Analyzing viral epitranscriptomes using nanopore direct RNA sequencing

Hong, Ari; Kim, Dong-Wan; Kim, V. Narry; Chang, Hyeshik

doi:10.1007/s12275-022-2324-4

Cited by 9 publications

(7 citation statements)

References 45 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Differential m 6 A sites between the siControl and siMETTL3 groups were detected using ELIGOS2 and DRUMMER ( 32 , 33 ). Both approaches were designed to identify RNA modifications through the comparative analysis of base-calling errors in the Nanopore DRS dataset ( 34 ). In our study, ELIGOS2 was utilized to assess the global difference in m 6 A modifications, employing strict threshold values of p<0.05 and odd ratio >1.2 ( https://gitlab.com/piroonj/eligos2 ) ( 32 ).…”

Section: Methodsmentioning

confidence: 99%

Dissecting the effects of METTL3 on alternative splicing in prostate cancer

Wang,

Shi,

Liang

et al. 2023

Front. Oncol.

View full text Add to dashboard Cite

Although the role of METTL3 has been extensively studied in many cancers, its role in isoform switching in prostate cancer (PCa) has been poorly explored. To investigate its role, we applied standard RNA-sequencing and long-read direct RNA-sequencing from Oxford Nanopore to examine how METTL3 affects alternative splicing (AS) in two PCa cell lines. By dissecting genome-wide METTL3-regulated AS events, we noted that two PCa cell lines (representing two different PCa subtypes, androgen-sensitive or resistant) behave differently in exon skipping and intron retention events following METTL3 depletion, suggesting AS heterogeneity in PCa. Moreover, we revealed that METTL3-regulated AS is dependent on N6-methyladenosine (m6A) and distinct splicing factors. Analysis of the AS landscape also revealed cell type specific AS signatures for some genes (e.g., MKNK2) involved in key functions in PCa tumorigenesis. Finally, we also validated the clinical relevance of MKNK2 AS events in PCa patients and pointed to the possible regulatory mechanism related to m6A in the exon14a/b region and SRSF1. Overall, we characterize the role of METTL3 in regulating PCa-associated AS programs, expand the role of METTL3 in tumorigenesis, and suggest that MKNK2 AS events may serve as a new potential prognostic biomarker.

show abstract

Section: Methodsmentioning

confidence: 99%

Dissecting the effects of METTL3 on alternative splicing in prostate cancer

Wang,

Shi,

Liang

et al. 2023

Front. Oncol.

View full text Add to dashboard Cite

show abstract

“…Direct RNA sequencing is in its infancy and promises to deliver a quick and reliable alternative method by eliminating amplification steps. While currently dedicated mostly to the analysis of epigenetic modifications of native RNA molecules, the technology is rapidly evolving with advancements in software and methods [ 8 , 9 , 10 , 11 , 12 , 13 ]. This has already led to applications as diverse as viral/viroid detection in grapevines [ 14 ] and to the definition of the soil transcriptome [ 15 ].…”

Section: Advantages Of Non-targeted Rna-based Metagenomics In Unveili...mentioning

confidence: 99%

Non-Targeted RNA Sequencing: Towards the Development of Universal Clinical Diagnosis Methods for Human and Veterinary Infectious Diseases

Spatz,

Afonso

2024

Veterinary Sciences

View full text Add to dashboard Cite

Metagenomics offers the potential to replace and simplify classical methods used in the clinical diagnosis of human and veterinary infectious diseases. Metagenomics boasts a high pathogen discovery rate and high specificity, advantages absent in most classical approaches. However, its widespread adoption in clinical settings is still pending, with a slow transition from research to routine use. While longer turnaround times and higher costs were once concerns, these issues are currently being addressed by automation, better chemistries, improved sequencing platforms, better databases, and automated bioinformatics analysis. However, many technical options and steps, each producing highly variable outcomes, have reduced the technology’s operational value, discouraging its implementation in diagnostic labs. We present a case for utilizing non-targeted RNA sequencing (NT-RNA-seq) as an ideal metagenomics method for the detection of infectious disease-causing agents in humans and animals. Additionally, to create operational value, we propose to identify best practices for the “core” of steps that are invariably shared among many human and veterinary protocols. Reference materials, sequencing procedures, and bioinformatics standards should accelerate the validation processes necessary for the widespread adoption of this technology. Best practices could be determined through “implementation research” by a consortium of interested institutions working on common samples.

