Identification of non-model mammal species using the MinION DNA sequencer from Oxford Nanopore.

Background: The Neotropics harbors the largest species richness of the planet; however, even in well-studied groups, there are potentially hundreds of species that lack a formal description, and likewise, many already described taxa are difficult to identify using morphology. Specifically in small mammals, complex morphological diagnoses have been facilitated by the use of molecular data, particularly from mitochondrial sequences, to obtain accurate species identifications. Obtaining mitochondrial markers implies the use of PCR and specific primers, which are largely absent for non-model organisms. Oxford Nanopore Technologies (ONT) is a new alternative for sequencing the entire mitochondrial genome without the need for specific primers. Only a limited number of studies have employed exclusively ONT long-reads to assemble mitochondrial genomes, and few studies have yet evaluated the usefulness of such reads in multiple non-model organisms. Methods: We implemented fieldwork to collect small mammals, including rodents, bats, and marsupials, in five localities in the northern extreme of the Cordillera Central of Colombia. DNA samples were sequenced using the MinION device and Flongle flow cells. Shotgun-sequenced data was used to reconstruct the mitochondrial genome of all the samples. In parallel, using a customized computational pipeline, species-level identifications were obtained based on sequencing raw reads (Whole Genome Sequencing). ONT-based identifications were corroborated using traditional morphological characters and phylogenetic analyses. Results: A total of 24 individuals from 18 species were collected, morphologically identified, and deposited in the biological collection of Universidad EAFIT. Our different computational pipelines were able to reconstruct mitochondrial genomes from exclusively ONT reads. We obtained three new mitochondrial genomes and eight new molecular mitochondrial sequences for six species. Our species identification pipeline was able to obtain accurate species identifications for up to 75% of the individuals in as little as 5 s. Finally, our phylogenetic analyses corroborated the identifications from our automated species identification pipeline and revealed important contributions to the knowledge of the diversity of Neotropical small mammals. Discussion: This study was able to evaluate different pipelines to reconstruct mitochondrial genomes from non-model organisms, using exclusively ONT reads, benchmarking these protocols on a multi-species dataset. The proposed methodology can be applied by non-expert taxonomists and has the potential to be implemented in real-time, without the need to euthanize the organisms and under field conditions. Therefore, it stands as a relevant tool to help increase the available data for non-model organisms, and the rate at which researchers can characterize life specially in highly biodiverse places as the Neotropics.

Exploring Microorganisms Associated to Acute Febrile Illness and Severe Neurological Disorders of Unknown Origin: A Nanopore Metagenomics Approach.

Acute febrile illness (AFI) and severe neurological disorders (SNDs) often present diagnostic challenges due to their potential origins from a wide range of infectious agents. Nanopore metagenomics is emerging as a powerful tool for identifying the microorganisms potentially responsible for these undiagnosed clinical cases. In this study, we aim to shed light on the etiological agents underlying AFI and SND cases that conventional diagnostic methods have not been able to fully elucidate. Our approach involved analyzing samples from fourteen hospitalized patients using a comprehensive nanopore metagenomic approach. This process included RNA extraction and enrichment using the SMART-9N protocol, followed by nanopore sequencing. Subsequent steps involved quality control, host DNA/cDNA removal, de novo genome assembly, and taxonomic classification. Our findings in AFI cases revealed a spectrum of disease-associated microbes, including Escherichia coli, Streptococcus sp., Human Immunodeficiency Virus 1 (Subtype B), and Human Pegivirus. Similarly, SND cases revealed the presence of pathogens such as Escherichia coli, Clostridium sp., and Dengue virus type 2 (Genotype-II lineage). This study employed a metagenomic analysis method, demonstrating its efficiency and adaptability in pathogen identification. Our investigation successfully identified pathogens likely associated with AFI and SNDs, underscoring the feasibility of retrieving near-complete genomes from RNA viruses. These findings offer promising prospects for advancing our understanding and control of infectious diseases, by facilitating detailed genomic analysis which is critical for developing targeted interventions and therapeutic strategies.

