Multiplexed long-read plasmid validation and analysis using OnRamp.

Recombinant plasmid vectors are versatile tools that have facilitated discoveries in molecular biology, genetics, proteomics, and many other fields. As the enzymatic and bacterial processes used to create recombinant DNA can introduce errors, sequence validation is an essential step in plasmid assembly. Sanger sequencing is the current standard for plasmid validation; however, this method is limited by an inability to sequence through complex secondary structure and lacks scalability when applied to full-plasmid sequencing of multiple plasmids owing to read-length limits. Although high-throughput sequencing does provide full-plasmid sequencing at scale, it is impractical and costly when used outside of library-scale validation. Here, we present Oxford nanopore-based rapid analysis of multiplexed plasmids (OnRamp), an alternative method for routine plasmid validation that combines the advantages of high-throughput sequencing's full-plasmid coverage and scalability with Sanger's affordability and accessibility by leveraging nanopore's long-read sequencing technology. We include customized wet-laboratory protocols for plasmid preparation along with a pipeline designed for analysis of read data obtained using these protocols. This analysis pipeline is deployed on the OnRamp web app, which generates alignments between actual and predicted plasmid sequences, quality scores, and read-level views. OnRamp is designed to be broadly accessible regardless of programming experience to facilitate more widespread adoption of long-read sequencing for routine plasmid validation. Here we describe the OnRamp protocols and pipeline and show our ability to obtain full sequences from pooled plasmids while detecting sequence variation even in regions of high secondary structure at less than half the cost of equivalent Sanger sequencing.

The Inducible lac Operator-Repressor System Is Functional in Zebrafish Cells.

BACKGROUND: Zebrafish are a foundational model organism for studying the spatio-temporal activity of genes and their regulatory sequences. A variety of approaches are currently available for editing genes and modifying gene expression in zebrafish, including RNAi, Cre/lox, and CRISPR-Cas9. However, the lac operator-repressor system, an E. coli lac operon component which has been adapted for use in many other species and is a valuable, flexible tool for inducible modulation of gene expression studies, has not been previously tested in zebrafish. RESULTS: Here we demonstrate that the lac operator-repressor system robustly decreases expression of firefly luciferase in cultured zebrafish fibroblast cells. Our work establishes the lac operator-repressor system as a promising tool for the manipulation of gene expression in whole zebrafish. CONCLUSION: Our results lay the groundwork for the development of lac-based reporter assays in zebrafish, and adds to the tools available for investigating dynamic gene expression in embryogenesis. We believe this work will catalyze the development of new reporter assay systems to investigate uncharacterized regulatory elements and their cell-type specific activities.