Efficient de novo assembly and modification of large DNA fragments

Synthetic genomics has provided new bottom-up platforms for the functional study of viral and microbial genomes. The construction of the large, gigabase (Gb)-sized genomes of higher organisms will deepen our understanding of genetic blueprints significantly. But for the synthesis and assembly of such large-scale genomes, the development of new or expanded methods is required. In this study, we develop an efficient pipeline for the construction of large DNA fragments sized 100 kilobases (kb) or above from scratches and describe an efficient method for “scar-free” engineering of the assembled sequences. Our method, therefore, should provide a standard framework for producing long DNA molecules, which are critical materials for synthetic genomics and metabolic engineering.