Orientational Pathways during Protein Translocation through Polymer-Modified Nanopores.

Protein translocation through nanopores holds significant promise for applications in biotechnology, biomolecular analysis, and medicine. However, the interpretation of signals generated by the translocation of the protein remains challenging. In this way, it is crucial to gain a comprehensive understanding on how macromolecules translocate through a nanopore and to identify what are the critical parameters that govern the process. In this study, we investigate the interplay between protein charge regulation, orientation, and nanopore surface modifications using a theoretical framework that allows us to explicitly take into account the acid-base reactions of the titrable amino acids in the proteins and in the polyelectrolytes grafted to the nanopore surface. Our goal is to thoroughly characterize the translocation process of different proteins (GFP, ß-lactoglobulin, lysozyme, and RNase) through nanopores modified with weak polyacids. Our calculations show that the charge regulation mechanism exerts a profound effect on the translocation process. The pH-dependent interactions between proteins and charged polymers within the nanopore lead to diverse free energy landscapes with barriers, wells, and flat regions dictating translocation efficiency. Comparison of different proteins allows us to identify the significance of protein isoelectric point, size, and morphology in the translocation behavior. Taking advantage of these insights, we propose pH-responsive nanopores that can load proteins at one pH and release them at another, offering opportunities for controlled protein delivery, separation, and sensing applications.

Electrochemical microfluidic immunosensor with graphene-decorated gold nanoporous for T-2 mycotoxin detection.

T-2 is one of the most potent cytotoxic food-borne mycotoxins. In this work, we have developed and characterized an electrochemical microfluidic immunosensor for T-2 toxin quantification in wheat germ samples. T-2 toxin detection was carried out using a competitive immunoassay method based on monoclonal anti-T-2 antibodies immobilized on the poly(methyl methacrylate) (PMMA) microfluidic central channel. The platinum wire working electrode at the end of the channel was in situ modified by a single-step electrodeposition procedure with reduced graphene oxide (rGO)-nanoporous gold (NPG). T-2 toxin in the sample was allowed to compete with T-2-horseradish peroxidase (HRP) conjugated for the specific recognizing sites of immobilized anti-T-2 monoclonal antibodies. The HRP, in the presence of hydrogen peroxide (H2O2), catalyzes the oxidation of 4-tert-butylcatechol (4-TBC), whose back electrochemical reduction was detected on the nanostructured electrode at -0.15 V. Thus, at low T-2 concentrations in the sample, more enzymatically conjugated T-2 will bind to the capture antibodies, and, therefore, a higher current is expected. The detection limits found for electrochemical immunosensor, and commercial ELISA procedure were 0.10 µg kg-1 and 10 µg kg-1, and the intra- and inter-assay coefficients of variation were below 5.35% and 6.87%, respectively. Finally, our microfluidic immunosensor to T-2 toxin will significantly contribute to faster, direct, and secure in situ analysis in agricultural samples.

First whole-genome sequence and assembly of the Ecuadorian brown-headed spider monkey (Ateles fusciceps fusciceps), a critically endangered species, using Oxford Nanopore Technologies.

The Ecuadorian brown-headed spider monkey (Ateles fusciceps fusciceps) is currently considered one of the most endangered primates in the world and is classified as critically endangered [International union for conservation of nature (IUCN)]. It faces multiple threats, the most significant one being habitat loss due to deforestation in western Ecuador. Genomic tools are keys for the management of endangered species, but this requires a reference genome, which until now was unavailable for A. f. fusciceps. The present study reports the first whole-genome sequence and assembly of A. f. fusciceps generated using Oxford Nanopore long reads. DNA was extracted from a subadult male, and libraries were prepared for sequencing following the Ligation Sequencing Kit SQK-LSK112 workflow. Sequencing was performed using a MinION Mk1C sequencer. The sequencing reads were processed to generate a genome assembly. Two different assemblers were used to obtain draft genomes using raw reads, of which the Flye assembly was found to be superior. The final assembly has a total length of 2.63 Gb and contains 3,861 contigs, with an N50 of 7,560,531 bp. The assembly was analyzed for annotation completeness based on primate ortholog prediction using a high-resolution database, and was found to be 84.3% complete, with a low number of duplicated genes indicating a precise assembly. The annotation of the assembly predicted 31,417 protein-coding genes, comparable with other mammal assemblies. A reference genome for this critically endangered species will allow researchers to gain insight into the genetics of its populations and thus aid conservation and management efforts of this vulnerable species.

