Inversion genotyping by droplet digital PCR (ddPCR). Homo sapiens

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. We validated 13 inversions predicted by different genome-wide techniques and generated new experimental human population information across 95 African, European and East-Asian individuals for 16 of them, including four already known inversions without high-throughput methods to determine orientation directly. 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 the additional data allowed us to define better the factors affecting recurrence levels and estimate the inversion rate across the genome. Finally, thanks to the generated genotypes, we have been able 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.