Direct RNA nanopore sequencing of SARS-CoV-2 extracted from critical material from swabs

In consideration of the increasing prevalence of COVID-19 cases in several countries and the resulting demand for unbiased sequencing approaches, we performed a direct RNA sequencing (direct RNA seq.) experiment using critical oropharyngeal swab samples collected from Italian patients infected with SARS-CoV-2 from the Palermo region in Sicily. Here, we identified the sequences SARS-CoV-2 directly in RNA extracted from critical samples using the Oxford Nanopore MinION technology without prior cDNA retrotranscription. Using an appropriate bioinformatics pipeline, we could identify mutations in the nucleocapsid (N) gene, which have been reported previously in studies conducted in other countries. In conclusion, to the best of our knowledge, the technique used in this study has not been used for SARS-CoV-2 detection previously owing to the difficulties in the extraction of RNA of sufficient quantity and quality from routine oropharyngeal swabs. Despite these limitations, this approach provides the advantages of true native RNA sequencing and does not include amplification steps that could introduce systematic errors. This study can provide novel information relevant to the current strategies adopted in SARS-CoV-2 next-generation sequencing.

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Direct RNA nanopore sequencing of SARS-CoV-2 extracted from critical material from swabs

10.1101/2020.12.21.20191346 ◽

2020 ◽

Author(s):

Davide Vacca ◽

Antonino Fiannaca ◽

Fabio Tramuto ◽

Valeria Cancila ◽

Laura La Paglia ◽

...

Keyword(s):

Rna Sequencing ◽

Gene Sequence ◽

N Gene ◽

Nanopore Sequencing ◽

Bioinformatics Pipeline ◽

Critical Material ◽

Oxford Nanopore ◽

Sequencing Experiment ◽

Oropharyngeal Swab ◽

Generation Sequencing

ABSTRACTBackgroundIn consideration of the increasing prevalence of COVID-19 cases in several countries and the resulting demand for unbiased sequencing approaches, we performed a direct RNA sequencing experiment using critical oropharyngeal swab samples collected from Italian patients infected with SARS-CoV-2 from the Palermo region in Sicily.MethodsHere, we identified the sequences SARS-CoV-2 directly in RNA extracted from critical samples using the Oxford Nanopore MinION technology without prior cDNA retro-transcription.ResultsUsing an appropriate bioinformatics pipeline, we could identify mutations in the nucleocapisid (N) gene, which have been reported previously in studies conducted in other countries.ConclusionTo the best of our knowledge, the technique used in this study has not been used for SARS-CoV-2 detection previously owing to the difficulties in the extraction of RNA of sufficient quantity and quality from routine oropharyngeal swabs.Despite these limitations, this approach provides the advantages of true native RNA sequencing, and does not include amplification steps that could introduce systematic errors.This study can provide novel information relevant to the current strategies adopted in SARS-CoV-2 next-generation sequencing.We deposited the gene sequence in the NCBI database under the following URL:https://www.ncbi.nlm.nih.gov/nuccore/MT457389

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Nanopore sequencing: direct RNA, cDNA, full-length reads with low bias, measuring poly(A) tail lengths, detecting modified bases, viral genomics and more – Libby Snell, Oxford Nanopore Technologies

10.26226/morressier.5ec5090eff91c32a887b363e ◽

2020 ◽

Author(s):

Libby Snell

Keyword(s):

Full Length ◽

Nanopore Sequencing ◽

Viral Genomics ◽

Oxford Nanopore ◽

Oxford Nanopore Technologies

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Faculty Opinions recommendation of Nanopore sequencing and assembly of a human genome with ultra-long reads.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.732576831.793542122 ◽

2018 ◽

Author(s):

James Coker

Keyword(s):

Human Genome ◽

Nanopore Sequencing ◽

Long Reads

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Novel Magnetic States in the Heavy-Fermion Quantum-Critical Material CeRhIn5 at High Magnetic Fields Studied by NMR

10.2172/1165175 ◽

2014 ◽

Author(s):

Andrew Mounce ◽

Joe Thompson ◽

Eric Bauer ◽

A. Reyes ◽

P. Kuhns

Keyword(s):

Magnetic Fields ◽

Heavy Fermion ◽

High Magnetic Fields ◽

Critical Material ◽

Quantum Critical ◽

Magnetic States

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Quality by Design: Concept to Applications

Current Drug Discovery Technologies ◽

10.2174/1570163815666180308142016 ◽

2019 ◽

Vol 16 (3) ◽

pp. 240-250 ◽

Cited By ~ 2

Author(s):

Suryakanta Swain ◽

Rabinarayan Parhi ◽

Bikash Ranjan Jena ◽

Sitty Manohar Babu

Keyword(s):

Quality By Design ◽

Pharmaceutical Products ◽

Assessment Tools ◽

Basic Knowledge ◽

Target Product ◽

Target Product Profile ◽

Critical Material ◽

Novel Approach ◽

Product Profile ◽

Performance Report

Background: Quality by Design (QbD) is associated with a modern, systematic, scientific and novel approach which is concerned with pre-distinct objectives that not only focus on product, process understanding but also lead to process control. It predominantly signifies the design and product improvement and the manufacturing process in order to fulfill the predefined manufactured goods or final products quality characteristics. It is quite essential to identify the desired and required product performance report, such as Target Product Profile, typical Quality Target Product Profile (QTPP) and Critical Quality Attributes (CQA). Methods: This review highlighted the concepts of QbD design space, for critical material attributes (CMAs) as well as the critical process parameters that can totally affect the CQAs within which the process shall be unaffected thus, consistently manufacturing the required product. Risk assessment tools and design of experiments are its prime components. Results: This paper outlines the basic knowledge of QbD, the key elements; steps as well as various tools for QbD implementation in pharmaceutics field are presented briefly. In addition to this, quite a lot of applications of QbD in numerous pharmaceutical related unit operations are discussed and summarized. Conclusion: This article provides a complete data as well as the roadmap for universal implementation and application of QbD for pharmaceutical products.

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