Detection of Biomarkers through Functionalized Polymers.

Over the past decade, there has been a rising interest in utilizing functionalized porous polymers for sensor applications. By incorporating functional groups into nanostructured materials like hydrogels, nanosheets, and nanopores, exciting new opportunities have emerged for biomarker detection. The ability of functionalized polymers to undergo physical changes and deformations makes them perfect for modulating optical signals. This chemical mechanism enables the creation of biocompatible sensors for in situ biomarker measurement. Here a comprehensive overview of the current publication trends is provided in functionalized polymers, encompassing functional groups that can induce measurable physical deformations. It explores various materials categorized based on their detection targets, which include proteins, carbohydrates, ions, and deoxyribonucleic acid. As such, this work serves as a valuable reference for the development of functionalized polymer-based sensors.

Blastocystis genetic diversity in animal and human samples from different departments of Colombia using complete sequencing of the 18S rRNA gene (SSU rRNA) by Oxford Nanopore Technologies (ONT).

Blastocystis is an intestinal microeukaryote that has raised attention due to its wide distribution in animals and humans. The risk of zoonotic circulation primarily arises from close contact with infected animals. Therefore, the following study aimed to evaluate the diversity and frequency of Blastocystis subtypes in Colombian human and animal samples using complete sequencing of the 18S rRNA gene. For this purpose, 341 human stool samples and 277 animal fecal samples (from cattle, sheep, goat, pigs, cats, and dogs), were collected from different Colombian regions and analyzed using PCR-based detection and full-length 18S SSU rRNA gene Next-Generation Sequencing (NGS). Among the 618 samples from both hosts, humans and animals, the results revealed widespread Blastocystis frequency, with 48.09% (n = 164) in humans and 31.4% (n = 87) detection in animals. Dogs, cats, sheep, pigs, and wild animals tested positive, aligning with global prevalence patterns. Also, 29 human samples and 23 animal samples were sequenced using ONT technology from which 11 long-read unique sequences were generated and cluster with their compared reference sequences. The subtype distribution varied within hosts, detecting ST1 and ST3 in both human and animal samples. Subtypes ST5, ST10, ST14, ST15, ST21, ST24, ST25 and ST26 were limited to animals hosts, some of which are considered to have zoonotic potential. On the other hand, ST2 was found exclusively in human samples from Bolivar region. Mixed infections occurred in both animal and humans, 60.86% and 27.58% respectively. Moreover, to our knowledge, this is the first study in Colombia identifying ST15 in pigs and ST25 in sheep. The subtypes (STs) identified in this study indicate that certain animals may serve as reservoirs with the potential for zoonotic transmission. The identification of zoonotic subtypes highlights the use of Next Generation Sequencing as the depth and resolution of the sequences increases providing insights into STs of medical and veterinarian significance. It also reveals the coexistence of diverse subtypes among hosts. Further research is essential for understanding transmission dynamics, health implications, and detection strategies for Blastocystis occurrence in animals and humans, mainly associated to the role of animals as reservoirs and their close interaction with humans.

