Rapid, robust plasmid verification by de novo assembly of short sequencing reads

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.

Home-made enzymatic premix and Illumina sequencing allow for one-step Gibson assembly and verification of virus infectious clones

“…Up to ~ 1-kb sequencing reads are obtained by Sanger; verification of large constructs such as potyvirid cDNA clones thus requires time-consuming primer walking strategies, multiple sequencing reactions, and custom-designed and synthesized oligonucleotides. High-throughput sequencing and read assembly pipelines have been developed to validate synthetic plasmids, including viral clones and vectors ( Shapland et al 2015 ; Pasin et al 2018 ; Saveliev et al 2018 ; Gallegos et al 2020 ). To overcome the limitations of Sanger sequencing, we used high-throughput sequencing on the Illumina platform for rapid, convenient verification of pLX-TVMV.…”

“…High-throughput sequencing is predicted to soon become the routine verification method for synthetic DNA constructs ( Shapland et al 2015 ; Currin et al 2019 ; Gallegos et al 2020 ). The full-length sequence of the plasmid assembled was verified using the Illumina platform in a single sequencing reaction that required no custom primers or data analysis pipelines, and avoided time-consuming primer walking strategies.…”

“…The full-length sequence of the plasmid assembled was verified using the Illumina platform in a single sequencing reaction that required no custom primers or data analysis pipelines, and avoided time-consuming primer walking strategies. The adoption of Illumina for sequencing of large viral clones as those of potyviruses is facilitated by data processing pipelines that require no or minimal bioinformatics skills ( Afgan et al 2018 ; Gallegos et al 2020 ), as well as by current availability of commercial services with competitive turn-around times and prices (e.g. ~ 35 USD/plasmid at seqWell™, < https://seqwell.com/ >, accessed October 20, 2020).…”

Imaging translational control by Argonaute with single-molecule resolution in live cells

“…Next, the entire open reading frame was inserted into the plasmid backbone from pFN24A HaloTag CMV d3 Flexi Vector (Promega) so that the ORF was under a CMVd3 minimal expression promoter. Lastly, we verified the plasmid sequence using whole-plasmid sequencing via de novo assembly 76 (Massachusetts General Hospital DNA sequencing core).…”

Imaging translational control by Argonaute with single-molecule resolution in live cells

“…Next, the entire open reading frame was inserted into the plasmid backbone from pFN24A HaloTag CMV d3 Flexi Vector (Promega) so that the ORF was under a CMV-d3 minimal expression promoter. Lastly, we verified the plasmid sequence using whole-plasmid sequencing via de novo assembly (Gallegos et al, 2020) (Massachusetts General Hospital DNA sequencing core).…”