scholarly journals A comprehensive examination of Nanopore native RNA sequencing for characterization of complex transcriptomes

2019 ◽  
Author(s):  
Charlotte Soneson ◽  
Yao Yao ◽  
Anna Bratus-Neuenschwander ◽  
Andrea Patrignani ◽  
Mark D. Robinson ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Direct Sequencing ◽  
Transcript Level ◽  
Human Cell Lines ◽  
Oxford Nanopore ◽  
Comprehensive Examination ◽  
Important Addition ◽  
Overall Performance ◽  
Oxford Nanopore Technologies

AbstractA platform for highly parallel direct sequencing of native RNA strands was recently described by Oxford Nanopore Technologies (ONT); in order to assess overall performance in transcript-level investigations, the technology was applied for sequencing sets of synthetic transcripts as well as a yeast transcriptome. However, despite initial efforts it remains crucial to further investigate characteristics of ONT native RNA sequencing when applied to much more complex transcriptomes. Here we thus undertook extensive native RNA sequencing of polyA+ RNA from two human cell lines, and thereby analysed ~5.2 million aligned native RNA reads which consisted of a total of ~4.6 billion bases. To enable informative comparisons, we also performed relevant ONT direct cDNA- and Illumina-sequencing. We find that while native RNA sequencing does enable some of the anticipated advantages, key unexpected aspects hamper its performance, most notably the quite frequent inability to obtain full-length transcripts from single reads, as well as difficulties to unambiguously infer their true transcript of origin. While characterising issues that need to be addressed when investigating more complex transcriptomes, our study highlights that with some defined improvements, native RNA sequencing could be an important addition to the mammalian transcriptomics toolbox.

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.

scholarly journals TagSeqTools: a flexible and comprehensive analysis pipeline for NAD tagSeq data

2020 ◽  
Author(s):  
Huan Zhong ◽  
Zongwei Cai ◽  
Zhu Yang ◽  
Yiji Xia
Keyword(s):  
Rna Sequencing ◽  
Comprehensive Analysis ◽  
Enzymatic Reactions ◽  
Computational Tool ◽  
Sequencing Data ◽  
Analysis Pipeline ◽  
Oxford Nanopore ◽  
Long Read ◽  
Identification And Characterization

AbstractNAD tagSeq has recently been developed for the identification and characterization of NAD+-capped RNAs (NAD-RNAs). This method adopts a strategy of chemo-enzymatic reactions to label the NAD-RNAs with a synthetic RNA tag before subjecting to the Oxford Nanopore direct RNA sequencing. A computational tool designed for analyzing the sequencing data of tagged RNA will facilitate the broader application of this method. Hence, we introduce TagSeqTools as a flexible, general pipeline for the identification and quantification of tagged RNAs (i.e., NAD+-capped RNAs) using long-read transcriptome sequencing data generated by NAD tagSeq method. TagSeqTools comprises two major modules, TagSeek for differentiating tagged and untagged reads, and TagSeqQuant for the quantitative and further characterization analysis of genes and isoforms. Besides, the pipeline also integrates some advanced functions to identify antisense or splicing, and supports the data reformation for visualization. Therefore, TagSeqTools provides a convenient and comprehensive workflow for researchers to analyze the data produced by the NAD tagSeq method or other tagging-based experiments using Oxford nanopore direct RNA sequencing. The pipeline is available at https://github.com/dorothyzh/TagSeqTools, under Apache License 2.0.

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 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 A comprehensive examination of Nanopore native RNA sequencing for characterization of complex transcriptomes

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Charlotte Soneson ◽  
Yao Yao ◽  
Anna Bratus-Neuenschwander ◽  
Andrea Patrignani ◽  
Mark D. Robinson ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Comprehensive Examination

scholarly journals New insights into Arabidopsis transcriptome complexity revealed by direct sequencing of native RNAs

2020 ◽  
Vol 48 (14) ◽  
pp. 7700-7711 ◽  
Author(s):  
Shoudong Zhang ◽  
Runsheng Li ◽  
Li Zhang ◽  
Shengjie Chen ◽  
Min Xie ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Transcription Initiation ◽  
Direct Sequencing ◽  
Functional Characterization ◽  
Read Length ◽  
Long Distance ◽  
Transcript Isoforms ◽  
Fusion Transcripts ◽  
Novel Transcript

Abstract Arabidopsis thaliana transcriptomes have been extensively studied and characterized under different conditions. However, most of the current ‘RNA-sequencing’ technologies produce a relatively short read length and demand a reverse-transcription step, preventing effective characterization of transcriptome complexity. Here, we performed Direct RNA Sequencing (DRS) using the latest Oxford Nanopore Technology (ONT) with exceptional read length. We demonstrate that the complexity of the A. thaliana transcriptomes has been substantially under-estimated. The ONT direct RNA sequencing identified novel transcript isoforms at both the vegetative (14-day old seedlings, stage 1.04) and reproductive stages (stage 6.00–6.10) of development. Using in-house software called TrackCluster, we determined alternative transcription initiation (ATI), alternative polyadenylation (APA), alternative splicing (AS), and fusion transcripts. More than 38 500 novel transcript isoforms were identified, including six categories of fusion-transcripts that may result from differential RNA processing mechanisms. Aided by the Tombo algorithm, we found an enrichment of m5C modifications in the mobile mRNAs, consistent with a recent finding that m5C modification in mRNAs is crucial for their long-distance movement. In summary, ONT DRS offers an advantage in the identification and functional characterization of novel RNA isoforms and RNA base modifications, significantly improving annotation of the A. thaliana genome.

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

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
M. Liem ◽  
T. Regensburg-Tuïnk ◽  
C. Henkel ◽  
H. Jansen ◽  
H. 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 Microbial diversity characterization of seawater in a pilot study using Oxford Nanopore Technologies long-read sequencing

2020 ◽  
Author(s):  
M. 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

ABSTRACTCurrently 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. 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

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