Capacidad de procesamiento de los nanofiltros empleados en la purificación de la proteína recombinante del dominio de unión al receptor del coronavirus tipo 2 del síndrome respiratorio agudo severo / Processability of nanofilters used in the purification of the recombinant receptor-binding domain protein of severe acute respiratory syndrome type 2 coronavirus

En el Centro de Inmunología Molecular se producen el ingrediente farmacéutico activo y la materia prima biológica empleados para la formulación de las vacunas SOBERANAS®. El antígeno de estas vacunas es la proteína del dominio de unión al receptor del coronavirus tipo 2 del síndrome respiratorio agudo severo. La producción de esta proteína recombinante se basa en el cultivo de células de ovario de hámster chino en biorreactores tipo tanque agitado. El proceso tecnológico a escala industrial consta de varias etapas: preparación de medios de cultivo y soluciones, fermentación, clarificación de sobrenadante y purificación. En los procesos biotecnológicos derivados de líneas celulares de origen animal, la contaminación viral endógena o adventicia constituye un riesgo potencial. Por tal motivo, en el proceso de purificación se emplea un paso específico para la remoción viral mediante la nanofiltración. Los nanofiltros empleados son materiales desechables que influyen significativamente en el costo del proceso. Actualmente se desconoce la capacidad de procesamiento de los nanofiltros en el proceso de purificación en cuestión, siendo el objetivo de la presente investigación con vistas a reducir los costos de producción. Se determinó la capacidad de procesamiento de los filtros Virosart CPV, siendo de 239,74 g/m2 (71,67 por ciento de saturación) y 1.259 g/m2 (67,82 por ciento de saturación) para la especie dímero y la mezcla, respectivamente. Se determinó la disminución del costo de producción de la etapa de nanofiltración, que representa una disminución del 54,85 por ciento del costo de filtración de la especie dímero y un 25 por ciento de la mezcla(AU)

The active pharmaceutical ingredient and the biological raw material, used for the formulation of the SOBERANA® vaccines, are produced at the Molecular Immunology Center. The antigen of these vaccines is the receptor-binding domain protein of the severe acute respiratory syndrome type 2 coronavirus. The production of this recombinant protein is based on the culture of Chinese hamster ovary cells in stirred tank bioreactors. The technological process on an industrial scale consists of several stages: preparation of culture media and solutions, fermentation, clarification of supernatant and purification. In biotechnological processes derived from cell lines of animal origin, endogenous or adventitious viral contamination is a potential risk. For this reason, a specific step for viral removal by nanofiltration is used in the purification process. The nanofilters used are disposable materials that significantly influence the cost of the process. The processing capacity of the nanofilters in the purification process in question is currently unknown, being the objective of the present investigation with a view to reducing production costs. The processing capacity of the Virosart CPV filters was determined to be 239.74 g/m2 (71.67percent saturation) and 1,259 g/m2 (67.82percent saturation) for the dimer species and the mixture, respectively. The decrease in the production cost of the nanofiltration stage was determined, representing a 54.85percent decrease in the filtration cost for the dimer species and a 25percent decrease for the mixture(AU)

Experimental and Theoretical Estimations of Atrazine's Adsorption in Mangosteen-Peel-Derived Nanoporous Carbons.

Nanoporous carbons were prepared via chemical and physical activation from mangosteen-peel-derived chars. The removal of atrazine was studied due to the bifunctionality of the N groups. Pseudo-first-order, pseudo-second-order, and intraparticle pore diffusion kinetic models were analyzed. Adsorption isotherms were also analyzed according to the Langmuir and Freundlich models. The obtained results were compared against two commercially activated carbons with comparable surface chemistry and porosimetry. The highest uptake was found for carbons with higher content of basic surface groups. The role of the oxygen-containing groups in the removal of atrazine was estimated experimentally using the surface density. The results were compared with the adsorption energy of atrazine theoretically estimated on pristine and functionalized graphene with different oxygen groups using periodic DFT methods. The energy of adsorption followed the same trend observed experimentally, namely the more basic the pH, the more favored the adsorption of atrazine. Micropores played an important role in the uptake of atrazine at low concentrations, but the presence of mesoporous was also required to inhibit the pore mass diffusion limitations. The present work contributes to the understanding of the interactions between triazine-based pollutants and the surface functional groups on nanoporous carbons in the liquid-solid interface.

