About: A novel method for the capture-based purification of whole viral native RNA genomes

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About: A novel method for the capture-based purification of whole viral native RNA genomes Goto Sponge NotDistinct Permalink

An Entity of Type : schema:ScholarlyArticle, within Data Space : covidontheweb.inria.fr associated with source document(s)

Attributes	Values
type	Academic Article research paper schema:ScholarlyArticle
isDefinedBy	Covid-on-the-Web dataset
has title	A novel method for the capture-based purification of whole viral native RNA genomes
Creator	Maurer-Stroh, Sebastian Wan, Yue Sessions, October Chih, Cedric Florez De Sessions, Paola Tan, Shen
Source	Medline; PMC
abstract	Current technologies for targeted characterization and manipulation of viral RNA primarily involve amplification or ultracentrifugation with isopycnic gradients of viral particles to decrease host RNA background. The former strategy is non-compatible for characterizing properties innate to RNA strands such as secondary structure, RNA–RNA interactions, and also for nanopore direct RNA sequencing involving the sequencing of native RNA strands. The latter strategy, ultracentrifugation, causes loss in genomic information due to its inability to retrieve unassembled viral RNA. To address this, we developed a novel application of current nucleic acid hybridization technologies for direct characterization of RNA. In particular, we modified a current enrichment protocol to capture whole viral native RNA genomes for downstream RNA assays to circumvent the abovementioned problems. This technique involves hybridization of biotinylated baits at 500 nucleotides (nt) intervals, stringent washes and release of free native RNA strands using DNase I treatment, with a turnaround time of about 6 h 15 min. RT-qPCR was used as the primary proof of concept that capture-based purification indeed removes host background. Subsequently, capture-based purification was applied to direct RNA sequencing as proof of concept that capture-based purification can be coupled with downstream RNA assays. We report that this protocol was able to successfully purify viral RNA by 561- to 791-fold. We also report that application of this protocol to direct RNA sequencing yielded a reduction in human host RNA background by 1580-fold, a 99.91% recovery of viral genome with at least 15× coverage, and a mean coverage across the genome of 120×. This report is, to the best of our knowledge, the first description of a capture-based purification method for assays that involve direct manipulation or characterisation of native RNA. This report also describes a successful application of capture-based purification as a direct RNA sequencing strategy that addresses certain limitations of current strategies in sequencing RNA viral genomes. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s13568-019-0772-y) contains supplementary material, which is available to authorized users.
has issue date	2019-04-08(xsd:dateTime)
bibo:doi	10.1186/s13568-019-0772-y
bibo:pmid	30963294
has license	cc-by
sha1sum (hex)	842863c4f79b4d13ef260ef46b9882e8afe2b8ee
schema:url	https://doi.org/10.1186/s13568-019-0772-y
resource representing a document's title	A novel method for the capture-based purification of whole viral native RNA genomes
has PubMed Central identifier	PMC6453989
has PubMed identifier	30963294
schema:publication	AMB Express
resource representing a document's body	covid:842863c4f79b4d13ef260ef46b9882e8afe2b8ee#body_text
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