title: The Effect of Nucleic Acid Extraction Platforms and Sample Storage on the Integrity of Viral RNA for Use in Whole Genome Sequencing.
abstract: Extraction of viral RNA and the storage of sample material are extremely important factors in the detection and whole genome sequencing (WGS) of viral pathogens. Although PCR-based detection methods focus on small amplicons, viral WGS applications require RNA of high quality and integrity for adequate sequence coverage and depth. This study examined the fitness of one manual and four automated RNA extraction platforms commonly used in diagnostic laboratories for use in metagenomic sequencing, how the practice of storing sample material in Qiagen buffer AVL before extraction affected the integrity of viral RNA and its suitability for use in amplicon-based WGS methods, and how the addition of Triton X-100 to buffer AVL affected the capability of the extraction platforms and the integrity of viral RNA in stored samples. This study found that the EZ1 platform gave the best performance of the automated platforms and gave comparable results to the frequently used manual Qiagen extraction protocol when extracted viral RNA was used in metagenomics sequencing. To maintain high levels of viral RNA integrity suitable for amplicon-based WGS, nucleic acid should be extracted from samples immediately, because even short storage periods in buffer AVL have a severe effect on integrity, and the addition of Triton X-100 had little effect on the quality of viral material for WGS.
authors.affiliation: National Infection Service, Public Health England, Salisbury, United Kingdom. Electronic address: kuiama.lewandowski@phe.gov.uk.
authors.forename: Kuiama
authors.initials: K
authors.lastname: Lewandowski
authors.affiliation: National Infection Service, Public Health England, Salisbury, United Kingdom.
authors.forename: Andrew
authors.initials: A
authors.lastname: Bell
authors.affiliation: National Infection Service, Public Health England, Salisbury, United Kingdom.
authors.forename: Rory
authors.initials: R
authors.lastname: Miles
authors.affiliation: National Infection Service, Public Health England, London, United Kingdom.
authors.forename: Simon
authors.initials: S
authors.lastname: Carne
authors.affiliation: National Infection Service, Public Health England, London, United Kingdom.
authors.forename: David
authors.initials: D
authors.lastname: Wooldridge
authors.affiliation: National Infection Service, Public Health England, London, United Kingdom.
authors.forename: Carmen
authors.initials: C
authors.lastname: Manso
authors.affiliation: National Infection Service, Public Health England, London, United Kingdom.
authors.forename: Nicola
authors.initials: N
authors.lastname: Hennessy
authors.affiliation: National Infection Service, Public Health England, Salisbury, United Kingdom.
authors.forename: Daniel
authors.initials: D
authors.lastname: Bailey
authors.affiliation: National Infection Service, Public Health England, Salisbury, United Kingdom.
authors.forename: Steven T
authors.initials: ST
authors.lastname: Pullan
authors.affiliation: National Infection Service, Public Health England, London, United Kingdom; NIHR Health Protection Research Unit in Emerging and Zoonotic Infections, Liverpool, United Kingdom.
authors.forename: Saheer
authors.initials: S
authors.lastname: Gharbia
authors.affiliation: National Infection Service, Public Health England, Salisbury, United Kingdom; NIHR Health Protection Research Unit in Emerging and Zoonotic Infections, Liverpool, United Kingdom.
authors.forename: Richard
authors.initials: R
authors.lastname: Vipond
mesh_terms: D016679:Genome, Viral; D006801:Humans; D056186:Metagenomics / Q000379:methods / Q000592:standards; D016133:Polymerase Chain Reaction / Q000379:methods / Q000592:standards; D012367:RNA, Viral / Q000235:genetics* / Q000302:isolation & purification*; D012015:Reference Standards; D017422:Sequence Analysis, DNA / Q000379:methods* / Q000592:standards*; D013048:Specimen Handling / Q000379:methods / Q000592:standards
chemical_list: D012367:RNA, Viral