Employing oxford nanopore technologies (ONT) for understanding the ecology and transmission dynamics of flaviviruses in mosquitoes (Diptera: Culicidae) from Eastern Colombia.

Studies focused on identifying the viral species of Flavivirus in vectors are scarce in Latin America and particularly in Colombia. Therefore, the frequency of infection of the Flavivirus genus and its feeding preferences were identified in the mosquito species circulating in the municipality of Puerto Carreño-Vichada, located in the Eastern Plains of Colombia. This was done by sequencing the viral NS5 and vertebrate 12S rRNA genes, respectively, using Oxford Nanopore Technologies (ONT). A total of 1,159 mosquitoes were captured, with the most abundant species being Aedes serratus at 73.6% (n = 853). All the mosquitoes were processed in 230 pools (2-6 individuals) and 51 individuals, where 37.01% (n = 104) were found to be infected with Flavivirus. In these samples, infection by arboviruses of epidemiological importance, such as dengue virus (DENV), Zika virus (ZIKV), and chikungunya virus (CHIKV), was ruled out by PCR. However, through sequencing, infection by different insect-specific viruses (ISFVs) and a medically important virus, West Nile virus (WNV), were identified in a mosquito of the Culex browni species. Additionally, the feeding patterns showed that most species present a generalist behavior. Given the above, conducting entomovirological surveillance studies is crucial, especially in areas of low anthropogenic intervention, due to the high probability that potentially pathogenic viruses could generate spillover events under deforestation scenarios.

Fast and cost-effective SARS-CoV-2 variant detection using Oxford Nanopore full-length spike gene sequencing.

Most biologically relevant and diagnostic mutations in the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) genome have been identified in the S gene through global genomic surveillance efforts. However, large-scale whole-genome sequencing (WGS) is still challenging in developing countries due to higher costs, reagent delays and limited infrastructure. Consequently, only a small fraction of SARS-CoV-2 samples are characterized through WGS in these regions. Here, we present a complete workflow consisting of a fast library preparation protocol based on tiled amplification of the S gene, followed by a PCR barcoding step and sequencing using Nanopore platforms. This protocol facilitates fast and cost-effective identification of main variants of concern and mutational surveillance of the S gene. By applying this protocol, report time and overall costs for SARS-CoV-2 variant detection could be reduced, contributing to improved genomic surveillance programmes, particularly in low-income regions.

Alphatoxin Nanopore Detection of Aflatoxin, Ochratoxin and Fumonisin in Aqueous Solution.

Mycotoxins are toxic and carcinogenic metabolites produced by groups of filamentous fungi that colonize food crops. Aflatoxin B1 (AFB1), ochratoxin A (OTA) and fumonisin B1 (FB1) are among the most relevant agricultural mycotoxins, as they can induce various toxic processes in humans and animals. To detect AFB1, OTA and FB1 in the most varied matrices, chromatographic and immunological methods are primarily used; however, these techniques are time-consuming and expensive. In this study, we demonstrate that unitary alphatoxin nanopore can be used to detect and differentiate these mycotoxins in aqueous solution. The presence of AFB1, OTA or FB1 inside the nanopore induces reversible blockage of the ionic current flowing through the nanopore, with distinct characteristics of blockage that are unique to each of the three toxins. The process of discrimination is based on the residual current ratio calculation and analysis of the residence time of each mycotoxin inside the unitary nanopore. Using a single alphatoxin nanopore, the mycotoxins could be detected at the nanomolar level, indicating that alphatoxin nanopore is a promising molecular tool for discriminatory analysis of mycotoxins in aqueous solution.

Microfluidic Platform Integrated with Carbon Nanofibers-Decorated Gold Nanoporous Sensing Device for Serum PSA Quantification.

