scholarly journals Complex DNA knots detected with a nanopore sensor

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Rajesh Kumar Sharma ◽  
Ishita Agrawal ◽  
Liang Dai ◽  
Patrick S. Doyle ◽  
Slaven Garaj
Keyword(s):  
High Throughput ◽  
Single Molecule ◽  
Equilibrium Structure ◽  
Biological Processes ◽  
Structure And Dynamics ◽  
Sequencing Technologies ◽  
Theoretical Assumptions ◽  
Biological Polymers ◽  
First Time ◽  
Nanopore Sensors

Abstract Equilibrium knots are common in biological polymers—their prevalence, size distribution, structure, and dynamics have been extensively studied, with implications to fundamental biological processes and DNA sequencing technologies. Nanopore microscopy is a high-throughput single-molecule technique capable of detecting the shape of biopolymers, including DNA knots. Here we demonstrate nanopore sensors that map the equilibrium structure of DNA knots, without spurious knot tightening and sliding. We show the occurrence of both tight and loose knots, reconciling previous contradictory results from different experimental techniques. We evidence the occurrence of two quantitatively different modes of knot translocation through the nanopores, involving very different tension forces. With large statistics, we explore the complex knots and, for the first time, reveal the existence of rare composite knots. We use parametrized complexity, in concert with simulations, to test the theoretical assumptions of the models, further asserting the relevance of nanopores in future investigation of knots.

scholarly journals Survey of the Bradysia odoriphaga Transcriptome Using PacBio Single-Molecule Long-Read Sequencing

Genes ◽  
2019 ◽  
Vol 10 (6) ◽  
pp. 481 ◽  
Author(s):  
Chen ◽  
Lin ◽  
Xie ◽  
Zhong ◽  
Zhang ◽  
...  
Keyword(s):  
Insecticide Resistance ◽  
Single Molecule ◽  
Functional Categories ◽  
Genetic Studies ◽  
Sequencing Technologies ◽  
Clusters Of Orthologous Groups ◽  
Long Read ◽  
Main Gene ◽  
First Time ◽  
Main Factor

The damage caused by Bradysia odoriphaga is the main factor threatening the production of vegetables in the Liliaceae family. However, few genetic studies of B. odoriphaga have been conducted because of a lack of genomic resources. Many long-read sequencing technologies have been developed in the last decade; therefore, in this study, the transcriptome including all development stages of B. odoriphaga was sequenced for the first time by Pacific single-molecule long-read sequencing. Here, 39,129 isoforms were generated, and 35,645 were found to have annotation results when checked against sequences available in different databases. Overall, 18,473 isoforms were distributed in 25 various Clusters of Orthologous Groups, and 11,880 isoforms were categorized into 60 functional groups that belonged to the three main Gene Ontology classifications. Moreover, 30,610 isoforms were assigned into 44 functional categories belonging to six main Kyoto Encyclopedia of Genes and Genomes functional categories. Coding DNA sequence (CDS) prediction showed that 36,419 out of 39,129 isoforms were predicted to have CDS, and 4319 simple sequence repeats were detected in total. Finally, 266 insecticide resistance and metabolism-related isoforms were identified as candidate genes for further investigation of insecticide resistance and metabolism in B. odoriphaga.

scholarly journals Perspectives and benefits of high-throughput long-read sequencing in microbial ecology

2021 ◽  
Author(s):  
Leho Tedersoo ◽  
Mads Albertsen ◽  
Sten Anslan ◽  
Benjamin Callahan
Keyword(s):  
Microbial Ecology ◽  
High Throughput ◽  
Single Molecule ◽  
High Throughput Sequencing ◽  
Environmental Dna ◽  
Nanopore Sequencing ◽  
High Quality ◽  
Short Read ◽  
Sequencing Technologies ◽  
Long Read

Short-read, high-throughput sequencing (HTS) methods have yielded numerous important insights into microbial ecology and function. Yet, in many instances short-read HTS techniques are suboptimal, for example by providing insufficient phylogenetic resolution or low integrity of assembled genomes. Single-molecule and synthetic long-read (SLR) HTS methods have successfully ameliorated these limitations. In addition, nanopore sequencing has generated a number of unique analysis opportunities such as rapid molecular diagnostics and direct RNA sequencing, and both PacBio and nanopore sequencing support detection of epigenetic modifications. Although initially suffering from relatively low sequence quality, recent advances have greatly improved the accuracy of long read sequencing technologies. In spite of great technological progress in recent years, the long-read HTS methods (PacBio and nanopore sequencing) are still relatively costly, require large amounts of high-quality starting material, and commonly need specific solutions in various analysis steps. Despite these challenges, long-read sequencing technologies offer high-quality, cutting-edge alternatives for testing hypotheses about microbiome structure and functioning as well as assembly of eukaryote genomes from complex environmental DNA samples.

