scholarly journals Highly parallel direct RNA sequencing on an array of nanopores

2016 ◽  
Author(s):  
Daniel R. Garalde ◽  
Elizabeth A. Snell ◽  
Daniel Jachimowicz ◽  
Andrew J. Heron ◽  
Mark Bruce ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Reverse Transcription ◽  
Biological Information ◽  
Rna Seq ◽  
Sequencing Technology ◽  
Rna Sequences ◽  
Oxford Nanopore ◽  
The Status ◽  
And Function ◽  
Oxford Nanopore Technologies

AbstractRibonucleic acid sequencing can allow us to monitor the RNAs present in a sample. This enables us to detect the presence and nucleotide sequence of viruses, or to build a picture of how active transcriptional processes are changing – information that is useful for understanding the status and function of a sample. Oxford Nanopore Technologies’ sequencing technology is capable of electronically analysing a sample’s DNA directly, and in real-time. In this manuscript we demonstrate the ability of an array of nanopores to sequence RNA directly, and we apply it to a range of biological situations. Nanopore technology is the only available sequencing technology that can sequence RNA directly, rather than depending on reverse transcription and PCR. There are several potential advantages of this approach over other RNA-seq strategies, including the absence of amplification and reverse transcription biases, the ability to detect nucleotide analogues and the ability to generate full-length, strand-specific RNA sequences. Direct RNA sequencing is a completely new way of analysing the sequence of RNA samples and it will improve the ease and speed of RNA analysis, while yielding richer biological information.

A Two-Stage Poisson Model for Testing RNA-Seq Data

2011 ◽  
Vol 10 (1) ◽  
Author(s):  
Paul L. Auer ◽  
Rebecca W Doerge
Keyword(s):  
Rna Sequencing ◽  
Statistical Approach ◽  
Poisson Model ◽  
Real Data ◽  
Rna Seq ◽  
Sequencing Data ◽  
Sequencing Technology ◽  
Two Stage ◽  
Individual Gene ◽  
Unique Nature

RNA sequencing technology is providing data of unprecedented throughput, resolution, and accuracy. Although there are many different computational tools for processing these data, there are a limited number of statistical methods for analyzing them, and even fewer that acknowledge the unique nature of individual gene transcription. We introduce a simple and powerful statistical approach, based on a two-stage Poisson model, for modeling RNA sequencing data and testing for biologically important changes in gene expression. The advantages of this approach are demonstrated through simulations and real data applications.

scholarly journals Nanopore native RNA sequencing of a human poly(A) transcriptome: RNA extraction, cDNA conversion and direct RNA and cDNA library preparation for Oxford Nanopore

2019 ◽  
Author(s):  
Rachael E. Workman ◽  
Alison D. Tang ◽  
Paul S. Tang ◽  
Miten Jain ◽  
John R. Tyson ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Rna Extraction ◽  
Human Cell Line ◽  
Cdna Libraries ◽  
Library Preparation ◽  
Sequencing Technologies ◽  
Oxford Nanopore ◽  
Sequencing Strategy ◽  
Oxford Nanopore Technologies ◽  
Cdna Library Preparation

Abstract High throughput cDNA sequencing technologies have dramatically advanced our understanding of transcriptome complexity and regulation. However, these methods lose information contained in biological RNA because the copied reads are often short and because modifications are not carried forward in cDNA. We address these limitations using a native poly(A) RNA sequencing strategy developed by Oxford Nanopore Technologies (ONT). Our study focused on poly(A) RNA from the human cell line GM12878, generating 9.9 million aligned sequence reads. These native RNA reads had an aligned N50 length of 1294 bases, and a maximum aligned length of over 21,000 bases. A total of 78,199 high-confidence isoforms were identified by combining long nanopore reads with short higher accuracy Illumina reads. We describe methods for extracting intact RNA, poly-A selection, cDNA conversion for a portion of sample, and library preparation for both direct RNA and cDNA libraries.

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 Direct RNA Nanopore Sequencing of SARS-CoV-2 Extracted from Critical Material from Swabs

Life ◽  
2022 ◽  
Vol 12 (1) ◽  
pp. 69
Author(s):  
Davide Vacca ◽  
Antonino Fiannaca ◽  
Fabio Tramuto ◽  
Valeria Cancila ◽  
Laura La Paglia ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Systematic Errors ◽  
N Gene ◽  
Nanopore Sequencing ◽  
Rna Seq ◽  
Bioinformatics Pipeline ◽  
Critical Material ◽  
Oxford Nanopore ◽  
Oropharyngeal Swab ◽  
Generation Sequencing

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.

scholarly journals A new protocol for single-cell RNA-seq reveals stochastic gene expression during lag phase in budding yeast