Prostate cancer is a disease with a high incidence and mortality rate in men worldwide. Serum prostate-specific antigens (PSA) are the main circulating biomarker for this disease in clinical practices. In this work, we present a portable and reusable microfluidic device for PSA quantification. This device comprises a polymethyl methacrylate microfluidic platform coupled with electrochemical detection. The platinum working microelectrode was positioned in the outflow region of the microchannel and was modified with carbon nanofibers (CNF)-decorated gold nanoporous (GNP) structures by the dynamic hydrogen bubble template method, through the simultaneous electrodeposition of metal precursors in the presence of CNF. CNF/GNP structures exhibit attractive properties, such as a large surface to volume ratio, which increases the antibody's immobilization capacity and the electroactive area. CNFs/GNP structures were characterized by scanning electron microscopy, energy dispersive spectrometry, and cyclic voltammetry. Anti-PSA antibodies and HRP were employed for the immune-electrochemical reaction. The detection limit for the device was 5 pg mL-1, with a linear range from 0.01 to 50 ng mL-1. The coefficients of variation within and between assays were lower than 4.40%, and 6.15%, respectively. Additionally, its clinical performance was tested in serum from 30 prostate cancer patients. This novel device was a sensitive, selective, portable, and reusable tool for the serological diagnosis and monitoring of prostate cancer.

Blocker Effect on Diffusion Resistance of a Membrane Channel: Dependence on the Blocker Geometry.

Being motivated by recent progress in nanopore sensing, we develop a theory of the effect of large analytes, or blockers, trapped within the nanopore confines, on diffusion flow of small solutes. The focus is on the nanopore diffusion resistance which is the ratio of the solute concentration difference in the reservoirs connected by the nanopore to the solute flux driven by this difference. Analytical expressions for the diffusion resistance are derived for a cylindrically symmetric blocker whose axis coincides with the axis of a cylindrical nanopore in two limiting cases where the blocker radius changes either smoothly or abruptly. Comparison of our theoretical predictions with the results obtained from Brownian dynamics simulations shows good agreement between the two.

Existence of Ti3+ and dislocation on nanoporous CdO-TiO2 heterostructure applicable for degrading chlorophenol pollutant.

This study addresses the significance of wastewater recuperation by a simple and facile treatment process known as photocatalyst technology using visible light. Titanium di-oxide (TiO2) is the most promising photocatalyst ever since longing decades, has good activity under UV light, owing to its small band gap. Hence, TiO2 has been modified with metal oxides for the positive response against visible light. Since this is an efficient process, the novelty has been made on nanometal oxide CdO (cadmium oxide) combined with TiO2 to acquire the best efficiency of degrading organic chlorophenol contaminant. Initially, the composites were synthesized by sol-gel and thermal decomposition methods and investigated for their various outstanding properties. The characterized outcomes have exhibited heterostructures with reduced crystallite size from the X-ray diffraction studies. Then, the determination of nanoporous feature was recognized through HR-TEM analysis which was also detected with some dislocations. The EDX spectrum was identified the perfect elemental composition. The nitrogen adsorption-desorption equilibrium was attained that offers many pores measured with high surface area. The XPS result convinced that Ti3+ was accessible along with TIO2/CdO composite. Further the absorption towards higher wavelength was obtained from UV-vis spectra. Finally, for the photocatalytic application of chlorophenol, the composite shows higher percentage of degrading efficiencies than the pristine TiO2. The photocatalytic mechanism was discussed in detail.

Highly sensitive and selective nanostructured microbiosensors for glucose and lactate simultaneous measurements in blood serum and in vivo in brain tissue.

