Latest RNA and DNA nanopore sequencing allows for rapid avian influenza profiling

Perlas, Albert; Reska, Tim; Croville, Guillaume; Tarrés-Freixas, Ferran; Guérin, Jean-Luc; Majó, Natàlia; Urban, Lara

doi:10.1101/2024.02.28.582540

Cited by 3 publications

(1 citation statement)

References 57 publications

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“…Rapid phenotypic proling of viruses has critical importance in pandemic prevention and response, for which nanopore sequencing oers unique benets [13]. Multiplexing samples on a single ow cell increases the speed and eciency of proling, while also reducing experimental variation.…”

Section: Rapid Phenotyping Of Sars-cov-2 Viruses On a Single Ow Cellmentioning

confidence: 99%

Demultiplexing and barcode-specific adaptive sampling for nanopore direct RNA sequencing

van der Toorn,

Bohn,

Liu-Wei

et al. 2024

Preprint

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Nanopore direct RNA sequencing (dRNA-seq) enables unique insights into (epi-)transcriptomics. However, applications are currently limited by the lack of accurate and cost-effective sample multiplexing. We introduce WarpDemuX, an ultra-fast and highly accurate adapter-barcoding and demultiplexing approach. WarpDemuX enhances speed and accuracy by fast processing of the raw nanopore signal, use of a light-weight machine-learning algorithm and design of optimized barcode sets. We demonstrate its utility by performing a rapid phenotypic profiling of different SARS-CoV-2 viruses, crucial for pandemic prevention and response, through multiplexed sequencing of longitudinal samples on a single flowcell. This identifies systematic differences in transcript abundance and poly(A) tail lengths during infection. Additionally, integrating WarpDemuX into sequencing control software enables real-time enrichment of target molecules through barcode-specific adaptive sampling, which we demonstrate by enriching low abundance viral RNA. In summary, WarpDemuX is a broadly applicable, high-performance, and economical multiplexing solution for nanopore dRNA-seq, facilitating advanced (epi-)transcriptomic research.

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Section: Rapid Phenotyping Of Sars-cov-2 Viruses On a Single Ow Cellmentioning

confidence: 99%

Demultiplexing and barcode-specific adaptive sampling for nanopore direct RNA sequencing

van der Toorn,

Bohn,

Liu-Wei

et al. 2024

Preprint

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Viral genome sequencing methods: benefits and pitfalls of current approaches

Jansz,

Faulkner

2024

Biochemical Society Transactions

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Whole genome sequencing of viruses provides high-resolution molecular insights, enhancing our understanding of viral genome function and phylogeny. Beyond fundamental research, viral sequencing is increasingly vital for pathogen surveillance, epidemiology, and clinical applications. As sequencing methods rapidly evolve, the diversity of viral genomics applications and catalogued genomes continues to expand. Advances in long-read, single molecule, real-time sequencing methodologies present opportunities to sequence contiguous, haplotype resolved viral genomes in a range of research and applied settings. Here we present an overview of nucleic acid sequencing methods and their applications in studying viral genomes. We emphasise the advantages of different viral sequencing approaches, with a particular focus on the benefits of third-generation sequencing technologies in elucidating viral evolution, transmission networks, and pathogenesis

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An optimized protocol for quality control of gene therapy vectors using nanopore direct RNA sequencing

Zeglinski,

Montellese,

Ritchie

et al. 2024

Genome Res.

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Despite recent advances made toward improving the efficacy of lentiviral gene therapies, a sizeable proportion of produced vector contains an incomplete and thus potentially nonfunctional RNA genome. This can undermine gene delivery by the lentivirus as well as increase manufacturing costs and must be improved to facilitate the widespread clinical implementation of lentiviral gene therapies. Here, we compare three long-read sequencing technologies for their ability to detect issues in vector design and determine nanopore direct RNA sequencing to be the most powerful. We show how this approach identifies and quantifies incomplete RNA caused by cryptic splicing and polyadenylation sites, including a potential cryptic polyadenylation site in the widely used Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE). Using artificial polyadenylation of the lentiviral RNA, we also identify multiple hairpin-associated truncations in the analyzed lentiviral vectors (LVs), which account for most of the detected RNA fragments. Finally, we show that these insights can be used for the optimization of LV design. In summary, nanopore direct RNA sequencing is a powerful tool for the quality control and optimization of LVs, which may help to improve lentivirus manufacturing and thus the development of higher quality lentiviral gene therapies.

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Latest RNA and DNA nanopore sequencing allows for rapid avian influenza profiling

Cited by 3 publications

References 57 publications

Demultiplexing and barcode-specific adaptive sampling for nanopore direct RNA sequencing

Demultiplexing and barcode-specific adaptive sampling for nanopore direct RNA sequencing

Viral genome sequencing methods: benefits and pitfalls of current approaches

An optimized protocol for quality control of gene therapy vectors using nanopore direct RNA sequencing

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