Hybrid Sequencing Facilitates Robust De Novo Plasmid Assembly.

Despite the wide use of plasmids in research and clinical production, the verification of plasmid sequences is a bottleneck that is too often overlooked in the manufacturing process. Although sequencing platforms continue to improve, the method and assembly pipeline chosen still influence the final plasmid assembly sequence. Furthermore, few dedicated tools exist for plasmid assembly, especially for de novo assembly. Here, we evaluated short-read, long-read, and hybrid (both short and long reads) de novo assembly pipelines across three replicates of a 24-plasmid library. Consistent with previous characterizations of each sequencing technology, short-read assemblies had frequent issues resolving GC-rich regions, and long-read assemblies commonly had small insertions and deletions, especially in repetitive regions. The hybrid approach facilitated the most consistent assembly generation. Although Sanger sequencing can be used to verify specific regions, it requires a reference sequence to design primers, emphasizing the need for accurate de novo plasmid assembly tools. Some GC-rich and repetitive regions were difficult to resolve using any methods, suggesting that easily sequenced genetic parts should be prioritized in the design of new genetic constructs. GRAPHICAL ABSTRACT: