scholarly journals Rapid Multiplex Small DNA Sequencing on the MinION Nanopore Sequencing Platform

2018 ◽  
Vol 8 (5) ◽  
pp. 1649-1657 ◽  
Author(s):  
Shan Wei ◽  
Zachary R. Weiss ◽  
Zev Williams
Keyword(s):  
Dna Sequencing ◽  
Nanopore Sequencing ◽  
Sequencing Platform

scholarly journals Rapid multiplex small DNA sequencing on the MinION nanopore sequencing platform

2018 ◽  
Author(s):  
Shan Wei ◽  
Zachary R. Weiss ◽  
Zev Williams
Keyword(s):  
Dna Sequencing ◽  
Genomic Dna ◽  
Great Promise ◽  
Nanopore Sequencing ◽  
Library Preparation ◽  
Short Read ◽  
Sequencing Platform ◽  
Sequencing Method ◽  
Novel Method ◽  
Clinical Diagnostic Test

AbstractReal-time sequencing of short DNA reads has a wide variety of clinical and research applications including screening for mutations, target sequences and aneuploidy. We recently demonstrated that MinION, a nanopore-based DNA sequencing device the size of a USB drive, could be used for short-read DNA sequencing. In this study, an ultra-rapid multiplex library preparation and sequencing method for the MinION is presented and applied to accurately test normal diploid and aneuploidy samples’ genomic DNA in under three hours, including library preparation and sequencing. This novel method shows great promise as a clinical diagnostic test for applications requiring rapid short-read DNA sequencing.

The mycobiome of the oral cavity in healthy dogs and dogs with periodontal disease

2022 ◽  
pp. 1-8
Author(s):  
Brook A. Niemiec ◽  
Jerzy Gawor ◽  
Shuiquan Tang ◽  
Aishani Prem ◽  
Janina A. Krumbeck
Keyword(s):  
Oral Cavity ◽  
Periodontal Disease ◽  
Dna Sequencing ◽  
Fungal Species ◽  
Next Generation ◽  
Data Set ◽  
Next Generation Dna Sequencing ◽  
Sequencing Platform ◽  
The Family ◽  
Healthy Dogs

Abstract OBJECTIVE To investigate the mycobiome of the oral cavity in healthy dogs and dogs with various stages of periodontal disease. ANIMALS 51 dogs without periodontal disease (n = 12) or with mild (10), moderate (19), or severe (10) periodontal disease. PROCEDURES The whole maxillary arcade of each dog was sampled with a sterile swab, and swabs were submitted for next-generation DNA sequencing targeting the internal transcribed spacer 2 region with a commercial sequencing platform. RESULTS Fungi were detected in all samples, with a total of 320 fungal species from 135 families detected in the data set. No single fungal species was found in all samples. The 3 most frequently found fungal species were Cladosporium sp (46/51 samples), Malassezia restricta (44/51 samples), and Malassezia arunalokei (36/51 samples). Certain fungi, specifically those of the family Didymellaceae, the family Irpicaceae, and the order Pleosporales, were significantly associated with different stages of periodontitis. Mycobial analysis indicated that Cladosporium sp could be considered part of the core oral cavity mycobiome. CONCLUSIONS AND CLINICAL RELEVANCE Results highlighted that fungi are present in the oral cavity of dogs and are characterized by substantial species diversity, with different fungal communities associated with various stages of periodontal disease. The next-generation DNA sequencing used in the present study revealed substantially more species of fungi than previous culture-based studies.

scholarly journals Development and Validation of Sex-Specific Markers in Pelodiscus Sinensis Using Restriction Site-Associated DNA Sequencing

Genes ◽  
2019 ◽  
Vol 10 (4) ◽  
pp. 302
Author(s):  
Liang ◽  
Wang ◽  
Sha ◽  
Zou
Keyword(s):  
Sexual Dimorphism ◽  
Dna Sequencing ◽  
Restriction Site ◽  
Large Body ◽  
Economic Traits ◽  
Pelodiscus Sinensis ◽  
Sequencing Platform ◽  
Determination System ◽  
Sex Determination System ◽  
Female Specific

