., -- under arbitrary experimental conditions starting from its crystal structure thus appears as an important challenge in contemporary theoretical research. The main obstacle for such description arises from the presence of the multiple non-negligible interactions in the system. These include ion-ion, ion-water, ion-ligands, ion-pore, and other interactions. Their self-consistent consideration is essential in narrow ion channels, where due to inter-ion interactions and atomic confinement, the ions move in a single-file highly-correlated manner. Molecular dynamics, the most detailed computational tool to date, does not allow one routinely to evaluate the properties of such channels, while continuous methods overlook the ion-ion interactions. Therefore, one needs a method that combines atomic details with the ability to estimate ionic currents. This thesis focuses on the classical treatment of ion channels. Namely, a Brownian Dynamics simulation is described where the interactions of the ion with other ions and the channel are incorporated via the multi-ion potential of the mean force (PMF). This allows one to model the channel’s behaviour under various experimental conditions, while preserving the details of the structure and nanoscale interactions with atomic precision. Secondly, we use the concept of a quasiparticle to describe the highly-correlated ionic motion in the selectivity filter of the KcsA channel. We derive the quasiparticle’s effective potential from the multi-ion atomic PMF, thus connecting the quasiparticle’s properties with the nanoscale features of the channel. We also evaluate the rates of transition between different quasiparticles by virtue of the BD simulation. These ingredients comprehensively describe the quasiparticle’s dynamics which hence serves as an intermediary between the crystal structure and the experimentally observed properties of a narrow ion channel. Lastly, an analytical method to describe the ion-solvent interaction is proposed. It incorporates the ion-solvent and ion-lattice radial density functions, and hence automatically accounts for the pore shape, the type of atoms comprising the lattice, the type of solvent, and the ion’s location near the pore entrance. This method paves the way to an analytical decomposition of single-ion PMFs, what is of fundamental importance in predicting the conductive and selective properties of mutated biological ion channels. This method can also find application in designing functionalized artificial nanopores with on-demand transport properties for efficient water desalination

Nanofluidic Pathways for Single Molecule Translocation and Sequencing -- Nanotubes and Nanopores

abstract: Driven by the curiosity for the secret of life, the effort on sequencing of DNAs and other large biopolymers has never been respited. Advanced from recent sequencing techniques, nanotube and nanopore based sequencing has been attracting much attention. This thesis focuses on the study of first and crucial compartment of the third generation sequencing technique, the capture and translocation of biopolymers, and discuss the advantages and obstacles of two different nanofluidic pathways, nanotubes and nanopores for single molecule capturing and translocation. Carbon nanotubes with its constrained structure, the frictionless inner wall and strong electroosmotic flow, are promising materials for linearly threading DNA and other biopolymers for sequencing. Solid state nanopore on the other hand, is a robust chemical, thermal and mechanical stable nanofluidic device, which has a high capturing rate and, to some extent, good controllable threading ability for DNA and other biomolecules. These two different but similar nanofluidic pathways both provide a good preparation of analyte molecules for the sequencing purpose. In addition, more and more research interests have move onto peptide chains and protein sensing. For proteome is better and more direct indicators for human health, peptide chains and protein sensing have a much wider range of applications on bio-medicine, disease early diagnoses, and etc. A universal peptide chain nanopore sensing technique with universal chemical modification of peptides is discussed in this thesis as well, which unifies the nanopore capturing process for vast varieties of peptides. Obstacles of these nanofluidic pathways are also discussed. In the end of this thesis, a proposal of integration of solid state nanopore and fixed-gap recognition tunneling sequencing technique for a more accurate DNA and peptide readout is discussed, together with some early study work, which gives a new direction for nanopore based sequencing.Dissertation/ThesisDoctoral Dissertation Physics 201