Highly Sensitive Detection of Paraquat with Pillar[5]arene as an aptamer in α-Hemolysin Nanopore

2021 ◽  
Author(s):  
Xiaojia Jiang ◽  
Mingsong Zang ◽  
Fei Li ◽  
Chunxi Hou ◽  
Quan Luo ◽  
...  
Keyword(s):  
Real Time ◽  
High Throughput ◽  
Single Molecule ◽  
Single Molecule Detection ◽  
Sensitive Detection ◽  
High Throughput Analysis ◽  
Throughput Analysis ◽  
The Real ◽  
Highly Sensitive ◽  
Highly Sensitive Detection

Biological nanopore-based techniques have attracted more and more attention recently in the field of single-molecule detection, because they allow the real-time, sensitive, high-throughput analysis. Herein, we report an engineered biological...

Polarization-resolved single-molecule tracking reveal strange dynamics of individual fluorescent tracers through a deep rubbery polymer network

2021 ◽  
Author(s):  
Jaladhar Mahato ◽  
Sukanya Bhattacharya ◽  
Dharmendar Kumar Sharma ◽  
Arindam Chowdhury
Keyword(s):  
Single Molecule ◽  
Local Structure ◽  
Biological Systems ◽  
Polymer Network ◽  
Soft Materials ◽  
Complex Environments ◽  
Fluorescent Tracers ◽  
Single Molecule Tracking ◽  
Structure And Dynamics

Tracking the movement of fluorescent single-molecule (SM) tracers has provided several new insights on the local structure and dynamics in complex environments such as soft materials and biological systems. However,...

scholarly journals Reconstruction of Microbial Haplotypes by Integration of Statistical and Physical Linkage in Scaffolding

2021 ◽  
Author(s):  
Chen Cao ◽  
Jingni He ◽  
Lauren Mak ◽  
Deshan Perera ◽  
Devin Kwok ◽  
...  
Keyword(s):  
Single Molecule ◽  
Human Genetics ◽  
Real Data ◽  
Sequencing Technologies ◽  
Bacterial Genomics ◽  
Physical Linkage ◽  
Pooled Sequencing ◽  
Computational Reconstruction ◽  
Host Genetic ◽  
Host Evolution

Abstract DNA sequencing technologies provide unprecedented opportunities to analyze within-host evolution of microorganism populations. Often, within-host populations are analyzed via pooled sequencing of the population, which contains multiple individuals or “haplotypes.” However, current next-generation sequencing instruments, in conjunction with single-molecule barcoded linked-reads, cannot distinguish long haplotypes directly. Computational reconstruction of haplotypes from pooled sequencing has been attempted in virology, bacterial genomics, metagenomics, and human genetics, using algorithms based on either cross-host genetic sharing or within-host genomic reads. Here, we describe PoolHapX, a flexible computational approach that integrates information from both genetic sharing and genomic sequencing. We demonstrated that PoolHapX outperforms state-of-the-art tools tailored to specific organismal systems, and is robust to within-host evolution. Importantly, together with barcoded linked-reads, PoolHapX can infer whole-chromosome-scale haplotypes from 50 pools each containing 12 different haplotypes. By analyzing real data, we uncovered dynamic variations in the evolutionary processes of within-patient HIV populations previously unobserved in single position-based analysis.

scholarly journals Hapo-G, haplotype-aware polishing of genome assemblies with accurate reads

2021 ◽  
Vol 3 (2) ◽  
Author(s):  
Jean-Marc Aury ◽  
Benjamin Istace
Keyword(s):  
Single Molecule ◽  
Direct Consequence ◽  
High Quality ◽  
Sequencing Errors ◽  
Coding Regions ◽  
Sequencing Technologies ◽  
Long Reads ◽  
Oxford Nanopore ◽  
Long Read ◽  
Genome Assemblies