show abstract

“…There are different RNA modification detecting approaches. Liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR) are the most fundamental techniques ( 9 , 12 ). They can detect almost all chemical modifications but do not allow the determination of sequence positions and are limited by the difficulty of purifying a particular RNA molecule ( 11 ).…”

Section: Introductionmentioning

confidence: 99%

Utilization of nanopore direct RNA sequencing to analyze viral RNA modifications

Tan,

Guo,

Wang

et al. 2024

mSystems

View full text Add to dashboard Cite

Modifications on viral RNAs (vRNAs), either genomic RNAs or RNA transcripts, have complex effects on the viral life cycle and cellular responses to viral infection. The advent of Oxford Nanopore Technologies Direct RNA Sequencing provides a new strategy for studying RNA modifications. To this end, multiple computational tools have been developed, but a systemic evaluation of their performance in mapping vRNA modifications is lacking. Here, 10 computational tools were tested using the Sindbis virus (SINV) RNAs isolated from infected mammalian (BHK-21) or mosquito (C6/36) cells, with in vitro -transcribed RNAs serving as modification-free control. Three single-mode approaches were shown to be inapplicable in the viral context, and three out of seven comparative methods required cutoff adjustments to reduce false-positive predictions. Utilizing optimized cutoffs, an integrated analysis of comparative tools suggested that the intersected predictions of Tombo_com and xPore were significantly enriched compared with the background. Consequently, a pipeline integrating Tombo_com and xPore was proposed for vRNA modification detection; the performance of which was supported by N 6 -methyladenosine prediction in severe acute respiratory syndrome coronavirus 2 RNAs using publicly available data. When applied to SINV RNAs, this pipeline revealed more intensive modifications in subgenomic RNAs than in genomic RNAs. Modified uridines were frequently identified, exhibiting substantive overlapping between vRNAs generated in different cell lines. On the other hand, the interpretation of other modifications remained unclear, underlining the limitations of the current computational tools despite their notable potential. IMPORTANCE Computational approaches utilizing Oxford Nanopore Technologies Direct RNA Sequencing data were almost exclusively designed to map eukaryotic epitranscriptomes. Therefore, extra caution must be exercised when using these tools to detect vRNA modifications, as in most cases, vRNA modification profiles should be regarded as unknown epitranscriptomes without prior knowledge. Here, we comprehensively evaluated the performance of 10 computational tools in detecting vRNA modification sites. All tested single-mode methods failed to differentiate native and in vitro- transcribed samples. Using optimized cutoff values, seven tested comparative tools generated very different predictions. An integrated analysis showed significant enrichment of Tombo_com and xPore predictions against the background. A pipeline for vRNA modification detection was proposed accordingly and applied to Sindbis virus RNAs. In conclusion, our study underscores the need for the careful application of computational tools to analyze viral epitranscriptomics. It also offers insights into alphaviral RNA modifications, although further validation is required.

show abstract

Analyzing viral epitranscriptomes using nanopore direct RNA sequencing

Cited by 9 publications

References 45 publications

Dissecting the effects of METTL3 on alternative splicing in prostate cancer

Dissecting the effects of METTL3 on alternative splicing in prostate cancer

Non-Targeted RNA Sequencing: Towards the Development of Universal Clinical Diagnosis Methods for Human and Veterinary Infectious Diseases

Utilization of nanopore direct RNA sequencing to analyze viral RNA modifications

Contact Info

Product

Resources

About