Protein engineering and synthetic biology applications increasingly rely on the assembly of modular libraries composed of thousands of different combinations of DNA building blocks. At present, the validation of such libraries is performed by Sanger sequencing analysis on a small subset of clones on an ad hoc basis. Here, we implement a systematic procedure for the comprehensive evaluation of combinatorial libraries, immediately after their creation in vitro, using long reads sequencing technology. After an initial step of nanopore sequencing, we use straightforward bioinformatics tools to tabulate the composition and synteny of the building blocks in each read. We subsequently use exploratory statistics to assess the library and validate its diversity before carrying downstream cloning and screening assays.
Keywords: DNA assembly; GoldenBraid assembly; GoldenGate assembly; VersaTile shuffling; combinatorial DNA library; constructs validation; nanopore sequencing; quality control; synthetic biology.