Abstract
Plasmids are a foundational tool for basic and applied research across all subfields of biology. Increasingly, researchers in synthetic biology are relying on and developing massive libraries of plasmids as vectors for directed evolution, combinatorial gene circuit tests, and for CRISPR multiplexing. Verification of plasmid sequences following synthesis is a crucial quality control step that creates a bottleneck in plasmid fabrication workflows. Crucially, researchers often elect to forego the cumbersome verification step, potentially leading to reproducibility and—depending on the application—security issues. In order to facilitate plasmid verification to improve the quality and reproducibility of life science research, we developed a fast, simple, and open source pipeline for assembly and verification of plasmid sequences from Illumina reads. We demonstrate that our pipeline, which relies on de novo assembly, can also be used to detect contaminating sequences in plasmid samples. In addition to presenting our pipeline, we discuss the role for verification and quality control in the increasingly complex life science workflows ushered in by synthetic biology.
Highlights
As synthetic biology programs increase in scale [1,2,3] workflows involving the high-throughput construction of dozens or even hundreds of plasmids are becoming increasingly common [4,5,6]
We have demonstrated the feasibility of rapidly producing highly accurate plasmid assemblies from short read sequencing data by de novo assembly
Our publicly available workflow relies on Unicycler [27], an open source tool that was developed for assembly of circular genomes
Summary
As synthetic biology programs increase in scale [1,2,3] workflows involving the high-throughput construction of dozens or even hundreds of plasmids are becoming increasingly common [4,5,6]. DNA sequencing is an integral part of fabrication workflows involving the assembly of synthetic DNA fragments [7,8,9,10]. Plasmids have been verified using Sanger sequencing. This sequencing method requires a short, known sequence to initiate an up to ~1000 nucleotide read. Many bioinformatics applications used to edit plasmid sequences have features that facilitate the visual inspection of the alignment of the sequence reads and the plasmid sequence. This approach is practical to verify the insert of a limited number of plasmids. The quality and length of Sanger sequencing reads simplify sequence assembly
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