Exploring the epitranscriptome by native RNA sequencing

Begik, Oguzhan; Mattick, John S.; Novoa, Eva María

doi:10.1261/rna.079404.122

Cited by 40 publications

(38 citation statements)

References 162 publications

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“…To reveal characteristics of the rRNA processing pathway(s) in archaea we applied long-read nanopore sequencing to the total RNA of three archaeal model organisms, namely the Euryarchaea Haloferax volcanii and Pyrococcus furiosus and the Crenarchaeon Sulfolobus acidocaldarius (Figure 1A). For maturation stage classification, amplification-free direct cDNA sequencing was performed in a poly(A)-independent way using a custom 3’-adapter to improve the accuracy of 3’ end detection (Figure 1B,C,D), while direct RNA sequencing was used for stage-dependent base modification analysis (Figure 1B,E) (14, 21, 34). Note that we previously showed that artificial polyadenylation of prokaryotic RNAs impairs the accuracy of 3’ detection (14).…”

Section: Resultsmentioning

confidence: 99%

“…Cleavage sites at the bulge-helix-bulge are indicated by orange triangles and dashed vertical lines. E, RNA base modifications can lead to deviations in the recorded current intensities from the expected signal and/or to systematic basecalling errors, which can be used to detect RNA modifications (34).…”

Section: Resultsmentioning

confidence: 99%

“…In turn, these signal changes might also lead to differences in the basecalling profiles (e.g. systematic errors or a drop in basecalling quality) (34).…”

Section: Resultsmentioning

confidence: 99%

“…Several recent analyses have explored different strategies to evaluate the capacity of nanopore sequencing to accurately detect RNA modifications (e.g. m 6 A) (13, 30, 34, 39–41, 54, 55).…”

Section: Discussionmentioning

confidence: 99%

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Insights into rRNA processing and modification mapping in Archaea using Nanopore-based RNA sequencing

Gruenberger

Ferreira-Cerca

Grohmann

2021

Preprint

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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 cDNA-seq offers improved yield and accuracy without bias in quantification compared to direct RNA sequencing. Notably, cDNA-seq can be readily used for simultaneous transcript quantification, accurate detection of transcript 5′ and 3′ boundaries, analysis of transcriptional units and transcriptional heterogeneity. In summary, we establish Nanopore RNA-seq to be a ready-to-use tool allowing rapid, cost-effective, and accurate annotation of multiple transcriptomic features thereby advancing it to become a standard method for RNA analysis in prokaryotes.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

“…In turn, these signal changes might also lead to differences in the basecalling profiles (e.g. systematic errors or a drop in basecalling quality) (34).…”

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Insights into rRNA processing and modification mapping in Archaea using Nanopore-based RNA sequencing

Gruenberger

Ferreira-Cerca

Grohmann

2021

Preprint

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“…Despite these advances, several major obstacles remain to detecting a broad repertoire of nucleic acid modifications by machine learning approaches. Developing robust algorithms for modified base calling requires DNA and RNA training sets that contain modified nucleotides in all possible sequence contexts, a non-trivial endeavor for nucleic acid modifications that cannot be readily generated by chemical synthesis or enzymatic modification ( Begik et al, 2022 ). This includes the majority of the 170+ endogenous modifications present on RNA molecules ( Boccaletto et al, 2022 ).…”

Section: Bioinformatic Strategies For De Novo Modi...mentioning

confidence: 99%

Modification mapping by nanopore sequencing

White

Hesselberth

2022

Front. Genet.

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Next generation sequencing (NGS) has provided biologists with an unprecedented view into biological processes and their regulation over the past 2 decades, fueling a wave of development of high throughput methods based on short read DNA and RNA sequencing. For nucleic acid modifications, NGS has been coupled with immunoprecipitation, chemical treatment, enzymatic treatment, and/or the use of reverse transcriptase enzymes with fortuitous activities to enrich for and to identify covalent modifications of RNA and DNA. However, the majority of nucleic acid modifications lack commercial monoclonal antibodies, and mapping techniques that rely on chemical or enzymatic treatments to manipulate modification signatures add additional technical complexities to library preparation. Moreover, such approaches tend to be specific to a single class of RNA or DNA modification, and generate only indirect readouts of modification status. Third generation sequencing technologies such as the commercially available “long read” platforms from Pacific Biosciences and Oxford Nanopore Technologies are an attractive alternative for high throughput detection of nucleic acid modifications. While the former can indirectly sense modified nucleotides through changes in the kinetics of reverse transcription reactions, nanopore sequencing can in principle directly detect any nucleic acid modification that produces a signal distortion as the nucleic acid passes through a nanopore sensor embedded within a charged membrane. To date, more than a dozen endogenous DNA and RNA modifications have been interrogated by nanopore sequencing, as well as a number of synthetic nucleic acid modifications used in metabolic labeling, structure probing, and other emerging applications. This review is intended to introduce the reader to nanopore sequencing and key principles underlying its use in direct detection of nucleic acid modifications in unamplified DNA or RNA samples, and outline current approaches for detecting and quantifying nucleic acid modifications by nanopore sequencing. As this technology matures, we anticipate advances in both sequencing chemistry and analysis methods will lead to rapid improvements in the identification and quantification of these epigenetic marks.

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Epigenetic Control of Cell Fate Decisions by Enhancer-Derived Long Noncoding RNAs

Mattick

2024

Epigenetics in Biological Communication

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Exploring the epitranscriptome by native RNA sequencing

Cited by 40 publications

References 162 publications

Insights into rRNA processing and modification mapping in Archaea using Nanopore-based RNA sequencing

Insights into rRNA processing and modification mapping in Archaea using Nanopore-based RNA sequencing

Modification mapping by nanopore sequencing

Epigenetic Control of Cell Fate Decisions by Enhancer-Derived Long Noncoding RNAs

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