Abstract Single-molecule sequencing technologies have recently been commercialized by Pacific Biosciences and Oxford Nanopore with the promise of sequencing long DNA fragments (kilobases to megabases order) and then, using efficient algorithms, provide high quality assemblies in terms of contiguity and completeness of repetitive regions. However, the error rate of long-read technologies is higher than that of short-read technologies. This has a direct consequence on the base quality of genome assemblies, particularly in coding regions where sequencing errors can disrupt the coding frame of genes. In the case of diploid genomes, the consensus of a given gene can be a mixture between the two haplotypes and can lead to premature stop codons. Several methods have been developed to polish genome assemblies using short reads and generally, they inspect the nucleotide one by one, and provide a correction for each nucleotide of the input assembly. As a result, these algorithms are not able to properly process diploid genomes and they typically switch from one haplotype to another. Herein we proposed Hapo-G (Haplotype-Aware Polishing Of Genomes), a new algorithm capable of incorporating phasing information from high-quality reads (short or long-reads) to polish genome assemblies and in particular assemblies of diploid and heterozygous genomes.

scholarly journals Sequoia: an interactive visual analytics platform for interpretation and feature extraction from nanopore sequencing datasets

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Ratanond Koonchanok ◽  
Swapna Vidhur Daulatabad ◽  
Quoseena Mir ◽  
Khairi Reda ◽  
Sarath Chandra Janga
Keyword(s):  
Single Molecule ◽  
Visual Analytics ◽  
Visual Analysis ◽  
Direct Sequencing ◽  
Visual Exploration ◽  
Nanopore Sequencing ◽  
Sequencing Data ◽  
Rna Sequences ◽  
Sequencing Technologies ◽  
Signal Features

Abstract Background Direct-sequencing technologies, such as Oxford Nanopore’s, are delivering long RNA reads with great efficacy and convenience. These technologies afford an ability to detect post-transcriptional modifications at a single-molecule resolution, promising new insights into the functional roles of RNA. However, realizing this potential requires new tools to analyze and explore this type of data. Result Here, we present Sequoia, a visual analytics tool that allows users to interactively explore nanopore sequences. Sequoia combines a Python-based backend with a multi-view visualization interface, enabling users to import raw nanopore sequencing data in a Fast5 format, cluster sequences based on electric-current similarities, and drill-down onto signals to identify properties of interest. We demonstrate the application of Sequoia by generating and analyzing ~ 500k reads from direct RNA sequencing data of human HeLa cell line. We focus on comparing signal features from m6A and m5C RNA modifications as the first step towards building automated classifiers. We show how, through iterative visual exploration and tuning of dimensionality reduction parameters, we can separate modified RNA sequences from their unmodified counterparts. We also document new, qualitative signal signatures that characterize these modifications from otherwise normal RNA bases, which we were able to discover from the visualization. Conclusions Sequoia’s interactive features complement existing computational approaches in nanopore-based RNA workflows. The insights gleaned through visual analysis should help users in developing rationales, hypotheses, and insights into the dynamic nature of RNA. Sequoia is available at https://github.com/dnonatar/Sequoia.

scholarly journals Assessing genotyping errors in mammalian museum study skins using high-throughput genotyping-by-sequencing

2021 ◽  
Author(s):  
Stella C. Yuan ◽  
Eric Malekos ◽  
Melissa T. R. Hawkins
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Massively Parallel Sequencing ◽  
Massively Parallel ◽  
Museum Specimens ◽  
Museum Specimen ◽  
Genotyping Errors ◽  
Allelic Dropout ◽  
Parallel Sequencing ◽  
Sequencing Technologies

AbstractThe use of museum specimens held in natural history repositories for population and conservation genetic research is increasing in tandem with the use of massively parallel sequencing technologies. Short Tandem Repeats (STRs), or microsatellite loci, are commonly used genetic markers in wildlife and population genetic studies. However, they traditionally suffered from a host of issues including length homoplasy, high costs, low throughput, and difficulties in reproducibility across laboratories. Massively parallel sequencing technologies can address these problems, but the incorporation of museum specimen derived DNA suffers from significant fragmentation and exogenous DNA contamination. Combatting these issues requires extra measures of stringency in the lab and during data analysis, yet there have not been any high-throughput sequencing studies evaluating microsatellite allelic dropout from museum specimen extracted DNA. In this study, we evaluate genotyping errors derived from mammalian museum skin DNA extracts for previously characterized microsatellites across PCR replicates utilizing high-throughput sequencing. We found it useful to classify samples based on DNA concentration, which determined the rate by which genotypes were accurately recovered. Longer microsatellites performed worse in all museum specimens. Allelic dropout rates across loci were dependent on sample quantity, with high concentration museum specimens performing as well and recovering quality metrics nearly as high as the frozen tissue sample. Based on our results, we provide a set of best practices for quality assurance and incorporation of reliable genotypes from museum specimens.

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