Nanopore-based DNA Sequencing Research Articles

Draft genome sequences of 53 Lactococcus and Leuconostoc strains isolated from two undefined DL-starter cultures.

This study presents the complete genomes of 53 strains of Lactococcus and Leuconostoc isolated from two undefined DL-starter cultures originating from Denmark, Tistrup, and P. The genomes were reconstructed using long-read, nanopore-based DNA sequencing, delivering comprehensive data set for comparative genomics and taxonomic classification, with potential utility in dairy fermentation processes.

CRISPR/Cas9 gene targeting plus nanopore DNA sequencing with the plasmid pBR322 in the classroom.

Both nanopore-based DNA sequencing and CRISPR/Cas-based gene editing represent groundbreaking innovations in molecular biology and genomics, offering unprecedented insights into and tools for working with genetic information. For students, reading, editing, and even writing DNA will be part of their everyday life. We have developed a laboratory procedure that includes (i) the biosynthesis of a guide RNA for, (ii) targeting Cas9 to specifically linearize the pBR322 plasmid, and (iii) the identification of the cutting site through nanopore DNA sequencing. The protocol is intentionally kept simple and requires neither living organisms nor biosafety laboratories. We divided the experimental procedures into separate activities to facilitate customization. Assuming access to a well-equipped molecular biology laboratory, an initial investment of approximately $2,700 is necessary. The material costs for each experiment group amount to around $130. Furthermore, we have developed a freely accessible website (https://dnalesen.hs-mittweida.de) for sequence read analysis and visualization, lowering the required computational skills to a minimum. For those with strong computational skills, we provide instructions for terminal-based data processing. With the presented activities, we aim to provide a hands-on experiment that engages students in modern molecular genetics and motivates them to discuss potential implications. The complete experiment can be accomplished within half a day and has been successfully implemented by us at high schools, in teacher training, and at universities. Our tip is to combine CRISPR/Cas gene targeting with nanopore-based DNA sequencing. As a tool, we provide a website that facilitates sequence data analysis and visualization.

A potentiostat readout array for nanopore-based DNA sequencing

A potentiostat readout array for nanopore-based DNA sequencing

Production of new functional coconut milk kefir with blueberry extract and microalgae: the comparison of the prebiotic potentials on lactic acid bacteria of kefir grain and biochemical characteristics

AbstractProbiotic foods are recognized for their importance on human health. Kefir is a versatile probiotic food that can be made from non-dairy sources for vegan diet. This study evaluated the addition of microalga Haematococcus pluvialis (0.50% w/v) and blueberry Vaccinium myrtillus (0.50% w/v) extracts to compare their influence on the biochemical properties and the bacterial community of coconut milk kefir through Nanopore-based DNA sequencing. Results revealed that the V. myrtillus increased the microbial diversity in coconut milk kefir with more abundant Proteobacteria species such as Lacticaseibacillus paracasei (22%) and Lactococcus lactis (6.3%). Microalga demonstrated the opposite effect on C, making Firmicutes represent the whole of the microbiota. Biochemical analysis revealed increased fat content in the kefir samples, with the C1 registering 1.62% and the 1.07% in C2, in contrast to the control group’s 0.87% fat content. The crude protein content exhibited a decrease in both samples compared to the control group (0.00% and 0.88% versus 1.07%). These findings suggest that fortifying vegan kefir with prebiotics has the potential to induce significant alterations in the kefir microbiota. Graphical abstract

Lokatt: a hybrid DNA nanopore basecaller with an explicit duration hidden Markov model and a residual LSTM network

BackgroundBasecalling long DNA sequences is a crucial step in nanopore-based DNA sequencing protocols. In recent years, the CTC-RNN model has become the leading basecalling model, supplanting preceding hidden Markov models (HMMs) that relied on pre-segmenting ion current measurements. However, the CTC-RNN model operates independently of prior biological and physical insights.ResultsWe present a novel basecaller named Lokatt: explicit duration Markov model and residual-LSTM network. It leverages an explicit duration HMM (EDHMM) designed to model the nanopore sequencing processes. Trained on a newly generated library with methylation-free Ecoli samples and MinION R9.4.1 chemistry, the Lokatt basecaller achieves basecalling performances with a median single read identity score of 0.930, a genome coverage ratio of 99.750%, on par with existing state-of-the-art structure when trained on the same datasets.ConclusionOur research underlines the potential of incorporating prior knowledge into the basecalling processes, particularly through integrating HMMs and recurrent neural networks. The Lokatt basecaller showcases the efficacy of a hybrid approach, emphasizing its capacity to achieve high-quality basecalling performance while accommodating the nuances of nanopore sequencing. These outcomes pave the way for advanced basecalling methodologies, with potential implications for enhancing the accuracy and efficiency of nanopore-based DNA sequencing protocols.

