Retrospective Investigation in Horses with Encephalitis Reveals Unnoticed Circulation of West Nile Virus in Brazil

During these past years, several studies have provided serological evidence regarding the circulation of West Nile virus (WNV) in Brazil. Despite some reports, much is still unknown regarding the genomic diversity and transmission dynamics of this virus in the country. Recently, genomic monitoring activities in horses revealed the circulation of WNV in several Brazilian regions. These findings on the paucity of genomic data reinforce the need for prompt investigation of WNV infection in horses, which may precede human cases of encephalitis in Brazil. Thus, in this study, we retrospectively screened 54 suspicious WNV samples collected between 2017 and 2020 from the spinal cord and brain of horses with encephalitis and generated three new WNV genomes from the Ceará and Bahia states, located in the northeastern region of Brazil. The Bayesian reconstruction revealed that at least two independent introduction events occurred in Brazil. The first introduction event appears to be likely related to the North American outbreak, and was estimated to have occurred in March 2013.The second introduction event appears to have occurred in September 2017 and appears to be likely related to the South American outbreak. Together, our results reinforce the importance of increasing the priority of WNV genomic monitoring in equines with encephalitis in order to track the dispersion of this emerging pathogen through the country.     

Targeted Virome Sequencing Enhances Unbiased Detection and Genome Assembly of Known and Emerging Viruses—The Example of SARS-CoV-2

Targeted virome enrichment and sequencing (VirCapSeq-VERT) utilizes a pool of oligos (baits) to enrich all known—up to 2015—vertebrate-infecting viruses, increasing their detection sensitivity. The hybridisation of the baits to the target sequences can be partial, thus enabling the detection and genomic reconstruction of novel pathogens with <40% genetic diversity compared to the strains used for the baits’ design. In this study, we deploy this method in multiplexed mixes of viral extracts, and we assess its performance in the unbiased detection of DNA and RNA viruses after cDNA synthesis. We further assess its efficiency in depleting various background genomic material. Finally, as a proof-of-concept, we explore the potential usage of the method for the characterization of unknown, emerging human viruses, such as SARS-CoV-2, which may not be included in the baits’ panel. We mixed positive samples of equimolar DNA/RNA viral extracts from SARS-CoV-2, coronavirus OC43, cytomegalovirus, influenza A virus H3N2, parvovirus B19, respiratory syncytial virus, adenovirus C and coxsackievirus A16. Targeted virome enrichment was performed on a dsDNA mix, followed by sequencing on the NextSeq500 (Illumina) and the portable MinION sequencer, to evaluate its usability as a point-of-care (PoC) application. Genome mapping assembly was performed using viral reference sequences. The untargeted libraries contained less than 1% of total reads mapped on most viral genomes, while RNA viruses remained undetected. In the targeted libraries, the percentage of viral-mapped reads were substantially increased, allowing full genome assembly in most cases. Targeted virome sequencing can enrich a broad range of viruses, potentially enabling the discovery of emerging viruses.     

Synthetic heparan sulfate standards and machine learning facilitate the development of solid-state nanopore analysis

The application of solid-state (SS) nanopore devices to single-molecule nucleic acid sequencing has been challenging. Thus, the early successes in applying SS nanopore devices to the more difficult class of biopolymer, glycosaminoglycans (GAGs), have been surprising, motivating us to examine the potential use of an SS nanopore to analyze synthetic heparan sulfate GAG chains of controlled composition and sequence prepared through a promising, recently developed chemoenzymatic route. A minimal representation of the nanopore data, using only signal magnitude and duration, revealed, by eye and image recognition algorithms, clear differences between the signals generated by four synthetic GAGs. By subsequent machine learning, it was possible to determine disaccharide and even monosaccharide composition of these four synthetic GAGs using as few as 500 events, corresponding to a zeptomole of sample. These data suggest that ultrasensitive GAG analysis may be possible using SS nanopore detection and well-characterized molecular training sets.

