LoopViz: A uLoop Assembly Clone Verification Tool for Nanopore Sequencing Reads

Abstract

Cloning has been an integral part of most laboratory research questions and continues to be an essential tool in defining the genetic elements determining life. Cloning can be difficult and time consuming as each plasmid is unique to a particular project and each sequence must be carefully selected, cloned and sequenced to determine correctness. Loop assembly (uLOOP) is a recursive, Golden Gate-like assembly method that allows rapid cloning of domesticated DNA fragments to robustly refactor novel pathways. With uLOOP methodologies, one can clone several sequences directionally to generate a library of transcriptional units (TUs) in plasmids within a single reaction but analysis of the plasmid population has been impeded by current sequencing and analysis methods. Here we develop LoopViz, a quality control tool that quantifies and visualizes results from assembly reactions using long-read Oxford Nanopore Technologies (ONT) sequencing. LoopViz identifies full length reads originating from a single plasmid in the population, and visualizes them in terms of a user input DNA fragments file, and provides QC statistics. This methodology enables validation and analysis of cloning and sequencing reactions in less than a day, determination of the entire plasmid’s sequence, and sequencing through repetitive meta-regions that cannot be meaningfully assembled. Finally, LoopViz represents a new paradigm in determining plasmid sequences that is rapid, cost-effective and performed in-lab. LoopViz is made publicly available at https://gitlab.com/marielelensink325/loopseq