scholarly journals Analysis of RNA Modifications by Second- and Third-Generation Deep Sequencing: 2020 Update

Genes ◽  
2021 ◽  
Vol 12 (2) ◽  
pp. 278
Author(s):  
Yuri Motorin ◽  
Virginie Marchand
Keyword(s):  
Single Molecule ◽  
Deep Sequencing ◽  
Rna Modification ◽  
Rna Modifications ◽  
Single Molecule Sequencing ◽  
Abasic Sites ◽  
Phosphate Chain ◽  
Adapter Ligation ◽  
Ribose Phosphate ◽  
Precise Mapping

The precise mapping and quantification of the numerous RNA modifications that are present in tRNAs, rRNAs, ncRNAs/miRNAs, and mRNAs remain a major challenge and a top priority of the epitranscriptomics field. After the keystone discoveries of massive m6A methylation in mRNAs, dozens of deep sequencing-based methods and protocols were proposed for the analysis of various RNA modifications, allowing us to considerably extend the list of detectable modified residues. Many of the currently used methods rely on the particular reverse transcription signatures left by RNA modifications in cDNA; these signatures may be naturally present or induced by an appropriate enzymatic or chemical treatment. The newest approaches also include labeling at RNA abasic sites that result from the selective removal of RNA modification or the enhanced cleavage of the RNA ribose-phosphate chain (perhaps also protection from cleavage), followed by specific adapter ligation. Classical affinity/immunoprecipitation-based protocols use either antibodies against modified RNA bases or proteins/enzymes, recognizing RNA modifications. In this survey, we review the most recent achievements in this highly dynamic field, including promising attempts to map RNA modifications by the direct single-molecule sequencing of RNA by nanopores.

scholarly journals Mapping the epigenetic modifications of DNA and RNA

2020 ◽  
Vol 11 (11) ◽  
pp. 792-808 ◽  
Author(s):  
Lin-Yong Zhao ◽  
Jinghui Song ◽  
Yibin Liu ◽  
Chun-Xiao Song ◽  
Chengqi Yi
Keyword(s):  
Single Molecule ◽  
Detection Methods ◽  
Rna Modification ◽  
Rna Modifications ◽  
Single Molecule Sequencing ◽  
Dna And Rna ◽  
Third Generation Sequencing ◽  
Technological Advances ◽  
Long Read ◽  
High Throughput Detection

Abstract Over 17 and 160 types of chemical modifications have been identified in DNA and RNA, respectively. The interest in understanding the various biological functions of DNA and RNA modifications has lead to the cutting-edged fields of epigenomics and epitranscriptomics. Developing chemical and biological tools to detect specific modifications in the genome or transcriptome has greatly facilitated their study. Here, we review the recent technological advances in this rapidly evolving field. We focus on high-throughput detection methods and biological findings for these modifications, and discuss questions to be addressed as well. We also summarize third-generation sequencing methods, which enable long-read and single-molecule sequencing of DNA and RNA modification.

scholarly journals Methods for RNA Modification Mapping Using Deep Sequencing: Established and New Emerging Technologies

Genes ◽  
2019 ◽  
Vol 10 (1) ◽  
pp. 35 ◽  
Author(s):  
Yuri Motorin ◽  
Mark Helm
Keyword(s):  
Deep Sequencing ◽  
Global Scale ◽  
High Rate ◽  
Rna Modification ◽  
Transcriptome Data ◽  
Rna Seq ◽  
Rna Modifications ◽  
Accuracy And Precision ◽  
A Cell ◽  
Mapping Techniques

