Nanopore DNA sequencing

The nanopore sequencing process is based on the transit of a DNA molecule through a nanoscopic pore, and since the 90s is considered as one of the most promising approaches to detect polymeric molecules. In 2014, Oxford Nanopore Technologies (ONT) launched a beta-testing program that supplied the scientific community with the first prototype of a nanopore se-quencer: the MinION. Thanks to this program, several research groups had the opportunity to evaluate the performance of this novel instrument and develop novel computational approaches for analyzing this new generation of data. Despite the short period of time from the release of the MinION, a large number of algorithms and tools have been developed for base calling, data handling, read mapping, de novo assembly and variant discovery. Here, we face the main computational challenges related to the analysis of nanopore data, and we carry out a comprehensive and up-to-date survey of the algorithmic solutions adopted by the bioinformatic community comparing performance and reporting limits and advantages of using this new generation of sequences for genomic analyses. Our analyses demonstrate that the use of nanopore data dramatically improves the de novo assembly of genomes and allows for the exploration of structural variants with an unprecedented accuracy and resolution. However, despite the impressive improvements reached by ONT in the past 2 years, the use of these data for small-variant calling is still challenging, and at present, it needs to be coupled with complementary short sequences for mitigating the intrinsic biases of nanopore sequencing technology.

This review revealed a new mechanism for gene regulation through
“gene silencing” at the transcriptional level (TGS) or at the post -transcriptional level (PTGS), which play a key role in many essential
cellular processes. Today dsRNA is used as a powerful tool to
experimentally elucidate the function of essentially any gene in a
cell. The immense impact of the discovery of RNA interference
(RNAi) on biomedical research and its novel medical applications in
the future are reviewed in this article, with particular stress on
therapeutic applications of radio -labeled antisense oligonucleotides
(RASONs) for diagnosis and treatment of various  cancers and
neurodegenerative diseases by “gene silencing”. Antisense
oligonucleotides (ASONs) can also modulate alternative splicing
which 74% of all human genes undergo.  Epigenetic changes affect
chromatin structure and thus regulate processes such as transcription,
X-chromosome inactivation, allele-specific expression of imprinted
genes, and inactivation of tumor suppressor genes. Treatment with
inhibitors of DNA methylation and histone deacetylation can
reactivate epigenetically silenced genes and has been shown to
restore normal gene function. In cancer cells, this results in
expression of tumor suppressor genes and other regulatory functions,
inducing growth arrest and apoptosis.

Solid-state nanopore-based sensors are promising platforms for next-generation sequencing technologies, featuring label-free single-molecule sensitivity, rapid detection, and low-cost manufacturing. In recent years, solid-state nanopores have been explored due to their miscellaneous fabrication methods and their use in a wide range of sensing applications. Here, we highlight a novel family of solid-state nanopores which have recently appeared, namely plasmonic nanopores. The use of plasmonic nanopores to engineer electromagnetic fields around a nanopore sensor allows for enhanced optical spectroscopies, local control over temperature, thermophoresis of molecules and ions to/from the sensor, and trapping of entities. This Mini Review offers a comprehensive understanding of the current state-of-the-art plasmonic nano-pores for single-molecule detection and biomolecular sequencing applications and discusses the latest advances and future perspectives on plasmonic nanopore-based technologies. S urface plasmon resonance (SPR) refers to the collective oscillations of the conduction electrons in metallic nanostructures. 1 Both the intensity and the energy of the SPR phenomenon strongly depend on the size, shape, and composition of the nanostructures, as well as on the dielectric properties of the surrounding environment. Various factors affect the plasmonic nanostructure response, which allow for a rationally guided design of plasmonic sensors with tunable sensitivity. These nanoscale detectors have found applications in several fields such as light harvesting, photocatalysis, subwavelength imaging, metamaterials, and nanomedicine. 2 Among others, one specific family of plasmonic platforms is based on plasmonic nanohole arrays and the more recently implemented plasmonic nanopores, sub-100 nm apertures connecting two compartments, for sensing applications. Although the reader can find exhaustive details on the principles, fabrication, and applications of plasmonic nanoholes and nanopores for biosensing in recent reviews by Dahlin 3 and by Spitzberg and co-workers, 4 here we focus our attention on the potential applications and integration of plasmonic nanopores as transducers in single-molecule detection, manipulation, and sequencing. During the last few decades a new generation of single-molecule information reader technologies have emerged, the most advanced example being single-molecule sequencing (SMS). 5 SMS involves threading single nucleic acid strands through elements (reader molecules, enzymes, nanopores, and other nanostructures) that can be used for detecting specific signals (e.g., electrical, optical, or mechanical). Individual base pairs, the monomer units of DNA/RNA polymers, are then detected sequentially one at a time as they interact with these elements. In particular, in the past several years SMS platforms, such as those by Pacific Biosciences and Oxford Nanopore Technologies, have become available to researchers and are currently being utilized in research and clinical settings. SMS nanotechnologies can be divided into two main categories: (i) Sequencing-by-synthesis in which a DNA polymerase repli-cates a DNA molecule and the added sequence is read out, either in real time 6 or by sequential addition of bases, 7 and (ii) nanopore-sequencing technologies in which single molecules of DNA (or RNA) are threaded through a nanopore or positioned in the vicinity of a nanopore, and k-mers of DNA bases (k = 4−5) are detected as they are translocated through the pore in single base steps. 8,9 However, nucleic acid sequencing goes beyond the four DNA/RNA bases, as various chemical modifications and damaged DNA sites produce additional signals that require orthogonal readout modes for correct modified base assignment. Therefore, at the core of nanopore sequencing technologies lies a challenge of producing solid-state nanopores with a quality that rivals their much more reliable biological pore counterparts. In particular, ion channels and bacterial toxin channels, for

Assessment of bacterial diversity through sequencing of 16S ribosomal RNA (16S rRNA) genes has been an approach widely used in environmental microbiology, particularly since the advent of highthroughput sequencing technologies. An additional innovation introduced by these technologies was the need of developing new strategies to manage and investigate the massive amount of sequencing data generated. This situation stimulated the rapid expansion of the field of bioinformatics with the release of new tools to be applied to the downstream analysis and interpretation of sequencing data mainly generated using Illumina technology. In recent years, a third generation of sequencing technologies has been developed and have been applied in parallel and complementarily to the former sequencing strategies. In particular, Oxford Nanopore Technologies (ONT) introduced nanopore sequencing which has become very popular among molecular ecologists. Nanopore technology offers a low price, portability and fast sequencing throughput. This powerful technology has been recently tested for 16S rRNA analyses showing promising results. However, compared with previous technologies, there is a scarcity of bioinformatic tools and protocols designed specifically for the analysis of Nanopore 16S sequences. Due its notable characteristics, researchers have recently started performing assessments regarding the suitability MinION on 16S rRNA sequencing studies, and have obtained remarkable results. Here we present a review of the state-of-the-art of MinION technology applied to microbiome studies, the current possible application and main challenges for its use on 16S rRNA metabarcoding.

Background: To demonstrate the bioinformatics capabilities of a low-cost computer, the Raspberry Pi, we present a comparison of the protein-coding gene content of two species in phylum Chlamydiae: Chlamydia trachomatis, a common sexually transmitted infection of humans, and Candidatus Protochlamydia amoebophila, a recently discovered amoebal endosymbiont. Identifying species-specific proteins and differences in protein families could provide insights into the unique phenotypes of the two species.

The nanopore sequencing process is based on the transit of a DNA molecule through a nanoscopic pore, and since the 90s is considered as one of the most promising approaches to detect polymeric molecules. In 2014, Oxford Nanopore Technologies (ONT) launched a beta-testing program that supplied the scientific community with the first prototype of a nanopore sequencer: the MinION. Thanks to this program, several research groups had the opportunity to evaluate the performance of this novel instrument and develop novel computational approaches for analyzing this new generation of data. Despite the short period of time from the release of the MinION, a large number of algorithms and tools have been developed for base calling, data handling, read mapping, de novo assembly and variant discovery. Here, we face the main computational challenges related to the analysis of nanopore data, and we carry out a comprehensive and up-to-date survey of the algorithmic solutions adopted by the bioinform...

