Sequencing Strategy to Ensure Accurate Plasmid Assembly

Despite the wide use of plasmids in research and clinical production, verifying plasmid sequences is a bottleneck often underestimated in the manufacturing process. While sequencing platforms continue to improve, the chosen method and assembly pipeline still significantly influence the final plasmid assembly sequence. Furthermore, few dedicated tools exist for plasmid assembly, particularly 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 faced challenges in resolving GC-rich regions, and long-read assemblies commonly exhibited small insertions and deletions, especially in repetitive regions. The hybrid approach facilitated the most accurate and consistent assembly generation, identifying mutations relative to the reference sequence. While Sanger sequencing can verify specific regions, some GC-rich and repetitive regions were challenging to resolve using any method, indicating that easily sequenced genetic parts should be prioritized in designing new genetic constructs.