The Effect of Nucleic Acid Extraction Platforms and Sample Storage on the Integrity of Viral RNA for Use in Whole Genome Sequencing
The Effect of Nucleic Acid Extraction Platforms and Sample Storage on the Integrity of Viral RNA for Use in Whole Genome Sequencing
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DOI：
10.1016/j.jmoldx.2016.10.005
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发表时间：
2017-03-01
影响因子：
4.1
通讯作者：
Vipond, Richard
中科院分区：
文献类型：
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作者：
Lewandowski, Kuiama;Bell, Andrew;Vipond, Richard
病毒RNA的提取和样本材料的保存是病毒检测和全基因组测序中极其重要的因素。病原体。尽管基于PCR的检测方法侧重于小扩增，但病毒WGS的应用需要高质量和完整性的RNA来获得足够的序列覆盖率和深度。本研究考察了诊断实验室常用的一种手动和四种自动RNA提取平台用于元基因组测序的适用性，提取前将样本材料保存在Qiagen Buffer AVL中的做法如何影响病毒RNA的完整性及其适用于基于扩增子的WGS方法，以及添加Triton X-100缓冲AVL如何影响提取平台的能力和存储样本中病毒RNA的完整性。这项研究发现，当提取的病毒RNA用于元基因组测序时，EZ1平台提供了自动化平台中最好的性能，并给出了与常用的手动Qigen提取方法相当的结果。为了保持适合于基于扩增子的WGS的高水平的病毒RNA完整性，应该立即从样本中提取核酸，因为即使在缓冲液AVL中短时间的储存也会严重影响完整性，而Triton X-100的添加对WGS的病毒材料的质量几乎没有影响。
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.