The western honeybee, Apis mellifera is a prominent model organism in the field of socioge-nomics and a recent upgrade substantially improved annotations of the reference genome. Nevertheless, genome assemblies based on short-sequencing reads suffer from problems in regions comprising e.g. multi-copy genes. We used single-molecule nanopore-based sequencing with extensive read-lengths to reconstruct the organization of the major royal jelly protein (mrjp) region in three species of the genus Apis. Long-amplicon sequencing provides evidence for lineage-specific evolutionary fates of Apis mrjps. Whereas the most basal species, A. florea, seems to encode ten mrjps, different patterns of gene loss and retention were observed for A. mellifera and A. dorsata. Furthermore, we show that a previously reported pseudogene in A. mellifera, mrjp2-like, is an assembly artefact arising from short read sequencing.

Background Macaque species share >93% genome homology with humans and develop many disease phenotypes similar to those of humans, making them valuable animal models for the study of human diseases (e.g., HIV and neurodegenerative diseases). However, the quality of genome assembly and annotation for several macaque species lags behind the human genome effort. Results To close this gap and enhance functional genomics approaches, we used a combination of de novo linked-read assembly and scaffolding using proximity ligation assay (HiC) to assemble the pig-tailed macaque (Macaca nemestrina) genome. This combinatorial method yielded large scaffolds at chromosome level with a scaffold N50 of 127.5 Mb; the 23 largest scaffolds covered 90% of the entire genome. This assembly revealed large-scale rearrangements between pig-tailed macaque chromosomes 7, 12, and 13 and human chromosomes 2, 14, and 15. We subsequently annotated the genome using transcriptome and proteomics data from personali...

Next-generation sequencing technologies are being rapidly adopted as a tool of choice for diagnostic and outbreak investigation in public health laboratories. However, costs of operation and the need for specialized staff remain major hurdles for laboratories with limited resources for implementing these technologies. This project aimed to assess the feasibility of using Oxford Nanopore MinION whole-genome sequencing data of Mycobacterium tuberculosis isolates for species identification, in silico spoligotyping, detection of mutations associated with antimicrobial resistance (AMR) to accurately predict drug susceptibility profiles, and phylogenetic analysis to detect transmission between cases.

Biological ion channels can realize delicate mass transport under complicated physiological conditions. Artificial nanochannels can achieve biomimetic ion current rectification (ICR), gating, and selectivity that are mostly performed in pure salt solutions. Synthetic nanochannels that can function under mixed ion systems are highly desirable, yet their performances are hard to be compared to those under pure systems. Seeking out the potential reasons by investigating the effect of mixed-system components on the ion-transport properties of the constructed nanochannels seems necessary and important. Herein, we report the effect of anions with different charges and sizes on the ICR properties of positively charged nanochannels. Among the investigated anions, the low-valent anions showed no impact on the ICR direction, while the high-valent component ferrocyanide [Fe(CN) 6 4− ] caused significant ICR inversion. The ICR inversion mechanism is evidenced to result from the adsorption of Fe(CN) 6 4−-induced surface charge reversal, which relates to solution concentration, pH conditions, and nanochannel sizes and applies to both aminated and quaternized nanochannels that are positively charged. Noticeably, Fe(CN) 6 4− is found to interfere with the transport of protein molecules in the nanochannel. This work points out that the ion species from mixed systems would potentially impact the intrinsic ICR properties of the nanochannels. Replacing highly charged counterions with organic components would be promising in building up future nanochannel-based mass transport systems running under mixed systems.

Surface-enhanced Raman spectroscopy (SERS) sensing of DNA bases by plasmonic nanopores could pave a way to novel methods for DNA analyses and new generation single-molecule sequencing platforms. The SERS discrimination of single DNA bases depends critically on the time that a DNA strand resides within the plasmonic hot spot. In fact, DNA molecules flow through the nanopores so rapidly that the SERS signals collected are not sufficient for single-molecule analysis. Here, we report an approach to control the residence time of molecules in the hot spot by an electro-plasmonic trapping effect. By directly adsorbing molecules onto a gold nanoparticle and then trapping the single nanoparticle in a plasmonic nanohole up to several minutes, we demonstrate single-molecule SERS detection of all four DNA bases as well as discrimination of single nucleobases in a single oligonu-cleotide. Our method can be extended easily to label-free sensing of single-molecule amino acids and proteins.

