Retroelements (REs) occupy a significant part of all eukaryotic genomes including humans. The majority of retroelements in the human genome are inactive and unable to retrotranspose. Dozens of active copies are repressed in most normal tissues by various cellular mechanisms. These copies can become active in normal germline and brain tissues or in cancer, leading to new retroposition events. The consequences of such events and their role in normal cell functioning and carcinogenesis are not yet fully understood. If new insertions occur in a small portion of cells they can be found only with the use of specific methods based on RE enrichment and high-throughput sequencing. The downside of the high sensitivity of such methods is the presence of various artifacts imitating real insertions, which in many cases cannot be validated due to lack of the initial template DNA. For this reason, adequate assessment of rare (< 1%) subclonal cancer specific RE insertions is complicated. Here we describe a new copy-capture technique which we implemented in a method called SeqURE for Sequencing Unknown of Retroposition Events that allows for efficient and reliable identification of new genomic RE insertions. The method is based on the capture of copies of target molecules (copy-capture), selective amplification and sequencing of genomic regions adjacent to active RE insertions from both sides. Importantly, the template genomic DNA remains intact and can be used for validation experiments. In addition, we applied a novel system for testing method sensitivity and precisely showed the ability of the developed method to reliably detect insertions present in 1 out of 100 cells and a substantial portion of insertions present in 1 out of 1000 cells. Using advantages of the method we showed the absence of somatic Alu insertions in colorectal cancer samples bearing tumor-specific L1HS insertions. This study presents the first description and implementation of the copy-capture technique and provides the first methodological basis for the quantitative assessment of RE insertions present in a small portion of cells.

中文翻译：

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SeqURE –一种新的基于拷贝捕获的方法，用于未知逆向事件的排序

反转录元件（REs）占据了包括人类在内的所有真核基因组的很大一部分。人类基因组中的大多数逆转录元件是不活跃的，不能逆转座。在大多数正常组织中，数十种活性拷贝通过各种细胞机制被抑制。这些副本可在正常生殖系和脑组织或癌症中活跃，从而导致新的逆转录事件。此类事件的后果及其在正常细胞功能和致癌作用中的作用尚未完全了解。如果新插入物出现在一小部分细胞中，则只能通过使用基于RE富集和高通量测序的特定方法来发现它们。这种方法灵敏度高的缺点是存在各种伪像，它们模仿了真实的插入，由于缺少初始模板DNA，在许多情况下无法对其进行验证。因此，对罕见的（<1％）亚克隆癌症特异性RE插入物进行充分评估非常复杂。在这里，我们描述了一种新的拷贝捕获技术，该技术已在一种称为SeqURE的方法中实现，用于对未知的逆序事件进行测序，从而可以高效，可靠地识别新的基因组RE插入片段。该方法基于靶分子拷贝的捕获（拷贝捕获），选择性扩增和从两侧对与活性RE插入相邻的基因组区域进行测序。重要的是，模板基因组DNA保持完整，可用于验证实验。此外，我们应用了一种新颖的系统来测试方法的灵敏度，并精确显示了开发方法能够可靠地检测100个细胞中有1个存在的插入和1000个细胞中有1个存在的大部分插入。利用该方法的优势，我们显示了在带有肿瘤特异性L1HS插入片段的大肠癌样本中没有体细胞Alu插入片段。这项研究提出了复制捕获技术的第一个描述和实施方法，并为定量评估存在于一小部分细胞中的RE插入提供了第一个方法学基础。利用该方法的优势，我们显示了在带有肿瘤特异性L1HS插入片段的大肠癌样本中没有体细胞Alu插入片段。这项研究提出了复制捕获技术的第一个描述和实施方法，并为定量评估存在于一小部分细胞中的RE插入提供了第一个方法学基础。利用该方法的优势，我们表明在带有肿瘤特异性L1HS插入片段的大肠癌样本中没有体细胞Alu插入片段。这项研究提出了复制捕获技术的第一个描述和实施方法，并为定量评估存在于一小部分细胞中的RE插入提供了第一个方法学基础。