scholarly journals Nanopore device-based fingerprinting of RNA oligos and microRNAs enhanced with an Osmium tag

2019 ◽  
Author(s):  
Madiha Sultan ◽  
Anastassia Kanavarioti
Keyword(s):  
Single Molecule ◽  
Osmium Tetroxide ◽  
Strong Interactions ◽  
Base Calling ◽  
Selective Labelling ◽  
Oxford Nanopore ◽  
Analytical Tools ◽  
Oxford Nanopore Technologies ◽  
Pore Protein

Nanopores, both protein and solid-state, are explored as single molecule analytical tools, but using an experimental platform is challenging. Here we show that a commercially available nanopore device, MinION from Oxford Nanopore Technologies (ONT), successfully accomplishes a task challenging for a conventional analytical tool. Specifically the MinION discriminates among 31 nucleotide (nt) long oligoriboadenylates with a single pyrimidine (Py) substitution, when this pyrimidine is tagged/labeled with a bulky group (Osmium tetroxide 2,2’-bipyridine or OsBp). This platform also discriminates between an osmylated Py (Py-OsBp) followed by a purine (Pu) and a Py-OsBp followed by a second Py-OsBp, leading to the conjecture that the bulky tag enables sensing of a two-nucleotide sequence. Two-nucleotide sensing could greatly improve base-calling accuracy in motor enzyme-assisted nanopore sequencing.We attribute the observed discrimination neither to the specific pore protein nor to OsBp, but to the tag’s bulkiness, that leads to markedly slower translocation and “touching” proximity at the pore’s constriction zone, that forces desolvation and reorganization, and enables strong interactions among the nanopore, the tagged pyrimidine, and the adjacent nucleobase. These results constitute proof-of-principle that size-suitable nanopores may be superior to traditional analytical tools, for the characterization of RNA oligos and microRNAs enhanced by selective labelling.

Dual Isoform Sequencing Reveals a Multifaceted Transcriptional Architecture of a Prototype Baculovirus

2021 ◽  
Author(s):  
Gábor Torma ◽  
Dóra Tombácz ◽  
Norbert Moldován ◽  
Ádám Fülöp ◽  
István Prazsák ◽  
...  
Keyword(s):  
Protein Coding ◽  
Rna Molecules ◽  
Non Coding Rna ◽  
Oxford Nanopore ◽  
The Pacific ◽  
Viral Genes ◽  
Long Read ◽  
Oxford Nanopore Technologies ◽  
Overlapping Transcripts

Abstract In this study, we used two long-read sequencing (LRS) techniques, Sequel from the Pacific Biosciences and MinION from Oxford Nanopore Technologies, for the transcriptional characterization of a prototype baculovirus, Autographacalifornica multiple nucleopolyhedrovirus. LRS is able to read full-length RNA molecules, and thereby to distinguish between transcript isoforms, mono- and polycistronic RNAs, and overlapping transcripts. Altogether, we detected 875 transcripts, of which 759 are novel and 116 have been annotated previously. These RNA molecules include 41 novel putative protein coding transcript (each containing 5’-truncated in-frame ORFs), 14 monocistronic transcripts, 99 multicistronic RNAs, 101 non-coding RNA, and 504 length isoforms. We also detected RNA methylation in 12 viral genes and RNA hyper-editing in the longer 5’-UTR transcript isoform of ORF 19 gene.

scholarly journals Microbial diversity characterization of seawater in a pilot study using Oxford Nanopore Technologies long-read sequencing

2020 ◽  
Author(s):  
Michael Liem ◽  
Tonny Regensburg-Tuïnk ◽  
Christiaan Henkel ◽  
Hans Jansen ◽  
Herman Spaink
Keyword(s):  
Microbial Diversity ◽  
Environmental Samples ◽  
Sea Water ◽  
Flow Cells ◽  
Oxford Nanopore ◽  
Challenging Tasks ◽  
Long Read ◽  
Close Relatives ◽  
Oxford Nanopore Technologies

Abstract Objective: Currently the majority of non-culturable microbes in sea water are yet to be discovered, Nanopore offers a solution to overcome the challenging tasks to identify the genomes and complex composition of oceanic microbiomes. In this study we evaluate the utility of Oxford Nanopore Technologies (ONT) sequencing to characterize microbial diversity in seawater from multiple locations. We compared the microbial species diversity of retrieved environmental samples from two different locations and time points.Results: With only three ONT flow cells we were able to identify thousands of organisms, including bacteriophages, from which a large part at species level. It was possible to assemble genomes from environmental samples with Flye. In several cases this resulted in >1 Mbp contigs and in the particular case of a Thioglobus singularis species it even produced a near complete genome. k-mer analysis reveals that a large part of the data represents species of which close relatives have not yet been deposited to the database. These results show that our approach is suitable for scalable genomic investigations such as monitoring oceanic biodiversity and provides a new platform for education in biodiversity.