Soil as a transdisciplinary research catalyst: from bioprospecting to biorespecting.

The vast microbial biodiversity of soils is beginning to be observed and understood by applying modern DNA sequencing techniques. However, ensuring this potentially valuable information is used in a fair and equitable way remains a challenge. Here, we present a public engagement project that explores this topic through collaborative research of soil microbiomes at six urban locations using nanopore-based DNA sequencing. The project brought together researchers from the disciplines of synthetic biology, environmental humanities and microbial ecology, as well as school students aged 14-16 years old, to gain a broader understanding of views on the use of data from the environment. Discussions led to the transformation of 'bioprospecting', a metaphor with extractive connotations which is often used to frame environmental DNA sequencing studies, towards a more collaborative approach-'biorespecting'. This shift in terminology acknowledges that genetic information contained in soil arises as a result of entire ecosystems, including the people involved in its creation. Therefore, any use of sequence information should be accountable to the ecosystems from which it arose. As knowledge can arise from ecosystems and communities, science and technology should acknowledge this link and reciprocate with care and benefit-sharing to help improve the wellbeing of future generations.

Synthetic Receptor Based on a Peptide Antibiotic-Functionalized Chimera for Hybridization-Based Polynucleotide Detection.

Nanopores offer highly sensitive, low-cost, and single-molecule sensing capabilities, and the societal impact of this approach is best captured by the advent of nanopore-based DNA detection and sequencing technologies, which extract genomic information without amplification. To address a critical difficulty plaguing such undertakings involving especially protein-based nanopores isolated in lipid bilayers, namely, the formation of a stable, long-lasting single nanopore, we pioneer herein an approach for generating functional nanostructures enabling small single-stranded DNA (ssDNA) detection. We designed a dynamic hybrid construct by appending extramembrane peptide nucleic acid (PNA) segments to the C-terminus of modified ion channel-forming alamethicin monomers. We found that the resulting chimeric molecules successfully coassemble in a voltage-dependent manner in planar lipid membranes generating diameter-variable oligomers. The subsequent interaction at the flexible extramembrane segment of such formed dynamic nanopores with aqueously added complementary ssDNA fragments leads to overall conformational alterations affecting the peptide assembly state kinetics and mediated ionic current. Such recognition events were found specific to the primary structure of target ssDNA and uninhibited the presence of serum. Our platform demonstrates the feasibility of designing an entirely new class of versatile chimeric biosensors, for which, dependent upon the nature of the attached receptor moiety and underlying recognition chemistry, the applicability area may extend to other analytes.

A primer on pollen assignment by nanopore-based DNA sequencing

The possibility to identify plants based on the taxonomic information coming from their pollen grains offers many applications within various biological disciplines. In the past and depending on the application or research in question, pollen origin was analyzed by microscopy, usually preceded by chemical treatment methods. This procedure for identification of pollen grains is both time-consuming and requires expert knowledge of morphological features. Additionally, these microscopically recognizable features usually have a low resolution at species-level. Since a few decades, DNA has been used for the identification of pollen taxa, as sequencing technologies evolved both in their handling and affordability. We discuss advantages and challenges of pollen DNA analyses compared to traditional methods. With readers with little experience in this field in mind, we present a hands-on primer for genetic pollen analysis by nanopore sequencing. As our lab mainly works with pollen collected within agroecological research projects, we focus on pollen collected by pollinating insects. We briefly consider sample collection, storage and processing in the laboratory as well as bioinformatic aspects. Currently, pollen metabarcoding is mostly conducted with next-generation sequencing methods that generate short sequence reads (<1 kb). Increasingly, however, pollen DNA analysis is carried out using the long-read generating (several kb), low-budget and mobile MinION nanopore sequencing platform by Oxford Nanopore Technologies. Therefore, we are focusing on aspects for palynology with the MinION DNA sequencing device.

