On-Ramp: a tool for rapid, multiplexed validation of plasmids using nanopore sequencing

The ability to generate vectors made of recombinant DNA has facilitated many achievements in molecular biology. As these vectors are assembled from many different parts and the enzymatic and bacterial processes used to create them can introduce errors, validation of the final product is an essential part of plasmid assembly. Sanger sequencing is the primary validation method available at a base-pair level, however it has many limitations. These include its inability to sequence through complex secondary structure, read length, and cost when validating the full sequence of plasmids or large numbers of plasmids. There is a need for a sequence validation method that can rapidly and affordably sequence the entirety of many plasmids simultaneously, using tools that are accessible to molecular cloning labs, without the need for the time and cost associated with next-generation sequencing. On-Ramp (Oxford-nanopore based Rapid Analysis of Multiplexed Plasmids) is a nanopore-based sequencing based approach that addresses this need by leveraging novel long-read technology. On-Ramp combines preparation protocols designed specifically for multi-plasmid pools and an analysis pipeline modified for accurate alignment of nanopore sequencing reads resulting from these plasmid pool methods. We demonstrate that through On-Ramp, large numbers of dissimilar or highly similar plasmids can be sequenced simultaneously and consensus sequences generated that capture single base-pair mutations, insertions or deletions, and highly repetitive region sequences can be resolved. Our tool allows for sequencing of multiple plasmids in their entirety at one-third the cost of Sanger sequencing, and through the use of our On-Ramp webapp, labs can obtain sequence results rapidly without the need for bioinformatic expertise. On-Ramp is available at http://OnRamp.BoyleLab.org. ### Competing Interest Statement The authors have declared no competing interest.