New analytics of post-transcriptional RNA modifications have paved the way for a tremendous upswing of the biological and biomedical research in this field. This especially applies to methods that included RNA-Seq techniques, and which typically result in what is termed global scale modification mapping. In this process, positions inside a cell`s transcriptome are receiving a status of potential modification sites (so called modification calling), typically based on a score of some kind that issues from the particular method applied. The resulting data are thought to represent information that goes beyond what is contained in typical transcriptome data, and hence the field has taken to use the term “epitranscriptome”. Due to the high rate of newly published mapping techniques, a significant number of chemically distinct RNA modifications have become amenable to mapping, albeit with variegated accuracy and precision, depending on the nature of the technique. This review gives a brief overview of known techniques, and how they were applied to modification calling.

scholarly journals Single-Molecule Sequencing Reveals Complex Genome Variation of Hepatitis B Virus during 15 Years of Chronic Infection following Liver Transplantation

2016 ◽  
Vol 90 (16) ◽  
pp. 7171-7183 ◽  
Author(s):  
B. D. Betz-Stablein ◽  
A. Töpfer ◽  
M. Littlejohn ◽  
L. Yuen ◽  
D. Colledge ◽  
...  
Keyword(s):  
Liver Transplantation ◽  
Drug Resistance ◽  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Single Molecule ◽  
Deep Sequencing ◽  
Wild Type ◽  
Single Molecule Sequencing ◽  
Treatment Regimens ◽  
B Virus

ABSTRACTChronic hepatitis B (CHB) is prevalent worldwide. The infectious agent, hepatitis B virus (HBV), replicates via an RNA intermediate and is error prone, leading to the rapid generation of closely related but not identical viral variants, including those that can escape host immune responses and antiviral treatments. The complexity of CHB can be further enhanced by the presence of HBV variants with large deletions in the genome generated via splicing (spHBV variants). Although spHBV variants are incapable of autonomous replication, their replication is rescued by wild-type HBV. spHBV variants have been shown to enhance wild-type virus replication, and their prevalence increases with liver disease progression. Single-molecule deep sequencing was performed on whole HBV genomes extracted from samples, including the liver explant, longitudinally collected from a subject with CHB over a 15-year period after liver transplantation. By employing novel bioinformatics methods, this analysis showed that the dynamics of the viral population across a period of changing treatment regimens was complex. The spHBV variants detected in the liver explant remained present posttransplantation, and a highly diverse novel spHBV population as well as variants with multiple deletions in the pre-S genes emerged. The identification of novel mutations outside the HBV reverse transcriptase gene that co-occurred with known drug resistance-associated mutations highlights the relevance of using full-genome deep sequencing and supports the hypothesis that drug resistance involves interactions across the full length of the HBV genome.IMPORTANCESingle-molecule sequencing allowed the characterization, in unprecedented detail, of the evolution of HBV populations and offered unique insights into the dynamics of defective and spHBV variants following liver transplantation and complex treatment regimens. This analysis also showed the rapid adaptation of HBV populations to treatment regimens with evolving drug resistance phenotypes and evidence of purifying selection across the whole genome. Finally, the new open-source bioinformatics tools with the capacity to easily identify potential spliced variants from deep sequencing data are freely available.

Hypoxia-induced alteration of RNA modifications in the mouse testis and sperm

2021 ◽  
Author(s):  
Tong He ◽  
Huanping Guo ◽  
Xipeng Shen ◽  
Xiao Wu ◽  
Lin Xia ◽  
...  
Keyword(s):  
Hypobaric Hypoxia ◽  
Environmental Changes ◽  
Epigenetic Inheritance ◽  
Transcriptional Regulators ◽  
Extreme Environment ◽  
Mouse Testis ◽  
Rna Modification ◽  
Rna Modifications ◽  
Sperm Counts ◽  
Health Studies

