Structural variations (SVs) on the genome can result in various sporadic diseases, while they are recognized to contribute to genetic evolution. However, the technologies to control SVs, such as generating new SVs, have not yet been developed. We have established a method to induce specific segmental duplication with high efficiency in the budding yeast genome using paired nicking by nCas9. This technology will provide a new approach to construct desired genomic SVs. Here, we provided the NGS data containing the long-read sequencing results which cover the entire duplicated segments, and short-read sequencing results used for Polymerase-Usage sequencing (Pu-seq) to investigate the DNA replication initiations of the target segment.