Transcriptome landscape of the developing olive fruit fly embryo delineated by Oxford Nanopore long-read RNA-Seq

Bayega, Anthony; Oikonomopoulos, Spyros; Zorbas, E; Yc, Wang; Gregoriou, Maria‐Eleni; Kt, Tsoumani; Kd, Mathiopoulos; Ragoussis, Jiannis

doi:10.1101/478172

Cited by 8 publications

(13 citation statements)

References 49 publications

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“…As ONT-based native RNA-seq holds the capacity to sequence full-length transcripts, to identify RNA base modifications and to detect molecular heterogeneity in a transcriptome, the technology found widespread attention 16 . Recently, the technology was exploited to sequence viral RNA genomes 17–20 to gain insights into viral and eukaryotic transcriptomes 19,21–23 and to detect RNA isoforms in eukaryotes 24,25 . However, prokaryotic transcriptomes have not been characterized on the genome-wide level by native RNA-seq approaches so far as prokaryotic RNAs lack a poly(A) tail which is required to capture the RNA and feed it into the nanopore.…”

Section: Introductionmentioning

confidence: 99%

Exploring prokaryotic transcription, operon structures, rRNA maturation and modifications using Nanopore-based native RNA sequencing

Grünberger

Knüppel

Jüttner

et al. 2019

Preprint

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The prokaryotic transcriptome is shaped by transcriptional and posttranscriptional events that define the characteristics of an RNA, including transcript boundaries, the base modification status, and processing pathways to yield mature RNAs. Currently, a combination of several specialised short-read sequencing approaches and additional biochemical experiments are required to describe all transcriptomic features. In this study, we present native RNA sequencing of bacterial (E. coli) and archaeal (H. volcanii, P. furiosus) transcriptomes employing the Oxford Nanopore sequencing technology. Based on this approach, we could address multiple transcriptomic characteristics simultaneously with single-molecule resolution. Taking advantage of long RNA reads provided by the Nanopore platform, we could (re-)annotate large transcriptional units and boundaries. Our analysis of transcription termination sites suggests that diverse termination mechanisms are in place in archaea. Moreover, we shed additional light on the poorly understood rRNA processing pathway in Archaea. One of the key features of native RNA sequencing is that RNA modifications are retained. We could confirm this ability by analysing the well-known KsgA-dependent methylation sites and mapping of N4-acetylcytosines modifications in rRNAs. Notably, we were able to follow the relative timely order of the installation of these modifications in the rRNA processing pathway.

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Section: Introductionmentioning

confidence: 99%

Exploring prokaryotic transcription, operon structures, rRNA maturation and modifications using Nanopore-based native RNA sequencing

Grünberger

Knüppel

Jüttner

et al. 2019

Preprint

View full text Add to dashboard Cite

show abstract

“…Nanopore sequencing, in contrast, has no theoretical upper limit to read length and is capable of sequencing transcripts from end to end at a single molecule level (Garalde et al 2018;Jenjaroenpun et al 2018;Workman et al 2019). Nanopore-based sequencing methods have been used to annotate transcriptome structure in a variety of organisms ranging from the relatively simple Saccharomyces cerevisiae to complex human cell lines (Byrne et al 2017;Bayega et al 2018;Garalde et al 2018;Jenjaroenpun et al 2018;Tang et al 2018;Volden et al 2018;Kadobianskyi et al 2019;Sessegolo et al 2019;Workman et al 2019). In nanoporebased direct RNA sequencing (dRNA-seq), RNA reads are captured by the 3 ′ end of their poly(A) tail and sequenced in the 3 ′ to 5 ′ direction natively, thus directly measuring the RNA molecule.…”