Highly sensitive and selective nanostructured lactate and glucose microbiosensors for their in vivo simultaneous determination in rat brain were developed based on carbon fiber microelectrodes (CFM) modified with nanoporous gold (NPG) using the Dynamic Hydrogen Bubble Template (DHBT) method. Electrodeposition of platinum nanoparticles (PtNP) onto the NPG film enhances the sensitivity and the electrocatalytic properties towards H2O2 detection. The nanostructured microelectrode platform was modified by glucose oxidase (GOx) and lactate oxidase (LOx) enzyme immobilization. High selective measurements were achieved by covering with a perm-selective layer of electropolymerized m-phenylenediamine, deposition of a Nafion® film and by using a null sensor. The morphological characteristics and electroanalytical performance of the microbiosensors were assessed, by scanning electron microscopy and electrochemical techniques, respectively. The PtNP/NPG/CFM shows a high sensitivity to H2O2 (5.96 A M-1 cm-2) at 0.36 V vs. Ag/AgCl, with a linear range from 0.2 to 200 µM, and an LOD of 10 nM. The microbiosensors were applied to the simultaneous determination of lactate and glucose in blood serum samples. Moreover, the basal extracellular concentrations of lactate and glucose were measured in vivo in four different rat brain structures. These results support the potential of the microbiosensor to be used as a valuable tool to investigate brain neurochemicals in vivo.

Nanopore Genome Sequencing and Variant Analysis of the Susceptible Candida auris Strain L1537/2020, Salvador, Brazil.

Candida auris has been reported worldwide, but only in December 2020, the first strain from a COVID-19 patient in Brazil was isolated. Here, we describe the genome sequence of this susceptible C. auris strain and performed variant analysis of the genetic relatedness with strains from other geographic localities.

Nanopore Sequencing Provides Rapid and Reliable Insight Into Microbial Profiles of Intensive Care Units.

Fast and accurate identification of pathogens is an essential task in healthcare settings. Second-generation sequencing platforms such as Illumina have greatly expanded the capacity with which different organisms can be detected in hospital samples, and third-generation nanopore-driven sequencing devices such as Oxford Nanopore's minION have recently emerged as ideal sequencing platforms for routine healthcare surveillance due to their long-read capacity and high portability. Despite its great potential, protocols and analysis pipelines for nanopore sequencing are still being extensively validated. In this work, we assess the ability of nanopore sequencing to provide reliable community profiles based on 16S rRNA sequencing in comparison to traditional Illumina platforms using samples collected from Intensive Care Units of a hospital in Brazil. While our results demonstrate that lower throughputs may be a shortcoming of the method in more complex samples, we show that the use of single-use Flongle flowcells in nanopore sequencing runs can provide insightful information on the community composition in healthcare settings.

Deep Learning of Nanopore Sensing Signals Using a Bi-Path Network.

Temporal changes in electrical resistance of a nanopore sensor caused by translocating target analytes are recorded as a sequence of pulses on current traces. Prevalent algorithms for feature extraction in pulse-like signals lack objectivity because empirical amplitude thresholds are user-defined to single out the pulses from the noisy background. Here, we use deep learning for feature extraction based on a bi-path network (B-Net). After training, the B-Net acquires the prototypical pulses and the ability of both pulse recognition and feature extraction without a priori assigned parameters. The B-Net is evaluated on simulated data sets and further applied to experimental data of DNA and protein translocation. The B-Net results are characterized by small relative errors and stable trends. The B-Net is further shown capable of processing data with a signal-to-noise ratio equal to 1, an impossibility for threshold-based algorithms. The B-Net presents a generic architecture applicable to pulse-like signals beyond nanopore currents.

Nanopore Long Read DNA Sequencing of Protozoan Parasites: Hybrid Genome Assembly of Trypanosoma cruzi.

Due to highly repetitive genome sequences, short-read-based Trypanosoma cruzi genomes are extremely fragmented. Contiguous trypanosomatid genomes assemblies have resulted in the advent of third-generation sequencing technologies. Long reads span several to hundreds of kbps allowing to correct assemblies of repeated and low complexity DNA regions. However, these techniques present higher error rates. Hybrid assembly strategies that combine error-prone long reads with much more accurate Illumina short reads represent a very convenient approach for enhancing genome completeness. Here, we describe how to perform a hybrid assembly for genomic analysis of protozoan pathogens using Illumina and Oxford Nanopore sequencing.