The Effect of Nucleic Acid Extraction Platforms and Sample Storage on the Integrity of Viral RNA for Use in Whole Genome Sequencing

Kuiama Lewandowski*, Andrew Bell, Rory Miles, Simon Carne, David Wooldridge, Carmen Manso, Nicola Hennessy, Daniel Bailey, Steven Pullan, Saheer Gharbia, Richard Vipond

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

8 Citations (Scopus)


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.

Original languageEnglish
Pages (from-to)303-312
Number of pages10
JournalJournal of Molecular Diagnostics
Issue number2
Publication statusPublished - 1 Mar 2017

Bibliographical note

Funding Information:
Supported by Public Health England.

Publisher Copyright:
© 2017

Copyright 2019 Elsevier B.V., All rights reserved.


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