Glycosaminoglycans (GAGs) are linear anionic polysaccharides found on cell surfaces and in the extracellular matrix in all animals. GAGs comprise an important class of biopolymers that are ubiquitous in nature and exhibit a number of critical functional roles including biological recognition and signaling (1–3). Such processes play critical roles in physiology, such as in development and wound healing, and pathophysiology, such as cancer and infectious disease. Sulfated GAGs result from template-independent synthesis in the Golgi of animal cells (4, 5) and are polydisperse, heteropolysaccharides comprising variable disaccharide repeating units that are classified by these repeating units. Like nucleic acids, sulfated GAGs are made up of repeating units that comprise a linear sequence (Fig. 1). Unlike the nucleic acids, GAGs have far more complicated structures and number of possible sequences and they present severe challenges to both synthesis and characterization. Thus, we undertook to chemoenzymatically synthesize defined GAGs and characterize these using solid-state nanopore analysis.Open in a separate windowFig. 1.Structures of four synthetic GAG samples. Polysaccharide NSH is made up of N-sulfoglucosamine (GlcNS) and glucuronic acid (GlcA),NS2S is made up with GlcNS and 2-O-sulfo-iduronic acid (IdoA2S), NS6S is made up with 6-O-sulfo-N-sulfoheparosan (GlcNS6S) and GlcA, and NS6S2S is made up with GlcNS6S and IdoA2S.Despite their structural complexities, sulfated GAGs often contain well-defined domain structures that are responsible for their diverse biological functions, yet even this level of structural complexity poses a significant general challenge to structural analysis and sequencing. The simple, short-chain, chondroitin sulfate GAG component of bikunin has been sequenced using liquid chromatography–tandem mass spectrometry (LC-MS/MS) (6). While LC-MS/MS is capable of sequencing such simple, short-chain GAGs, it is not yet able to distinguish all of the many isobaric isomers of the variably sulfated saccharide residues and uronic acid epimers commonly encountered in more structurally complex GAGs, such as heparan sulfate (HS) (7). NMR has been applied to determine GAG structures but often requires milligram amounts of samples. HS/heparin is made up of →4)-β-d-glucuronic acid (GlcA) [or α-l-iduronic acid (IdoA)] (1→4)-α-d-glucosamine (GlcN) [1→ repeating units with 2-O-sulfo (S) groups on selected uronic acid residues and 3- and/or 6-O-S and N-S or N-acetyl (Ac) group substitutions on the glucosamine residues] (Fig. 1). GAG structural analysis presents challenges beyond their chemical complexity. There are no amplification methods to detect small numbers of GAG chains, whereas nucleic acid analysis can rely on PCR. Similarly, there are few GAG-specific antibodies or aptamers (8), and no natural GAG chromophores or fluorophores (9), in contrast to the many used for protein sensing. Ultrasensitive (zeptomole) detection methods of modified GAGs, based on fluorescence resonance energy transfer (FRET) (10), DNA bar coding (11), and dye-based nanosensors (12) have been demonstrated, but their application to sequencing is particularly challenging because of the high level of structural complexity of sulfated GAGs.Nanopore single-molecule detection is now routinely applied to DNA (13, 14) and RNA (15–17) biopolymers, and is increasingly applied to protein characterization (18–22). In brief, a nanopore is a nanofluidic channel ∼10 nm long and <100 nm in diameter, serving as the sole fluid connection between two reservoirs of electrolyte separated by an otherwise impermeable membrane (Fig. 2A). On applying a voltage across this nanopore, the passage of supporting electrolyte ions results in a “baseline,” or open-pore current, i₀. The passage of a biopolymer analyte through this nanopore disrupts the flow of supporting electrolyte ions, often as a current blockage. This temporary reduction in ionic current is called an “event,” and its magnitude (mean blockage ratio over the dwell time, ⟨f_b⟩=⟨i⟩_Td/⟨i₀⟩) and its temporal features [dwell time (Td)] (Fig. 2 B and C) depend on the size and shape of the nanopore, the biopolymer analyte, and the applied voltage and interfacial charge distributions. Indeed, the passage of DNA through engineered protein nanopore devices produces current blockages that can be applied in sequencing, and the widespread use of these commercial protein nanopore DNA sequencing devices is increasing (23, 24). Despite this success with protein nanopores, the potential benefits of (abiotic) solid-state (SS) nanopores have continued to drive development efforts. Such a transition to the freely size-tunable SS platform (25, 26), however, is vital for the application of nanopores to the characterization of branched glycans (27). Yet the use of SS nanopores in even the better-established DNA sensing regime remains challenging. The application of nanopore sensing to glycans, while promising, remains profoundly exploratory using nanopores of any kind. The transition to the SS nanopores is accompanied by significant changes in pore geometry, chemistry, characteristics, and potential analyte–pore interactions and sensing modalities, so that there is a critical need for studies in the realm of nanopore glycomics (27, 28). For example, outcomes of early nanopore studies on a structurally simple unsulfated GAG, hyaluronan (HA, →4)- β -GlcA (1 → 3)- β -GlcNAc (1→), while providing some information on HA size does not provide definitive structural information (29, 30). SS nanopore analysis of two sulfated GAGs, heparin and a heparin contaminant, oversulfated chondroitin sulfate, using a silicon nitride SS nanopore was able to qualitatively identify these GAGs by either the magnitude or duration of characteristic current blockages (28). SS nanopore data on GAGs, analyzed using a machine-learning (ML) algorithm (i.e., a support vector machine [SVM]), distinguished heparin and chondroitin sulfate oligosaccharides and unfractionated heparin and low molecular weight heparin with >90% accuracy (31).Open in a separate windowFig. 2.Nanopore characteristics of four samples. (A) Schematic of the nanopore configuration. Anionic GAGs driven by electrophoresis to and through the pore with a negative applied voltage would be detected if they perturbed the open-pore current. (B) A representative current trace and events from polysaccharide NS6S2S test using an ∼6-nm-diameter nanopore. Measurements were collected using a −150-mV applied-voltage (details in Results and Discussion, and Materials and Methods) (C) Scatter plots of dwell time vs. current blockage ratio for four polysaccharides. To remove the bias of event numbers in human image recognition, all plots contain only the first 2,475 events. (D) PCA visualization of the embedded images from the four unique GAGs. The blue circles and region represent NSH, the red X and region represents NS2S, the green triangle and region represents NS6S, and the brown cross and region represents NS6S2S. The algorithm clusters signals from each GAG based on scatter plot images. Each insert shows one 500-events image from each sample class. All 500-events images are in SI Appendix, Fig. S12.Nanopore studies on GAGs, and glycans more broadly, have been severely limited by the lack of a library of structurally defined standards. The uniformity of sulfated GAGs prepared from animal sources is difficult to control and exhibits significant sequence heterogeneity and polydispersity (32). HS is particularly problematic as even for a small HS hexasaccharide, composed of an IdoA/GlcA:GlcNS/GlcNAc sequence with 12 available sites for random sulfation, there are 32,768 possible sequences. Recently, chemoenzymatic synthesis has made inroads in the preparation of high-purity sulfated HS GAGs from heparosan (→4)- β -GlcA (1→4)- β -GlcNAc (1→) (33). HS GAGs of approximately the same chain length and polydispersity and having a single repeating disaccharide unit (SI Appendix, Table S1) including, NSH (→4)- β -GlcA (1→4)- β -GlcNS (1→), NS2S (→4)-α α -IdoA2S (1 → 4)- β -GlcNS (1→), NS6S (→4)- β -GlcA (1→4)- β -GlcNS6S(1→)), NS6S2S (→4)- α -IdoA2S (1→4)- β -GlcNS6S (1→) have been prepared (see Materials and Methods and ref. 34) (Fig. 1). Here we use our recently developed synthetic technique, which has proven difficult to benchmark, in conjunction with a nanopore technique, which has only just begun to be applied to glycomics and has been severely challenged by the lack of available high-quality samples, to develop a fully integrated approach for the nanopore analysis of complex carbohydrates.     