Development of an Artificially Intelligent Nanopore for High-Throughput DNA Sequencing with a Machine-Learning-Aided Quantum-Tunneling Approach.

Solid-state nanopore-based single-molecule DNA sequencing with quantum tunneling technology poses formidable challenges to achieve long-read sequencing and high-throughput analysis. Here, we propose a method for developing an artificially intelligent (AI) nanopore that does not require extraction of the signature transmission function for each nucleotide of the whole DNA strand by integrating supervised machine learning (ML) and transverse quantum transport technology with a graphene nanopore. The optimized ML model can predict the transmission function of all other nucleotides after training with data sets of all the orientations of any nucleotide inside the nanopore with a root-mean-square error (RMSE) of as low as 0.062. Further, up to 96.01% accuracy is achieved in classifying the unlabeled nucleotides with their transmission readouts. We envision that an AI nanopore can alleviate the experimental challenges of the quantum-tunneling method and pave the way for rapid and high-precision DNA sequencing by predicting their signature transmission functions.

Unpicking DNA translocation in nanopores with simultaneous single-molecule fluorescence and optical single channel recording.

Nanopore-based DNA sequencing offers high throughput and long reads by measuring blockades in current as a strand is translocated through the pore, providing a single-molecule readout with high time resolution. However, electrical measurements alone offer only information regarding the current block itself; to improve nanopore sensing, a molecular picture of the steps of analyte binding, capture and translocation is required. Optical single channel recording of calcium flux through the nanopore enables parallel single-molecule capture events to be distinguished. We combine optical single channel recording with a single molecule fluorescence readout of DNA unzipping to correlate the steps of individual translocations and image the location of the system components. By annealing short oligomers bearing fluorophore-quencher pairs to the translocating DNA strand, we map the process of nanopore capture, threading and unzipping.

A review and analysis of current-mode biosensing front-end ICs for nanopore-based DNA sequencing

Bio-sensors connect the biological world with electronic devices, widely used in biomedical applications. The combination of microelectronic and medical technologies makes biomedical diagnosis more rapid, accurate, and efficient. In this article, the current-mode biosensing front-end integrated circuits (ICs) for nanopore-based DNA sequencing are reviewed and analyzed, aiming to present their operation theories, advantages, limitations, and performances including gain, bandwidth, noise, and power consumption. Because biological information and external interference are contained in extremely weak sensing current, usually at the pA or nA level, it is challenging to accurately detect and restore the desired signals. Based on the requirements of DNA sequencing, this paper shows three circuit topologies of biosensing front-end, namely, discrete-time, continuous-time, and current-to-frequency conversion types. This paper also makes an introduction to the current-mode sensor array for DNA sequencing. To better review and evaluate the research of the state-of-the-art, the most relevant published works are summarized and compared. The review and analysis would help the researchers be familiar with the requirements, constraints, and methods for current-mode biosensing front-end IC designs for nanopore-based DNA sequencing.

THE SUCCESSFUL DEVELOPMENT AND CLINICAL VALIDATION OF STORK, A RAPID ANEUPLOIDY DETECTION METHOD USING NANOPORE SEQUENCING

Aneuploid pregnancies are a significant cause of pregnancy loss, fetal structural anomalies, and developmental delays. Consequently, the identification of genetic abnormalities is an important component of prenatal and fertility care. However, existing tests for aneuploidy have shortcomings that reduce access to reproductive care, delay diagnosis and treatment, and increase costs. We developed a nanopore-based DNA sequencing technology that enables low-cost, same-day, point-of-care, aneuploidy testing of reproductive tissues that we termed STORK (Short-read Transpore Rapid Karyotyping). Here we report on the first clinical validation of STORK in a CLIA-approved laboratory.

Molecular Dynamics Simulation of a Single Carbon Chain through an Asymmetric Double-Layer Graphene Nanopore for Prolonging the Translocation Time.