PSXIV-24 Real-time, on-site whole genome sequencing with oxford nanopore technologies’ MinION

2021 ◽  
Vol 99 (Supplement_3) ◽  
pp. 257-258
Author(s):  
Hanna Ostrovski ◽  
Rodrigo Pelicioni Savegnago ◽  
Wen Huang ◽  
Cedric Gondro
Keyword(s):  
Real Time ◽  
Ease Of Use ◽  
Whole Genome Sequence ◽  
Genomic Sequencing ◽  
Whole Genome ◽  
Illumina Hiseq ◽  
Base Calling ◽  
Oxford Nanopore ◽  
Oxford Nanopore Technologies

Abstract Most quantitative geneticists are traditionally trained for data analysis in genetic evaluation and genomic prediction, but rarely have extensive knowledge of molecular genetics or experience in experimental labs. Recent products, such as those launched by Oxford Nanopore Technologies (ONT), give those quantitative geneticists a comprehensible and hands-on toolkit to explore DNA sequencing. The ‘MinION’, a small DNA sequencer, is of interest for quantitative geneticists due to both the minimal learning curve and the non-proprietary USB connectivity. This device is small enough to be portable, allowing for potential real-time, on-farm sequencing. The objective of this project is to compare the whole genome sequence (WGS) output of the MinION sequencer to that of the Illumina HiSeq 4000. Blood was collected from a 6-month-old Akaushi calf born on a Michigan State University farm. DNA was extracted from the sample using the QIAamp DNA Blood Kit from Qiagen, and library DNA ligation preparation (SQK-LSK109) from ONT was used. After base-calling with guppy software (provided by ONT), the data were preprocessed and experimental runs with the MinION were compared using quality control. Finally, the data were aligned with guppy software, and was compared to the aligned WGS obtained with Illumina HiSeq. Quality results from each MinION indicate that, despite the low amount of sequence collected in each run (~225,303 reads per run), the quality of bases sequenced was high (Q≥7). The aligned data from the Illumina sequencer provided 40x coverage of the genome, with a total of 739,339,742 reads. Although the amount of data obtained with MinION is much smaller than that of Illumina HiSeq, the high quality of MinION’s data combined with its ease of use give an opportunity of genomic sequencing for users who are either inexperienced or do not have access to large genomic sequencing devices.

scholarly journals Nanopore device-based fingerprinting of RNA oligos and microRNAs enhanced with an Osmium tag

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Madiha Sultan ◽  
Anastassia Kanavarioti
Keyword(s):  
Solid State ◽  
Nucleotide Sequence ◽  
Nucleic Acids ◽  
Rna Sequencing ◽  
Single Molecule ◽  
Selective Labeling ◽  
Base Calling ◽  
Alpha Hemolysin ◽  
Oxford Nanopore ◽  
Single Molecule Analysis

Abstract Protein and solid-state nanopores are used for DNA/RNA sequencing as well as for single molecule analysis. We proposed that selective labeling/tagging may improve base-to-base resolution of nucleic acids via nanopores. We have explored one specific tag, the Osmium tetroxide 2,2′-bipyridine (OsBp), which conjugates to pyrimidines and leaves purines intact. Earlier reports using OsBp-tagged oligodeoxyribonucleotides demonstrated proof-of-principle during unassisted voltage-driven translocation via either alpha-Hemolysin or a solid-state nanopore. Here we extend this work to RNA oligos and a third nanopore by employing the MinION, a commercially available device from Oxford Nanopore Technologies (ONT). Conductance measurements demonstrate that the MinION visibly discriminates oligoriboadenylates with sequence A15PyA15, where Py is an OsBp-tagged pyrimidine. Such resolution rivals traditional chromatography, suggesting that nanopore devices could be exploited for the characterization of RNA oligos and microRNAs enhanced by selective labeling. The data also reveal marked discrimination between a single pyrimidine and two consecutive pyrimidines in OsBp-tagged AnPyAn and AnPyPyAn. This observation leads to the conjecture that the MinION/OsBp platform senses a 2-nucleotide sequence, in contrast to the reported 5-nucleotide sequence with native nucleic acids. Such improvement in sensing, enabled by the presence of OsBp, may enhance base-calling accuracy in enzyme-assisted DNA/RNA sequencing.