Computer-Aided Design of Boron Nitride-Based Membranes with Armchair and Zigzag Nanopores for Efficient Water Desalination

Recent studies have shown that the use of membranes based on artificial nanoporous materials can be effective for desalination and decontamination of water, separation of ions and gases as well as for solutions to other related problems. Before the expensive stages of synthesis and experimental testing, the search of the optimal dimensions and geometry of nanopores for the water desalination membranes can be done using computer-aided design. In the present study, we propose and examine the assumption that rectangular nanopores with a high aspect ratio would demonstrate excellent properties in terms of water permeation rate and ion rejection. Using the non-equilibrium molecular dynamic simulations, the properties of promising hexagonal boron nitride (h-BN) membranes with rectangular nanopores were predicted. It has been found that not only the nanopore width but also its design (“armchair” or “zigzag”) determines the permeability and ion selectivity of the h-BN-based membrane. The results show that membranes with a zigzag-like design of nanopores of ~6.5 Å width and the armchair-like nanopores of ~7.5 Å width possess better efficiency compared with other considered geometries. Moreover, the estimated efficiency of these membranes is higher than that of any commercial membranes and many other previously studied single-layer model membranes with other designs of the nanopores.     

Xdrop: Targeted sequencing of long DNA molecules from low input samples using droplet sorting

Long‐read sequencing can resolve regions of the genome that are inaccessible to short reads, and therefore are ideal for genome‐gap closure, solving structural rearrangements and sequencing through repetitive elements. Here we introduce the Xdrop technology: a novel microfluidic‐based system that allows for targeted enrichment of long DNA molecules starting from only a few nanograms of DNA. Xdrop is based on the isolation of long DNA fragments in millions of droplets, where the droplets containing a target sequence of interest are fluorescently labeled and sorted using flow cytometry. The final product from the Xdrop procedure is an enriched population of long DNA molecules that can be investigated by sequencing. To demonstrate the capability of Xdrop, we performed enrichment of the human papilloma virus 18 integrated into the genome of human HeLa cells. Analysis of the sequencing reads resolved three HPV18‐chr8 integrations at base‐pair resolution, and the captured fragments extended up to 30 kb into the human genome at the integration sites. Further, we enriched the complete TP53 locus in a leukemia cell line and could successfully phase coexisting mutations using PacBio sequencing. In summary, our results show that Xdrop is an efficient enrichment technology for studying complex genomic regions.     