In recent years, sensing technology based on nanopores has become one of the trustworthy options for characterization and even identification of a single biomolecule. In nanopore based DNA sequencing technology, the DNA strand in the electrolyte solution passes through the nanopore under an applied bias electric field. Commonly, the ionic current signals carrying the sequence information are difficult to detect effectively due to the fast translocation speed of the DNA strand, so that slowing down the translocation speed is expected to make the signals easier to distinguish and improve the sequencing accuracy. Modifying the nanopore structure is one of the effective methods. Through all-atom molecular dynamics simulations, we designed an asymmetric double-layer graphene nanopore structure to regulate the translocation speed of a single carbon chain. The structure consists of two nanopores with different sizes located on two layers. The simulation results indicate that the asymmetric nanopore structure will affect the chain’s translocation speed and the ionic current value. When the single carbon chain passes from the smaller pore to the larger pore, the translocation time is significantly prolonged, which is about three times as long as the chain passing from the larger pore to the smaller pore. These results provide a new idea for designing more accurate and effective single-molecule solid-state nanopore sensors.

Nanopore-based DNA sequencing sensors and CMOS readout approaches

PurposeNanopore-based molecular sensing and measurement, specifically DNA sequencing, is advancing at a fast pace. Some embodiments have matured from coarse particle counters to enabling full human genome assembly. This evolution has been powered not only by improvements in the sensors themselves, but also in the assisting microelectronic CMOS readout circuitry closely interfaced to them. In this light, this paper aims to review established and emerging nanopore-based sensing modalities considered for DNA sequencing and CMOS microelectronic methods currently being used.Design/methodology/approachReadout and amplifier circuits, which are potentially appropriate for conditioning and conversion of nanopore signals for downstream processing, are studied. Furthermore, arrayed CMOS readout implementations are focused on and the relevant status of the nanopore sensor technology is reviewed as well.FindingsIon channel nanopore devices have unique properties compared with other electrochemical cells. Currently biological nanopores are the only variants reported which can be used for actual DNA sequencing. The translocation rate of DNA through such pores, the current range at which these cells operate on and the cell capacitance effect, all impose the necessity of using low-noise circuits in the process of signal detection. The requirement of using in-pixel low-noise circuits in turn tends to impose challenges in the implementation of large size arrays.Originality/valueThe study presents an overview on the readout circuits used for signal acquisition in electrochemical cell arrays and investigates the specific requirements necessary for implementation of nanopore-type electrochemical cell amplifiers and their associated readout electronics.

Simulation and Analysis of Bionanopore Dna Sequencing Signals for Genetic Mutations Detection

The application of genomic signal processing methods to the problem of modeling and analysis of nanoporous DNA sequencing signals is considered in the paper. Based on the nucleotide sequences in the norm and in the case of mutations, 1200 signals are simulated, which represent 4 classes: norm, missense mutation, insertion mutation and deletion mutation. Correlation analysis was used to determine the similarity of nanoporous DNA sequencing signals using a cross-correlation function between two current signals in the protein nanopore, specifically signal in norm and in the presence of mutation. The location of the correlation peak determines the type of mutation (insertion or deletion), as well as the alignment of the same nucleotide sequences using a defined signal shift. The results of applying machine learning methods to the problem of classification of nanoporous DNA sequencing signals significantly depend on the noise level of the registered current signals through the protein nanopore and the type of mutation. Given a relatively low noise level, when the values of the ion current through a protein nanopore for different nucleotides do not intersect, the classification accuracy reaches 100%. In the case of increasing the standard deviation of the law of distribution of noise components, there is an overlap of the levels of current values in the nanopore in the case of its blocking by nucleotides of the close size. As a result, errors in the definition of normal and single nucleotide mutations (missense or nonsense) often occur, especially if the levels of current steps in the nanopore for two nucleotides are similar (for example, guanine and thymine, thymine and adenine, adenine and cytosine) and noise masks their contribution to reduction current in the nanopore. Mutations of insertion and deletion of a certain nucleotide sequence are often classified without errors, because these mutations are characterized by a shift of several nucleotides between normal signals and pathology, which increases the distance between these signals. Among the machine learning methods that have demonstrated the high accuracy of classification of the signals of nanopore-based DNA sequencing, the methods of linear discriminant, k-nearest neighbors classifier (with Euclidean distance and the sufficient number of nearest neighbors), as well as the method of reference vectors should be mentioned. The best results were obtained for the classification method of support vector machines. The use of linear, quadratic and cubic kernel functions shows the high accuracy of correctly classified signals - from 93 to 100%.

