Comparison of direct RNA sequencing of Orthoavulavirus javaense using two different chemistries on the MinION platform DOI Creative Commons
Megan C. Mears, Quentin D. Read, Abhijeet Bakre

et al.

Journal of Virological Methods, Journal Year: 2024, Volume and Issue: unknown, P. 115103 - 115103

Published: Dec. 1, 2024

Rapidly identifying and sequencing viral pathogens in poultry flocks can substantially reduce economic loss especially during disease outbreaks. Current next generation technologies require multi-step laboratory-intensive workflows to generate sequence data which precludes field adaptation. In this study, we hypothesized that direct RNA (DRS) using an Oxford Nanopore Technology (ONT) MinION device would enable of the full-length genome Orthoavulavirus javaense (OAVJ), causative Newcastle disease, a major challenge. The demonstrate custom OAVJ-specific adapter paired with ONT DRS kits enables capture OAVJ RNAs. Further, new SQK-RNA004 chemistry flow cells, associated super accurate base calling workflow improves on read quality length compared previous SQK-RNA002 chemistry. This is first report method near member Paramyxoviridae family. While additional improvements are needed before widespread adaptation for rapid sequencing, has potential further studies into epitranscriptome its role infection pathogenesis.

Language: Английский

Analysis of bacterial transcriptome and epitranscriptome using nanopore direct RNA sequencing DOI Creative Commons
Lu Tan, Zhihao Guo, Yanwen Shao

et al.

Nucleic Acids Research, Journal Year: 2024, Volume and Issue: 52(15), P. 8746 - 8762

Published: July 16, 2024

Abstract Bacterial gene expression is a complex process involving extensive regulatory mechanisms. Along with growing interests in this field, Nanopore Direct RNA Sequencing (DRS) provides promising platform for rapid and comprehensive characterization of bacterial biology. However, the DRS currently deficient yield mRNA-mapping reads has yet to be exploited transcriptome-wide modification mapping. Here, we showed that pre-processing total (size selection followed by ribosomal depletion polyadenylation) guaranteed high throughputs sequencing data considerably increased amount mRNA reads. This way, transcriptome architectures were reconstructed Escherichia coli Staphylococcus aureus extended boundaries 225 known E. operons 89 defined S. operons. Utilizing unmodified vitro-transcribed (IVT) libraries as negative control, several Nanopore-based computational tools globally detected putative sites transcriptomes. Combined Next-Generation Sequencing-based N6-methyladenosine (m6A) detection methods, 75 high-confidence m6A candidates identified protein-coding transcripts, while none aureus. Altogether, demonstrated potential systematic convenient epitranscriptome analysis.

Language: Английский

Citations

6
Wolbachia elevates host methyltransferase expression and alters the m6A methylation landscape in Aedes aegypti mosquito cells DOI Creative Commons
Michael Leitner, Valentine Murigneux, Kayvan Etebari

et al.

BMC Microbiology, Journal Year: 2025, Volume and Issue: 25(1)

Published: March 25, 2025

Wolbachia pipientis is an intracellular endosymbiotic bacterium that blocks the replication of several arboviruses in transinfected Aedes aegypti mosquitoes, yet its antiviral mechanism remains unknown. For first time, we employed Nanopore direct RNA sequencing technology to investigate impact wAlbB strain on host's N6-methyladenosine (m6A) machinery and post-transcriptional modification landscape. Our study revealed infection elevates expression genes involved mosquito's m6A methyltransferase complex. However, knocking down these m6A-related did not affect density. identified 1,392 differentially modified DRACH motifs mosquito transcripts, with 776 showing increased 616 decreased levels due Wolbachia. These sites were predominantly enriched coding sequences 3′-untranslated regions. Gene Ontology analysis reduced over-represented functional GO terms associated purine nucleotide binding functions critical process m6A. Differential gene data uncovered a total 643 protein-coding significantly expressed, 427 downregulated, 216 upregulated. Several classical non-classical immune-related amongst downregulated DEGs. Notably, it host factor, transmembrane protein 41B (TMEM41B), which required for flavivirus infection, was upregulated methylated presence Indeed, there strong correlation between being both modification, respectively. findings underscore Wolbachia's ability modulate many aspects by influencing modifications expression, unveils potential link behind properties.

Language: Английский

Citations

0
Quantitative profiling N1-methyladenosine (m1A) RNA methylation from Oxford nanopore direct RNA sequencing data DOI

Shenglun Chen,

Jia Meng, Yuxin Zhang

et al.

Methods, Journal Year: 2024, Volume and Issue: 228, P. 30 - 37

Published: May 18, 2024

Language: Английский

Citations

2
Comparison of direct RNA sequencing of Orthoavulavirus javaense using two different chemistries on the MinION platform DOI Creative Commons
Megan C. Mears, Quentin D. Read, Abhijeet Bakre

et al.

Journal of Virological Methods, Journal Year: 2024, Volume and Issue: unknown, P. 115103 - 115103

Published: Dec. 1, 2024

Rapidly identifying and sequencing viral pathogens in poultry flocks can substantially reduce economic loss especially during disease outbreaks. Current next generation technologies require multi-step laboratory-intensive workflows to generate sequence data which precludes field adaptation. In this study, we hypothesized that direct RNA (DRS) using an Oxford Nanopore Technology (ONT) MinION device would enable of the full-length genome Orthoavulavirus javaense (OAVJ), causative Newcastle disease, a major challenge. The demonstrate custom OAVJ-specific adapter paired with ONT DRS kits enables capture OAVJ RNAs. Further, new SQK-RNA004 chemistry flow cells, associated super accurate base calling workflow improves on read quality length compared previous SQK-RNA002 chemistry. This is first report method near member Paramyxoviridae family. While additional improvements are needed before widespread adaptation for rapid sequencing, has potential further studies into epitranscriptome its role infection pathogenesis.

Language: Английский

Citations

0