scholarly journals Plasmidome analysis of carbapenem-resistant Enterobacteriaceae isolated in Vietnam

2020 ◽  
Author(s):  
Aki Hirabayashi ◽  
Koji Yahara ◽  
Satomi Mitsuhashi ◽  
So Nakagawa ◽  
Tadashi Imanishi ◽  
...  
Keyword(s):  
Carbapenem Resistance ◽  
Genomic Epidemiology ◽  
Carbapenem Resistant ◽  
Oxford Nanopore ◽  
Carbapenemase Gene ◽  
Long Read ◽  
Severe Infections ◽  
Oxford Nanopore Technologies ◽  
Carbapenem Resistant Enterobacteriaceae

Carbapenem-resistant Enterobacteriaceae (CRE) represent a serious threat to public health due to limited management of severe infections and high mortality. The rate of resistance of Enterobacteriaceae isolates to major antimicrobials, including carbapenems, is much higher in Vietnam than in Western countries, but the reasons remain unknown due to the lack of genomic epidemiology research. A previous study suggested that carbapenem resistance genes, such as the carbapenemase gene bla NDM-1 , spread via plasmids among Enterobacteriaceae in Vietnam. In this study, we performed detection and molecular characterization of bla NDM-1 -carrying plasmids in CRE isolated in Vietnam, and identified several possible cases of horizontal transfer of plasmids both within and among species of bacteria. Twenty-five carbapenem-resistant isolates from Enterobacteriaceae clinically isolated in a reference medical institution in Hanoi were sequenced on Illumina short-read sequencers, and 12 isolates harboring bla NDM-1 were sequenced on an Oxford Nanopore Technologies long-read sequencer to obtain complete plasmid sequences. Most of the plasmids co-carried genes conferring resistance to clinically relevant antimicrobials, including third-generation cephalosporins, aminoglycosides, and fluoroquinolones, in addition to bla NDM-1 , leading to multidrug resistance of their bacterial hosts. These results provide insight into the genetic basis of CRE in Vietnam, and could help control nosocomial infections.

scholarly journals Microbial diversity characterization of seawater in a pilot study using Oxford Nanopore Technologies long-read sequencing

2020 ◽  
Author(s):  
Michael Liem ◽  
A.J.G. Regensburg-Tuïnk ◽  
C.V. Henkel ◽  
H.P. Spaink
Keyword(s):  
Microbial Diversity ◽  
Environmental Samples ◽  
Sea Water ◽  
Flow Cells ◽  
Oxford Nanopore ◽  
Challenging Tasks ◽  
Long Read ◽  
Close Relatives ◽  
Oxford Nanopore Technologies

Abstract Objective Currently the majority of non-culturable microbes in sea water are yet to be discovered, Nanopore offers a solution to overcome the challenging tasks to identify the genomes and complex composition of oceanic microbiomes. In this study we evaluate the utility of Oxford Nanopore Technologies (ONT) sequencing to characterize microbial diversity in seawater from multiple locations. We compared the microbial species diversity of retrieved environmental samples from two different locations and time points. Results With only three ONT flow cells we were able to identify thousands of organisms, including bacteriophages, from which a large part at species level. It was possible to assemble genomes from environmental samples with Flye. In several cases this resulted in >1 Mbp contigs and in the particular case of a Thioglobus singularis species it even produced a near complete genome. k-mer analysis reveals that a large part of the data represents species of which close relatives have not yet been deposited to the database. These results show that our approach is suitable for scalable genomic investigations such as monitoring oceanic biodiversity and provides a new platform for education in biodiversity.