Rapid profiling of drug-resistant bacteria using DNA-binding dyes and a nanopore-based DNA sequencer

Spread of drug-resistant bacteria is a serious problem worldwide. We thus designed a new sequence-based protocol that can quickly identify bacterial compositions of clinical samples and their drug-resistance profiles simultaneously. Here we utilized propidium monoazide (PMA) that prohibits DNA amplifications from dead bacteria, and subjected the original and antibiotics-treated samples to 16S rRNA metagenome sequencing. We tested our protocol on bacterial mixtures, and observed that sequencing reads derived from drug-resistant bacteria were significantly increased compared with those from drug-sensitive bacteria when samples were treated by antibiotics. Our protocol is scalable and will be useful for quickly profiling drug-resistant bacteria.

A Scalable Discrete-Time Integrated CMOS Readout Array for Nanopore Based DNA Sequencing

This paper introduces a high-speed mixed-signal readout array in 130-nm CMOS for the amplification and digitization of picoampere-range signals. Its design is inspired by the needs of emerging DNA sequencing technologies based on biological nanopore sensors. To overcome switching and substrate noise this system adopts an in-pixel analog-to-digital converter (ADC) architecture and a novel readout technique while consuming 10x less power than similar designs described in the literature. The in-pixel ADC architecture is inherently scalable and immune to electrical interference which can be extended to 100s of channels. With a 5 pF input capacitance, the amplifiers achieve a maximum bandwidth of 100 kHz and demonstrate a noise floor as low as 4 fA/ $\sqrt {\text{Hz}}$ and a gain in the range of $\text{G}\Omega $ at 10 kHz. Circuit noise behaviour and theoretical maximum performance estimates using behavioural models are also discussed.

Preparation of Fragaceatoxin C (FraC) Nanopores.

Biological nanopores are an emerging class of biosensors with high-end precision owing to their reproducible fabrication at the nanometer scale. Most notably, nanopore-based DNA sequencing applications are currently being commercialized, while nanopore-based proteomics may become a reality in the near future.Although membrane proteins often prove to be difficult to purify, we describe a straightforward protocol for the preparation of Fragaceatoxin C (FraC) nanopores, which may have applications for DNA analysis and nanopore-based proteomics. Recombinantly expressed FraC nanopores are purified via two rounds of Ni-NTA affinity chromatography before and after oligomerization on sphingomyelin-containing liposomes. Starting from a plasmid vector containing the FraC gene, our method allows the production of purified nanopores within a week. Afterward, the FraC nanopores can be stored at +4°C for several months, or frozen.

Human short tandem repeat identification using a nanopore-based DNA sequencer: a pilot study.

Short tandem repeats (STRs) are repetitive DNA sequences that are highly polymorphic and widely used for personal identification in the field of forensic medicine. The standard method for determining the repeat number of STRs is capillary electrophoresis of PCR products; however, the use of DNA sequencing has increased because it can identify same-sized alleles with nucleotide substitutions (iso-alleles). In this study, we performed human STR genotyping using a portable nanopore-based DNA sequencer, the MinION, and evaluated its performance. Because the sequence quality obtained by MinION is considerably lower than those obtained with other DNA sequencers, we developed an original scoring scheme for judging the genotypes from MinION reads. Analysis of seven human samples for 21-45 STR loci yielded an average of 857 thousand reads per sample, and the accuracy of genotyping and iso-allele identification reached 75.7% and 82%, respectively. Although the accuracy is higher than that reported previously, further improvements are required before this method can be practically applied.

DNA Nucleobases Sensing by Localized Plasmon Resonances in Graphene Quantum Dots with Nanopore: A First Principle Approach

Taking advantage of a nanopore-based DNA sequencing concept, a variety of recognition approaches have been intensively explored. We have recently presented a potential mechanism for DNA sequencing based on interband π plasmons of graphene nanopores. In this paper, a realistic ab initio analysis of the proposed method based on π and also π+σ plasmons is investigated making use of graphene quantum dots (GQDs) with a nanopore. The plasmonic properties are studied by post processing the density functional theory (DFT) calculations. The first principle study provides an unprecedented fully theoretical description of the proposed structure. The critical features such as passivating atoms, structure relaxation, DNA-graphene interactions, and nucleobase rotations are considered, which result in a more accurate and realistic description of the presented method. Our calculations show a 0.04 to 0.28 eV shift to the energy of the plasmonic modes related to each inserted nucleobase in the nanopore of GQD, which demons...