Short-read, high throughput sequencing technology cannot identify the chromosomal position of repetitive insertion sequences that typically flank horizontally acquired genes such as bacterial virulence genes and antibiotic resistance genes. The MinION™ nanopore sequencer can produce long sequencing reads on a device

We report the fabrication of devices in which one single-walled carbon nanotube (SWCNT) spans a barrier between two fluid reservoirs, enabling direct electrical measurement of ion transport through the tube. A fraction of the tubes pass anomalously high ionic currents. Electrophoretic transport of small single stranded DNA oligomers through these tubes is marked by large transient increases in ion current and was confirmed by PCR analysis. Each current pulse contains about 10 7 charges, an enormous amplification of the translocated charge. Carbon nanotubes simplify the construction of nanopores, permit new types of electrical measurements, and may open avenues for control of DNA translocation.

The western honeybee, Apis mellifera is a prominent model organism in the field of sociogenomics and a recent upgrade substantially improved annotations of the reference genome. Nevertheless, genome assemblies based on short-sequencing reads suffer from problems in regions comprising e.g. multi-copy genes. We used single-molecule nanopore-based sequencing with extensive read-lengths to reconstruct the organization of the major royal jelly protein (mrjp) region in three species of the genus Apis. Long-amplicon sequencing provides evidence for lineage-specific evolutionary fates of Apis mrjps. Whereas the most basal species, A. florea, seems to encode ten mrjps, different patterns of gene loss and retention were observed for A. mellifera and A. dorsata. Furthermore, we show that a previously reported pseudogene in A. mellifera, mrjp2-like, is an assembly artefact arising from short read sequencing.

Nanopore resistive pulse techniques are based on analysis of current or voltage spikes in the recorded signal. These spikes result from translocation of nanometer sized analytes through a nanopore. The most important information that needs to be extracted is the duration, amplitude and number of the translocation spikes. The recorded signal is usually considerably noisy, with a huge baseline drift and hundreds of translocation spikes. Thus, incorporation of suitable signal processing algorithms is necessary for correct and fast detection of all the translocation spikes and to accurately measure their amplitude and duration. Generally, low-pass filtering is used for denoising, averaging is used for baseline detection, and thresholding is used for spike detection and measurement. Here we present novel algorithms and specifically developed software for nanopore signal processing that are significantly improving the accuracy of the nanopore measurements. It includes an improved method f...

This technique enables the scalable production of independently addressable nanopores. By confining the electric field within the microfluidic architecture, nanopore fabrication is precisely localized and electrical noise is significantly reduced. Both DNA and protein molecules are detected to validate the performance of this sensing platform.

Biomarker-binding nucleotide sequences, like aptamers, have gained recent attention in cancer cell isolation and detection works. Self-assembly and 3D conformation of aptamers enable them to selectively capture and bind diseased cells and related biomarkers. One mode of utilizing such an extraordinary selective property of the aptamers is by grafting these in nanopores. Coating the inside walls of the nanopore with biomarker specific ligands, like DNA, changes the statistics of the dynamic translocation events. When the target protein passes through the nanopore, it interacts with ligand coated inside the nanopore, and the process alters the overall potential energy profile which is essentially specific to the protein detected. The fundamental goal in this process is to ensure that these detection motifs hold their structure and functionality under applied electric field and experimental conditions. We report here all-atom molecular dynamics simulations of the effects of external electric field on the 3D conformation of such DNA structures. The simulations demonstrate how the grafted moieties affect the translocation time, velocity, and detection frequency of the target molecule. We also investigated a novel case of protein translocation, where DNA is prebound to the protein. As model, a thrombin-specific Gquartet and thrombin pair was used for this study.