1. There are few available tools to comprehensively and economically identify uncharacterized flanking regions that are not extremely labour intensive and which exploit the advantages of emerging long‐read sequencing platforms.
2. We describe SIP; a sonication‐based inverse PCR high‐throughput sequencing strategy to investigate uncharacterized flanking region sequences, including those flanking mobile DNA. SIP combines unbiased fragmentation by sonication and target enrichment by coupling outward facing PCR priming with long‐read sequencing technologies.
3. We demonstrate the effectiveness of SIP by determining retroviral integrations which are high copy and challenging to characterize. We further describe SIP's workflow, examine retroviral (proviral) enrichment and characterize viral structural variants identified. When SIP was coupled with long‐read sequencing using the PacBio RS II platform, proviral integration was extensively characterized at high sequence depth per integration. By interrogating the sequence data, we were also able to test several intrinsic factors including SIP's propensity to form chimeric sequences and adapter ligation efficiencies.
4. SIP is an adaption of a traditional molecular biology technique that can be used to characterize any unknown genomic flanking sequence or to extend any sequence for which only minimal sequence information is available. SIP can be applied broadly to study complex biological systems such as mobile genetic elements with high throughput.

中文翻译：

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DNA超声逆向PCR用于未表征侧翼序列的基因组规模分析

1. 很少有可用的工具来全面，经济地识别劳动力不足不是很强的未表征的侧翼区域，它们利用了新兴的长读测序平台的优势。
2. 我们描述SIP；一种基于超声处理的反向PCR高通量测序策略，用于研究未表征的侧翼区域序列，包括侧翼可移动DNA的序列。SIP通过将面向外的PCR引物与长读测序技术相结合，将超声处理中的无偏片段化和靶标富集相结合。
3. 我们通过确定高拷贝且难以表征的逆转录病毒整合来证明SIP的有效性。我们进一步描述SIP的工作流程，检查逆转录病毒（实验性）富集并鉴定已鉴定的病毒结构变异。当使用PacBio RS II平台将SIP与长时间测序结合时，原病毒整合的特征是每次整合的序列深度很高。通过查询序列数据，我们还能够测试几个内在因素，包括SIP形成嵌合序列的倾向性和衔接子连接效率。
4. SIP是对传统分子生物学技术的一种改编，可用于表征任何未知的基因组侧翼序列或扩展任何仅可获得最小序列信息的序列。SIP可以广泛地用于研究复杂的生物系统，例如具有高通量的移动遗传元件。