Application of targeted enrichment to next-generation sequencing of retroviruses integrated into the host human genome.
Application of targeted enrichment to next-generation sequencing of retroviruses integrated into the host human genome.
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DOI：
10.1038/srep28324
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发表时间：
2016-06-20
影响因子：
4.6
通讯作者：
Satou Y
中科院分区：
文献类型：
--
作者：
Miyazato P;Katsuya H;Fukuda A;Uchiyama Y;Matsuo M;Tokunaga M;Hino S;Nakao M;Satou Y
下一代测序（NGS）技术的最新发展和进步使得能够以极高的分辨率表征人类基因组。在逆转录病毒学领域，NGS技术已被应用于整合位点分析和病毒基因组的深度测序，并结合使用病毒特异性引物的PCR扩增。然而，病毒特异性引物无法用于某些表观遗传学分析，例如染色质免疫沉淀测序（ChIP-seq）测定。病毒序列在没有特异性PCR扩增的情况下检测很差，因为与人类基因组DNA相比，前病毒DNA非常稀少。在这里，我们已经开发并评估了使用生物素化的DNA探针从为NGS制备的文库中捕获病毒遗传片段。我们的研究结果表明，病毒序列检测在富集后的灵敏度提高了数百倍或数千倍，使我们能够减少在试图通过NGS分析前病毒的表观遗传景观时产生的经济负担。此外，该方法是通用的，足以分析与DNA探针相比具有错配的前病毒。综上所述，我们认为这种方法是一个强大的工具，以澄清转录和表观遗传调控的逆转录病毒前病毒的机制，到目前为止，仍然难以捉摸。
The recent development and advancement of next-generation sequencing (NGS) technologies have enabled the characterization of the human genome at extremely high resolution. In the retrovirology field, NGS technologies have been applied to integration-site analysis and deep sequencing of viral genomes in combination with PCR amplification using virus-specific primers. However, virus-specific primers are not available for some epigenetic analyses, like chromatin immunoprecipitation sequencing (ChIP-seq) assays. Viral sequences are poorly detected without specific PCR amplification because proviral DNA is very scarce compared to human genomic DNA. Here, we have developed and evaluated the use of biotinylated DNA probes for the capture of viral genetic fragments from a library prepared for NGS. Our results demonstrated that viral sequence detection was hundreds or thousands of times more sensitive after enrichment, enabling us to reduce the economic burden that arises when attempting to analyze the epigenetic landscape of proviruses by NGS. In addition, the method is versatile enough to analyze proviruses that have mismatches compared to the DNA probes. Taken together, we propose that this approach is a powerful tool to clarify the mechanisms of transcriptional and epigenetic regulation of retroviral proviruses that have, until now, remained elusive.