The Effect of Nucleic Acid Extraction Platforms and Sample Storage on the Integrity of Viral RNA for Use in Whole Genome Sequencing
| Autor: | Richard Vipond, Nicola Hennessy, Saheer E. Gharbia, Kuiama Lewandowski, Carmen F. Manso, Rory W. Miles, Andrew Bell, Daniel Bailey, Steven T. Pullan, Simon Carne, David J. Wooldridge |
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| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Genetics Whole genome sequencing Sequence analysis RNA Genome Viral Sequence Analysis DNA Computational biology Reference Standards Biology Amplicon Polymerase Chain Reaction Genome Specimen Handling Pathology and Forensic Medicine 03 medical and health sciences 030104 developmental biology Metagenomics Nucleic acid Humans RNA Viral Molecular Medicine RNA extraction |
| Zdroj: | The Journal of Molecular Diagnostics. 19:303-312 |
| ISSN: | 1525-1578 |
| DOI: | 10.1016/j.jmoldx.2016.10.005 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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