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Abbas Jariani ◽  
Lieselotte Vermeersch ◽  
Bram Cerulus ◽  
Gemma Perez-Samper ◽  
Karin Voordeckers ◽  
...  
Keyword(s):  
Single Cell ◽  
Rna Sequencing ◽  
Mammalian Cells ◽  
Yeast Cells ◽  
Lag Phase ◽  
Rna Seq ◽  
Molecular Processes ◽  
Sequencing Technology ◽  
Single Cell Rna Sequencing ◽  
Order Of Magnitude

Current methods for single-cell RNA sequencing (scRNA-seq) of yeast cells do not match the throughput and relative simplicity of the state-of-the-art techniques that are available for mammalian cells. In this study, we report how 10x Genomics’ droplet-based single-cell RNA sequencing technology can be modified to allow analysis of yeast cells. The protocol, which is based on in-droplet spheroplasting of the cells, yields an order-of-magnitude higher throughput in comparison to existing methods. After extensive validation of the method, we demonstrate its use by studying the dynamics of the response of isogenic yeast populations to a shift in carbon source, revealing the heterogeneity and underlying molecular processes during this shift. The method we describe opens new avenues for studies focusing on yeast cells, as well as other cells with a degradable cell wall.

scholarly journals RNA Sequencing to Explore Dominant Isoform Switch During CCR6+ Memory T Cell Activation

2021 ◽  
Author(s):  
Shanrong Zhao ◽  
Alexander Barron ◽  
Ken Dower
Keyword(s):  
Gene Expression ◽  
T Cell ◽  
Rna Sequencing ◽  
T Cell Activation ◽  
Cell Cycle Progression ◽  
Cell Activation ◽  
Cell Function ◽  
Control Cell ◽  
Biological Information ◽  
Rna Seq

Abstract Alternative splicing (AS) is an essential, but under-investigated component of T-cell function during immune responses. Recent developments in RNA sequencing (RNA-seq) technologies, combined with the advent of computational tools, have enabled transcriptome-wide studies of AS at an unprecedented scale and resolution. In this paper, we analysed AS in an RNA-seq dataset previously generated to investigate the expression changes during T-cell maturation and antigen stimulation. Eight genes were identified with their most dominant isoforms switched during T cell activation. Of those, seven genes either directly control cell cycle progression or are oncogenes. We selected CDKN2C, FBXO5, NT5E and NET1 for discussion of the functional importance of AS of these genes. Our case study demonstrates that combining AS and gene expression analyses derives greater biological information and deeper insights from RNA-seq datasets than gene expression analysis alone.

scholarly journals Comparing total RNA sequencing and metagenomics pipelines for multi-domain taxonomic profiling: implications for ecological assessments

2021 ◽  
Vol 4 ◽  
Author(s):  
Christopher Hempel ◽  
Julia Harvie ◽  
Jose Hleap Lozano ◽  
Natalie Wright ◽  
Sarah Adamowicz ◽  
...  
Keyword(s):  
Microbial Communities ◽  
Rna Sequencing ◽  
Sample Type ◽  
Rna Seq ◽  
Total Rna ◽  
Taxonomic Profiling ◽  
Dna And Rna ◽  
Accuracy And Precision ◽  
The Status ◽  
Unicellular Organisms

Ecological assessments are necessary to evaluate the status of our deteriorating ecosystems, however, assessment methods traditionally omit most microbes because unicellular organisms are challenging to identify. This omission is not ideal, as microbes might be better indicators for changes in environmental conditions than taxa traditionally used. DNA- and RNA-based techniques are increasingly applied for ecological assessments to overcome this challenge but require more testing and optimization. In this study, we compare metagenomics and total RNA sequencing (total RNA-Seq) for their taxonomic profiling performance for microbial communities. We applied both techniques on two sample sets, 1) a commercially available microbial mock community consisting of eight bacterial and two eukaryotic species, and 2) a display tank water sample. We processed the data using 1,532 bioinformatics pipelines and evaluated each workflow, i.e., the combination of sample type (metagenomics or total RNA-Seq) and pipeline, in terms of their accuracy and precision. This talk will showcase preliminary results and highlight differences in workflow performances. A recommended workflow to maximize taxonomic profiling accuracy of microbial communities will also be presented.

scholarly journals Application of monomolecular sequencing technology to the diagnostics of hypertrophic cardiomyopathy

2020 ◽  
pp. 9-10
Author(s):  
Р.Р. Салахов ◽  
М.В. Голубенко ◽  
Е.Н. Павлюкова ◽  
А.В. Марков ◽  
Н.П. Бабушкина ◽  
...  
Keyword(s):  
Data Analysis ◽  
Hypertrophic Cardiomyopathy ◽  
Sequencing Technology ◽  
Missense Variants ◽  
Oxford Nanopore ◽  
Oxford Nanopore Technologies