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confidence: 99%

The full-length transcriptome of C. elegans using direct RNA sequencing

et al. 2020

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Current transcriptome annotations have largely relied on short read lengths intrinsic to the most widely used high-throughput cDNA sequencing technologies. For example, in the annotation of the Caenorhabditis elegans transcriptome, more than half of the transcript isoforms lack full-length support and instead rely on inference from short reads that do not span the full length of the isoform. We applied nanopore-based direct RNA sequencing to characterize the developmental polyadenylated transcriptome of C. elegans. Taking advantage of long reads spanning the full length of mRNA transcripts, we provide support for 23,865 splice isoforms across 14,611 genes, without the need for computational reconstruction of gene models. Of the isoforms identified, 3452 are novel splice isoforms not present in the WormBase WS265 annotation. Furthermore, we identified 16,342 isoforms in the 3 ′ untranslated region (3 ′ UTR), 2640 of which are novel and do not fall within 10 bp of existing 3 ′-UTR data sets and annotations. Combining 3 ′ UTRs and splice isoforms, we identified 28,858 fulllength transcript isoforms. We also determined that poly(A) tail lengths of transcripts vary across development, as do the strengths of previously reported correlations between poly(A) tail length and expression level, and poly(A) tail length and 3 ′-UTR length. Finally, we have formatted this data as a publicly accessible track hub, enabling researchers to explore this data set easily in a genome browser.

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“…The most widely approach for cDNA synthesis is the highly sensitive Smart-seq2 protocol (Picelli et al, 2013), which uses template switching and preamplification (Figure 1). It utilizes a combination of custom reagents and kits and is similar to the methodology tailored to long-read sequencing we recently published (Bayega et al, 2018b). It is important to consider the reverse transcriptase used in cDNA synthesis.…”

Section: Cdna Synthesismentioning

confidence: 99%

Methodologies for Transcript Profiling Using Long-Read Technologies

et al. 2020

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RNA sequencing using next-generation sequencing technologies (NGS) is currently the standard approach for gene expression profiling, particularly for large-scale highthroughput studies. NGS technologies comprise high throughput, cost efficient shortread RNA-Seq, while emerging single molecule, long-read RNA-Seq technologies have enabled new approaches to study the transcriptome and its function. The emerging single molecule, long-read technologies are currently commercially available by Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT), while new methodologies based on short-read sequencing approaches are also being developed in order to provide long range single molecule level information-for example, the ones represented by the 10x Genomics linked read methodology. The shift toward longread sequencing technologies for transcriptome characterization is based on current increases in throughput and decreases in cost, making these attractive for de novo transcriptome assembly, isoform expression quantification, and in-depth RNA species analysis. These types of analyses were challenging with standard short sequencing approaches, due to the complex nature of the transcriptome, which consists of variable lengths of transcripts and multiple alternatively spliced isoforms for most genes, as well as the high sequence similarity of highly abundant species of RNA, such as rRNAs. Here we aim to focus on single molecule level sequencing technologies and single-cell technologies that, combined with perturbation tools, allow the analysis of complete RNA species, whether short or long, at high resolution. In parallel, these tools have opened new ways in understanding gene functions at the tissue, network, and pathway levels, as well as their detailed functional characterization. Analysis of the epi-transcriptome, including RNA methylation and modification and the effects of such modifications on biological systems is now enabled through direct RNA sequencing instead of classical indirect approaches. However, many difficulties and challenges remain, such as methodologies to generate full-length RNA or cDNA libraries from all different species of RNAs, not only poly-A containing transcripts, and the identification of allele-specific transcripts due to current error rates of single molecule technologies, while the bioinformatics analysis on long-read data for accurate identification of 5 and 3 UTRs is still in development.

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Transcriptome landscape of the developing olive fruit fly embryo delineated by Oxford Nanopore long-read RNA-Seq

Abstract: characterization of the isoform complexity and the transcriptional dynamics of the first embryonic stages of the B. oleae.

Cited by 8 publications

References 49 publications

Exploring prokaryotic transcription, operon structures, rRNA maturation and modifications using Nanopore-based native RNA sequencing

Exploring prokaryotic transcription, operon structures, rRNA maturation and modifications using Nanopore-based native RNA sequencing

The full-length transcriptome of C. elegans using direct RNA sequencing

Methodologies for Transcript Profiling Using Long-Read Technologies

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