scholarly journals Fast-Bonito: A Faster Basecaller for Nanopore Sequencing

2020 ◽  
Author(s):  
Zhimeng Xu ◽  
Yuting Mai ◽  
Denghui Liu ◽  
Wenjun He ◽  
Xinyuan Lin ◽  
...  
Keyword(s):  
Network Architecture ◽  
Original Version ◽  
Neuron Network ◽  
Base Calling ◽  
Oxford Nanopore ◽  
Speed Up ◽  
Downstream Analysis ◽  
Next Generation Sequencing Ngs ◽  
Oxford Nanopore Technologies ◽  
Generation Sequencing

AbstractOxford Nanopore Technologies (ONT) is a promising sequencing technology that could generate relatively longer sequencing reads compared to the next generation sequencing (NGS) technology. The base calling process is very important for TGS. It translates the original electrical signals from the sequencer to the nucleotide sequence. By doing that, the base calling could significantly influence the accuracy of downstream analysis. Bonito is a recently developed basecaller based on deep neuron network, the neuron network architecture of which is composed of a single convolutional layer followed by three stacked bidirectional GRU layers. Although Bonito achieved the state-of-the-art accuracy, its speed is so slow that it is not likely to be used in production. We therefore implement Fast-Bonito, which introduces systematic optimization to speed up Bonito. Fast-Bonito archives 53.8% faster than the original version on NVIDIA V100 and could be further speed up by HUAWEI Ascend 910 NPU, achieving 565% faster than the original version. The accuracy of Fast-Bonito is also slightly higher than the original Bonito.

scholarly journals Accurate detection of m6A RNA modifications in native RNA sequences

2019 ◽  
Author(s):  
Huanle Liu ◽  
Oguzhan Begik ◽  
Morghan C Lucas ◽  
Christopher E. Mason ◽  
Schraga Schwartz ◽  
...  
Keyword(s):  
Rna Modifications ◽  
Rna Sequences ◽  
Single Nucleotide ◽  
Rna Molecules ◽  
Base Calling ◽  
Oxford Nanopore ◽  
Nucleotide Resolution ◽  
The Universe ◽  
Oxford Nanopore Technologies ◽  
Single Nucleotide Resolution

ABSTRACTThe field of epitranscriptomics has undergone an enormous expansion in the last few years; however, a major limitation is the lack of generic methods to map RNA modifications transcriptome-wide. Here we show that using Oxford Nanopore Technologies, N6-methyladenosine (m6A) RNA modifications can be detected with high accuracy, in the form of systematic errors and decreased base-calling qualities. Our results open new avenues to investigate the universe of RNA modifications with single nucleotide resolution, in individual RNA molecules.

scholarly journals Ready-to-use nanopore platform for the detection of any DNA/RNA oligo at attomole range using an Osmium tagged complementary probe

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Albert S. W. Kang ◽  
Janette G. Bernasconi ◽  
William Jack ◽  
Anastassia Kanavarioti
Keyword(s):  
Single Molecule ◽  
Point Of Care ◽  
Complex Mixture ◽  
Channel Measurements ◽  
Single Digit ◽  
Acquisition Rate ◽  
Oxford Nanopore ◽  
Strong Hybridization ◽  
Technology Probes ◽  
Oxford Nanopore Technologies

AbstractNanopores can serve as single molecule sensors. We exploited the MinION, a portable nanopore device from Oxford Nanopore Technologies, and repurposed it to detect any DNA/RNA oligo (target) in a complex mixture by conducting voltage-driven ion-channel measurements. The detection and quantitation of the target is enabled by the use of a unique complementary probe. Using a validated labeling technology, probes are tagged with a bulky Osmium tag (Osmium tetroxide 2,2′-bipyridine), in a way that preserves strong hybridization between probe and target. Intact oligos traverse the MinION’s nanopore relatively quickly compared to the device’s acquisition rate, and exhibit count of events comparable to the baseline. Counts are reported by a publicly available software, OsBp_detect. Due to the presence of the bulky Osmium tag, probes traverse more slowly, produce multiple counts over the baseline, and are even detected at single digit attomole (amole) range. In the presence of the target the probe is “silenced”. Silencing is attributed to a 1:1 double stranded (ds) complex that does not fit and cannot traverse this nanopore. This ready-to-use platform can be tailored as a diagnostic test to meet the requirements for point-of-care cell-free tumor DNA (ctDNA) and microRNA (miRNA) detection and quantitation in body fluids.

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