The sex of an animal influences its economic traits, especially in species displaying sexual dimorphism. The Chinese soft-shelled turtle, Pelodiscus sinensis, is an economically important aquatic species that shows significant male sexual dimorphism, with a large body size, faster growth, a thick and wide calipash, and lower body fat. In this study, ten male and ten female turtles were subjected to restriction site-associated DNA sequencing (RAD-seq) using the Hi-Seq 4000 sequencing platform to isolate female-specific DNA fragments. We identified 5967 bp and 6532 bp fragments using genome walking. Three female-specific markers designed from these two fragments were confirmed to separate the sexes of Pelodiscus sinensis perfectly. One of the female-specific markers showed dosage association in female and male individuals. Individuals from different populations (n = 296) were used to validate that the female-specific markers could identify the genetic sex of Pelodiscus sinensis with 100% accuracy. The results of the present study demonstrated that RAD-seq was useful to develop sex-related markers in animals, and verified that the sex determination system of Pelodiscus sinensis belonged to the ZZ/ZW heterogametic system. Importantly, the developed markers could lead to a method for sex-controlled breeding in the Chinese soft-shelled turtle.

scholarly journals Genetic Biomonitoring and Biodiversity Assessment Using Portable Sequencing Technologies: Current Uses and Future Directions

Genes ◽  
2019 ◽  
Vol 10 (11) ◽  
pp. 858 ◽  
Author(s):  
Krehenwinkel ◽  
Pomerantz ◽  
Prost
Keyword(s):  
Dna Sequencing ◽  
Biodiversity Loss ◽  
Taxonomic Composition ◽  
Great Promise ◽  
Sequencing Platform ◽  
Biological Communities ◽  
Sequencing Technologies ◽  
Oxford Nanopore ◽  
Sequencing Studies ◽  
High Throughput Dna Sequencing

We live in an era of unprecedented biodiversity loss, affecting the taxonomic composition of ecosystems worldwide. The immense task of quantifying human imprints on global ecosystems has been greatly simplified by developments in high-throughput DNA sequencing technology (HTS). Approaches like DNA metabarcoding enable the study of biological communities at unparalleled detail. However, current protocols for HTS-based biodiversity exploration have several drawbacks. They are usually based on short sequences, with limited taxonomic and phylogenetic information content. Access to expensive HTS technology is often restricted in developing countries. Ecosystems of particular conservation priority are often remote and hard to access, requiring extensive time from field collection to laboratory processing of specimens. The advent of inexpensive mobile laboratory and DNA sequencing technologies show great promise to facilitate monitoring projects in biodiversity hot-spots around the world. Recent attention has been given to portable DNA sequencing studies related to infectious organisms, such as bacteria and viruses, yet relatively few studies have focused on applying these tools to Eukaryotes, such as plants and animals. Here, we outline the current state of genetic biodiversity monitoring of higher Eukaryotes using Oxford Nanopore Technology’s MinION portable sequencing platform, as well as summarize areas of recent development.

scholarly journals Peptide Synthesis on a Next-Generation DNA Sequencing Platform

ChemBioChem ◽  
2016 ◽  
Vol 17 (17) ◽  
pp. 1628-1635 ◽  
Author(s):  
Nina Svensen ◽  
Olve B. Peersen ◽  
Samie R. Jaffrey
Keyword(s):  
Dna Sequencing ◽  
Peptide Synthesis ◽  
Next Generation ◽  
Next Generation Dna Sequencing ◽  
Sequencing Platform

scholarly journals Genomic resources for Pseudomonas savastanoi pv. phaseolicola races 5 and 8

2020 ◽  
Author(s):  
Bret Cooper ◽  
Ronghui Yang
Keyword(s):  
Phaseolus Vulgaris ◽  
Dna Sequencing ◽  
Common Bean ◽  
Genome Sequence ◽  
Genomic Information ◽  
Genome Sequences ◽  
Sequencing Platform ◽  
Halo Blight ◽  
Long Read ◽  
Blight Disease

Pseudomonas savastanoi pv. phaseolicola causes halo blight disease on Phaseolus vulgaris. Using a long-read DNA sequencing platform, we assembled the genome sequences for P. savastanoi pv. phaseolicola races 5 and 8 that have distinguishable avirulent and virulent phenotypes on P. vulgaris PI G19833, a common bean with an annotated genome sequence. The twelve race 5 assemblies comprise two major 4.5 Mb and 1.4 Mb chromosome-like contigs and ten smaller contigs. The four race 8 assemblies comprise a major 6.1 Mb chromosome and 3 smaller contigs. Annotation yielded 5,890 genes for race 5 and 5,919 genes for race 8. These data will enable the discovery of the genetic and proteomic differences between these two races and allow comparisons to other races for which genomic information already exists.

scholarly journals A Multiplex Pharmacogenetics Assay using the MinION Nanopore Sequencing Device

2019 ◽  
Author(s):  
Yusmiati Liau ◽  
Simone L. Cree ◽  
Simran Maggo ◽  
Allison L. Miller ◽  
John F. Pearson ◽  
...  
Keyword(s):  
Dna Sequencing ◽  
Genetic Polymorphisms ◽  
Point Of Care ◽  
Variant Calling ◽  
Cost Effective ◽  
Response Variability ◽  
Multiplex Assay ◽  
Nanopore Sequencing ◽  
Three Samples ◽  
Screening Assays

AbstractAimThe MinION nanopore sequencing device opens the opportunity to cost-effective and point-of-care DNA sequencing. We developed a multiplex assay targeting pharmacogenetic variants related to clopidogrel and warfarin, two commonly used drugs that show response variability due to genetic polymorphisms.Materials & MethodsSix reference and 78 clinical DNA samples were amplified by PCR to generate 15 amplicons targeting key variants. These products were then barcoded to enable sample multiplexing. Three variant calling tools were used to compare genotyping accuracy.Results and ConclusionsAll but three samples were successfully sequenced and genotyped. Nanopolish software achieved accuracy > 90 % for all except one variant. While minor mis-genotyping issues exist, this work demonstrates that drug-specific or broad pharmacogenetic screening assays are possible on the MinION sequencing device.

scholarly journals Nanopore sequencing of the glucocerebrosidase (GBA) gene in a New Zealand Parkinson’s disease cohort

2019 ◽  
Author(s):  
O.E.E. Graham ◽  
T.L. Pitcher ◽  
Y. Liau ◽  
A.L. Miller ◽  
J.C. Dalrymple-Alford ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
New Zealand ◽  
Gaucher Disease ◽  
Nanopore Sequencing ◽  
Lysosomal Storage ◽  
Coding Region ◽  
Structural Variations ◽  
Sequencing Platform ◽  
Nanopore Dna Sequencing

AbstractIntroductionBi-allelic mutations in the gene for glucocerebrosidase (GBA) cause Gaucher disease, an autosomal recessive lysosomal storage disorder. Gaucher disease causing GBA mutations in the heterozygous state are also high risk factors for Parkinson’s disease (PD). GBA analysis is challenging due to a related pseudogene and structural variations (SVs) that can occur at this locus. We have applied and refined a recently developed nanopore DNA sequencing method to analyze GBA variants in a clinically assessed New Zealand longitudinal cohort of PD.MethodWe examined amplicons encompassing the coding region of GBA (8.9kb) from 229 PD cases and 50 healthy controls using the GridION nanopore sequencing platform, and Sanger validation.ResultsWe detected 23 variants in 21 PD cases (9.2% of patients). We detected modest PD risk variant p.N409S (rs76763715) in one case, p.E365K (rs2230288) in 12 cases, and p.T408M (rs75548401) in seven cases, one of whom also had p.E365K. We additionally detected the possible risk variants p.R78C (rs146774384) in one case, p.D179H (rs147138516) in one case which occurred on the same haplotype as p.E365K, and one novel variant c.335C>T or p.(L335=), that potentially impacts splicing of GBA transcripts. Additionally, we found a higher prevalence of dementia among patients with GBA variants.ConclusionThis work confirmed the utility of nanopore sequencing as a high-throughput method to identify known and novel GBA variants, and to assign precise haplotypes. Our observations may contribute to improved understanding of the effects of variants on disease pathogenesis, and to the development of more targeted treatments.

Nanotrap Particles Improve Nanopore Sequencing of SARS-CoV-2 and Other Respiratory Viruses

2021 ◽  
Author(s):  
Patrick Daniel Andersen ◽  
Stephanie Barksdale ◽  
Robert Alex Barclay ◽  
Natalie Smith ◽  
Justin Fernandes ◽  
...  
Keyword(s):  
Rna Extraction ◽  
Nanopore Sequencing ◽  
Sequencing Platform ◽  
Magnetic Hydrogel ◽  
Oxford Nanopore ◽  
Input Sample ◽  
Lower Titer ◽  
Syncytial Virus ◽  
Hydrogel Particle ◽  
Oxford Nanopore Technologies

Presented here is a magnetic hydrogel particle enabled workflow for capturing and concentrating SARS-CoV-2 from diagnostic remnant swab samples that significantly improves sequencing results using the Oxford Nanopore Technologies MinION sequencing platform. Our approach utilizes a novel affinity-based magnetic hydrogel particle, circumventing low input sample volumes and allowing for both rapid manual and automated high throughput workflows that are compatible with nanopore sequencing. This approach enhances standard RNA extraction protocols, providing up to 40x improvements in viral mapped reads, and improves sequencing coverage by 20-80% from lower titer diagnostic remnant samples. Furthermore, we demonstrate that this approach works for contrived influenza virus and respiratory syncytial virus samples, suggesting that it can be used to identify and improve sequencing results of multiple viruses in VTM samples. These methods can be performed manually or on a KingFisher Apex system.

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