Abstract Hypobaric hypoxia as an extreme environment in a plateau may have deleterious effects on human health. Studies have indicated that rush entry into a plateau may reduce male fertility and manifest in decreased sperm counts and weakened sperm motility. RNA modifications are sensitive to environmental changes and have recently emerged as novel post-transcriptional regulators in male spermatogenesis and intergenerational epigenetic inheritance. In the present study, we generated a mouse hypoxia model simulating the environment of 5500 meters in altitude for 35 days, which led to compromised spermatogenesis, decreased sperm counts, and an increased sperm deformation rate. Using this hypoxia model, we further applied our recently developed high-throughput RNA modification quantification platform based on LC–MS/MS, which exhibited the capacity to simultaneously examine 25 types of RNA modifications. Our results revealed an altered sperm RNA modifications signature in the testis (6 types) and mature sperm (11 types) under the hypoxia model, with 4 types showing overlap (Am, Gm, m7G, and m22G). Our data first drew the signature of RNA modification profiles and comprehensively analyzed the alteration of RNA modification levels in mouse testis and sperm under a mouse hypoxia model. These data may be highly related to human conditions under a similar hypoxia environment.

scholarly journals RNA Modification Level Estimation with pulseR

Genes ◽  
2018 ◽  
Vol 9 (12) ◽  
pp. 619
Author(s):  
Etienne Boileau ◽  
Christoph Dieterich
Keyword(s):  
Experimental Approach ◽  
High Efficiency ◽  
Rna Modification ◽  
Model Parameters ◽  
Metabolic Labeling ◽  
Rna Seq ◽  
Rna Modifications ◽  
Log Odds ◽  
Pros And Cons ◽  
Wide Scale

RNA modifications regulate the complex life of transcripts. An experimental approach called LAIC-seq was developed to characterize modification levels on a transcriptome-wide scale. In this method, the modified and unmodified molecules are separated using antibodies specific for a given RNA modification (e.g., m6A). In essence, the procedure of biochemical separation yields three fractions: Input, eluate, and supernatent, which are subjected to RNA-seq. In this work, we present a bioinformatics workflow, which starts from RNA-seq data to infer gene-specific modification levels by a statistical model on a transcriptome-wide scale. Our workflow centers around the pulseR package, which was originally developed for the analysis of metabolic labeling experiments. We demonstrate how to analyze data without external normalization (i.e., in the absence of spike-ins), given high efficiency of separation, and how, alternatively, scaling factors can be derived from unmodified spike-ins. Importantly, our workflow provides an estimate of uncertainty of modification levels in terms of confidence intervals for model parameters, such as gene expression and RNA modification levels. We also compare alternative model parametrizations, log-odds, or the proportion of the modified molecules and discuss the pros and cons of each representation. In summary, our workflow is a versatile approach to RNA modification level estimation, which is open to any read-count-based experimental approach.

scholarly journals Genome assembly of the maize inbred line A188 provides a new reference genome for functional genomics

2021 ◽  
Author(s):  
Fei Ge ◽  
Jingtao Qu ◽  
Peng Liu ◽  
Lang Pan ◽  
Chaoying Zou ◽  
...  
Keyword(s):  
Single Molecule ◽  
Inbred Line ◽  
Genome Mapping ◽  
Maize Inbred Line ◽  
Sequencing Data ◽  
Structural Variations ◽  
Single Molecule Sequencing ◽  
Maize Genetic ◽  
Induction Ratio ◽  
Phenotypic Variations

Heretofore, little is known about the mechanism underlying the genotype-dependence of embryonic callus (EC) induction, which has severely inhibited the development of maize genetic engineering. Here, we report the genome sequence and annotation of a maize inbred line with high EC induction ratio, A188, which is assembled from single-molecule sequencing and optical genome mapping. We assembled a 2,210 Mb genome with a scaffold N50 size of 11.61 million bases (Mb), compared to those of 9.73 Mb for B73 and 10.2 Mb for Mo17. Comparative analysis revealed that ~30% of the predicted A188 genes had large structural variations to B73, Mo17 and W22 genomes, which caused considerable protein divergence and might lead to phenotypic variations between the four inbred lines. Combining our new A188 genome, previously reported QTLs and RNA sequencing data, we reveal 8 large structural variation genes and 4 differentially expressed genes playing potential roles in EC induction.

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