Despite the interest in characterizing genomic variation, the presence of large repeats at the breakpoints hinders the analysis of many structural variants. This is especially problematic for inversions, since there is typically no gain or loss of DNA. Here, we tested novel linkage-based droplet digital PCR (ddPCR) assays to study 20 inversions ranging from 3.1 to 742 kb flanked by inverted repeats (IRs) up to 134 kb long. Of those, we validated 13 inversions predicted by different genome-wide techniques. In addition, we obtained new experimental human population information across 95 African, European, and East Asian individuals for 16 inversions, including four already validated variants without high-throughput genotyping methods. Through comparison with previous data, independent replicates and both inversion breakpoints, we demonstrate that the technique is highly accurate and reproducible. Most studied inversions are widespread across continents, and their frequency is negatively correlated with genetic length. Moreover, all except two show clear signs of being recurrent, and we could better define the factors affecting recurrence levels and estimate the inversion rate across the genome. Finally, the generated genotypes have allowed us to check inversion functional effects, validating gene expression differences reported before for two inversions and finding new candidate associations. Therefore, the developed methodology makes it possible to screen these and other complex genomic variants quickly in a large number of samples for the first time, highlighting the importance of direct genotyping to assess their potential consequences and clinical implications.

中文翻译：

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通过液滴数字PCR确定人类基因组中未表征的反演的影响。

尽管感兴趣的是表征基因组变异，但在断点处存在大的重复序列阻碍了许多结构变异的分析。这对于倒置尤其成问题，因为通常不存在DNA的得失。在这里，我们测试了新的基于连锁的液滴数字PCR（ddPCR）分析方法，以研究20个倒位，范围从3.1到742 kb，两侧是长达134 kb的反向重复（IR）。其中，我们验证了通过不同的全基因组技术预测的13个倒置。此外，我们获得了针对95个非洲，欧洲和东亚个体的16个倒位实验的新实验人群信息，包括4个已经验证的变体，没有高通量基因分型方法。通过与以前的数据，独立的复制和两个反转断点进行比较，我们证明了该技术是高度准确且可重复的。大多数研究的反演都分布在各大洲，其频率与遗传长度负相关。此外，除了两个以外，所有其他迹象均显示出明显的复发迹象，因此我们可以更好地定义影响复发水平的因素，并估计整个基因组的转化率。最后，产生的基因型使我们能够检查反演功能的作用，验证两次反演之前报道的基因表达差异，并找到新的候选关联。因此，发达的方法学使得首次在大量样品中快速筛查这些和其他复杂的基因组变体成为可能，突出了直接基因分型评估其潜在后果和临床意义的重要性。