В работе представлены результаты секвенирования пяти генов, ассоциированных с гипертрофической кардиомиопатией, с использованием технологии мономолекулярного секвенирования компании Oxford Nanopore Technologies. В результате анализа данных с помощью различных алгоритмов были выявлены миссенс-варианты в исследованных генах, которые могут являться причиной заболевания у пациентов. The paper presents the results of sequencing of five genes associated with hypertrophic cardiomyopathy, using monomolecular sequencing (Oxford Nanopore Technologies). As a result of data analysis with various algorithms, missense variants were identified in the studied genes that may be the cause of the disease in the patients.

scholarly journals RNA Sequencing by Direct Tagmentation of RNA/DNA Hybrids

2019 ◽  
Author(s):  
Lin Di ◽  
Yusi Fu ◽  
Yue Sun ◽  
Jie Li ◽  
Lu Liu ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Reverse Transcription ◽  
Single Cells ◽  
Pcr Amplification ◽  
Transcriptome Profiling ◽  
Rna Seq ◽  
New Approach ◽  
Double Stranded Dna ◽  
Wide Range ◽  
Tn5 Transposase

AbstractTranscriptome profiling by RNA sequencing (RNA-seq) has been widely used to characterize cellular status but it relies on second strand cDNA synthesis to generate initial material for library preparation. Here we use bacterial transposase Tn5, which has been increasingly used in various high-throughput DNA analyses, to construct RNA-seq libraries without second strand synthesis. We show that Tn5 transposome can randomly bind RNA/DNA heteroduplexes and add sequencing adapters onto RNA directly after reverse transcription. This method, Sequencing HEteRo RNA-DNA-hYbrid (SHERRY), is versatile and scalable. SHERRY accepts a wide range of starting materials, from bulk RNA to single cells. SHERRY offers a greatly simplified protocol, and produces results with higher reproducibility and GC uniformity compared with prevailing RNA-seq methods.Significance StatementRNA sequencing is widely used to measure gene expression in biomedical research; therefore, improvements in the simplicity and accuracy of the technology are desirable. All existing RNA sequencing methods rely on the conversion of RNA into double-stranded DNA through reverse transcription followed by second strand synthesis. The latter step requires additional enzymes and purification, and introduces sequence-dependent bias. Here, we show that Tn5 transposase, which randomly binds and cuts double-stranded DNA, can directly fragment and prime the RNA/DNA heteroduplexes generated by reverse transcription. The primed fragments are then subject to PCR amplification. This provides a new approach for simple and accurate RNA characterization and quantification.

scholarly journals Nanopore sequencing of RNA and cDNA molecules in Escherichia coli

RNA ◽  
2021 ◽  
pp. rna.078937.121
Author(s):  
Felix Grünberger ◽  
Sébastien Ferreira-Cerca ◽  
Dina Grohmann
Keyword(s):  
Escherichia Coli ◽  
Rna Sequencing ◽  
Single Molecule ◽  
High Throughput Sequencing ◽  
Model Organism ◽  
Cost Effective ◽  
Rna Seq ◽  
Sequencing Platform ◽  
Quantitative Measurements ◽  
Oxford Nanopore

High-throughput sequencing dramatically changed our view of transcriptome architectures and allowed for ground-breaking discoveries in RNA biology. Recently, sequencing of full-length transcripts based on the single-molecule sequencing platform from Oxford Nanopore Technologies (ONT) was introduced and is widely employed to sequence eukaryotic and viral RNAs. However, experimental approaches implementing this technique for prokaryotic transcriptomes remain scarce. Here, we present an experimental and bioinformatic workflow for ONT RNA-seq in the bacterial model organism Escherichia coli, which can be applied to any microorganism. Our study highlights critical steps of library preparation and computational analysis and compares the results to gold standards in the field. Furthermore, we comprehensively evaluate the applicability and advantages of different ONT-based RNA sequencing protocols, including direct RNA, direct cDNA, and PCR-cDNA. We find that (PCR)-cDNA-seq offers improved yield and accuracy compared to direct RNA sequencing. Notably, (PCR)-cDNA-seq is suitable for quantitative measurements and can be readily used for simultaneous and accurate detection of transcript 5'and 3' boundaries, analysis of transcriptional units and transcriptional heterogeneity. In summary, based on our comprehensive study, we show that Nanopore RNA-seq to be a ready-to-use tool allowing rapid, cost-effective, and accurate annotation of multiple transcriptomic features. Thereby Nanopore RNA-seq holds the potential to become a valuable alternative method for RNA analysis in prokaryotes.

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