Genome Profiling of SARS-CoV-2 in Indonesia,ASEAN and the Neighbouring East Asian Countries: Features,Challenges and Achievements

Whole-genome sequencing (WGS) has played a significant role in understanding the epidemiology and biology of SARS-CoV-2 virus. Here, we investigate the use of SARS-CoV-2 WGS in Southeast and East Asian countries as a genomic surveillance during the COVID-19 pandemic. Nottingham–Indonesia Collaboration for Clinical Research and Training (NICCRAT) initiative has facilitated collaboration between the University of Nottingham and a team in the Research Center for Biotechnology, National Research and Innovation Agency (BRIN), to carry out a small number of SARS-CoV-2 WGS in Indonesia using Oxford Nanopore Technology (ONT). Analyses of SARS- CoV-2 genomes deposited on GISAID reveal the importance of clinical and demographic metadata collection and the importance of open access and data sharing. Lineage and phylogenetic analyses of two periods defined by the Delta variant outbreak reveal that: (1) B.1.466.2 variants were the most predominant in Indonesia before the Delta variant outbreak, having a unique spike gene mutation N439K at more than 98% frequency, (2) Delta variants AY.23 sub-lineage took over after June 2021, and (3) the highest rate of virus transmissions between Indonesia and other countries was through interactions with Singapore and Japan, two neighbouring countries with a high degree of access and travels to and from Indonesia.     

Dynamics of intestinal and intratumoral microbiome signatures in genetically engineered mice and human pancreatic ductal adenocarcinoma

BackgroundEmerging evidence has recently revealed a prominent role of the microbiome in pancreatic ductal adenocarcinoma (PDAC). However, while most observations were made in patients, mouse models still require a precise characterization of their disease-related microbiome to employ them for mechanistic and interventional preclinical studies.MethodsTo investigate the fecal and tumoral microbiome of LSL-Kras^G12D/+;LSL-Trp53^R172H/+;Pdx-1-Cre (KPC) and control (CTRL) mice, Oxford Nanopore sequencing was applied. Feces were collected from 10 KPC mice and 10 CTRLs at 3 timepoints (6 weeks, 12 weeks, and when tumor-bearing (KPC) or 6 months (CTRL), respectively). Metagenomic sequencing was performed on feces DNA. KPC tumor and healthy pancreas DNA samples were subjected to 16S rRNA gene sequencing. Bacterial marker components were detected in KPC tumor tissue over time by fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC).ResultsMurine fecal samples showed a significantly different microbiome compared to age-matched healthy CTRLs regarding beta diversity (p = 0.001, R2 = 0.2–0.25 for Bray-Curtis). Adjusted human PDAC classifiers predicted disease status from feces of KPC mice achieving area under the receiver operating characteristic (AUROC) values of 80%. Furthermore, KPC tumors harbored significantly more bacterial components than healthy pancreas. Also the microbial composition differs significantly between KPC tumors and healthy pancreas tissue (p = 0.042 for Bray-Curtis). Microbiota found highly abundant in human PDAC samples were considerably more abundant in KPC tumors as compared to healthy pancreas samples (p-value <0.001).ConclusionKPC fecal samples show similarities with the microbial composition of stool samples from human PDAC patients.     

Fabrication of Solid State Nanopore in Thin Silicon Membrane Using Low Cost Multistep Chemical Etching

Nanopore-based analysis is currently an area of great interest in many disciplines with the potential for exceptionally versatile applications in medicine. This work presents a novel step towards fabrication of a single solid-state nanopore (SSSN) in a thin silicon membrane. Silicon nanopores are realized using multistep processes on both sides of n-type silicon-on-insulator (SOI) <100> wafer with resistivity 1–4 Ω·cm. An electrochemical HF etch with low current density (0.47 mA/cm²) is employed to produce SSSN. Blue LED is considered to emit light in a narrow band region which facilitates the etching procedure in a unilateral direction. This helps in production of straight nanopores in n-type Si. Additionally, a variety of pore diameters are demonstrated using different HF concentrations. Atomic force microscopy is used to demonstrate the surface morphology of the fabricated pores in non-contact mode. Pore edges exhibit a pronounced rounded shape and can offer high stability to fluidic artificial lipid bilayer to study membrane proteins. Electrochemically-fabricated SSSN has excellent smoothness and potential applications in diagnostics and pharmaceutical research on transmembrane proteins and label free detection.