Immunocapture of virions with virus-specific antibodies prior to high-throughput sequencing effectively enriches for virus-specific sequences
Immunocapture of virions with virus-specific antibodies prior to high-throughput sequencing effectively enriches for virus-specific sequences
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DOI：
10.1371/journal.pone.0216713
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发表时间：
2019-05-09
期刊：
影响因子：
3.7
通讯作者：
Winter, Stephan
中科院分区：
文献类型：
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作者：
Knierim, Dennis;Menzel, Wulf;Winter, Stephan
基于高通量测序的病毒发现依赖于在文库制备之前富集病毒序列以获得足够数量的病毒读段。通常，经处理以去除rRNA的双链RNA或总RNA制备物的制备物用于序列富集。我们使用病毒特异性抗体从植物汁液中免疫捕获病毒体进行cDNA合成，然后进行文库制备和HTS。对于四种马铃薯病毒PLRV，PVY，PVA和PYV，通过病毒粒子免疫捕获的模板制备提供了一个简单和便宜的方法比富集总RNA的核糖体消耗。在没有中间扩增步骤的情况下实现了病毒序列的特异性富集，并且这种跨病毒基因组的序列的高覆盖对于鉴定罕见的序列变异是重要的。利用这种方法，本研究首次测定了马铃薯黄化病毒分离物（PYV，DSMZ PV-0706）的全基因组序列。PYV可以被确信地指定为Ilarvirus属中的独特种。
Virus discovery based on high-throughput sequencing relies on enrichment for virus sequences prior to library preparation to achieve a sufficient number of viral reads. In general, preparations of double-stranded RNA or total RNA preparations treated to remove rRNA are used for sequence enrichment. We used virus-specific antibodies to immunocapture virions from plant sap to conduct cDNA synthesis, followed by library preparation and HTS. For the four potato viruses PLRV, PVY, PVA and PYV, template preparation by virion immunocapture provided a simpler and less expensive method than the enrichment of total RNA by ribosomal depletion. Specific enrichment of viral sequences without an intermediate amplification step was achieved, and this high coverage of sequences across the viral genomes was important to identify rare sequence variations. Using this approach, the first complete genome sequence of a potato yellowing virus isolate (PYV, DSMZ PV-0706) was determined in this study. PYV can be confidently assigned as a distinct species in the genus Ilarvirus.