Quality control and statistical evaluation of combinatorial DNA libraries using nanopore sequencing

METHOD SUMMARY Nanopore sequencing enables a comprehensive assessment of combinatorial libraries of DNA sequences assembled in single tube reactions. The inspection of such DNA libraries after their creationin vitroallows investigating potential biases in the DNA assembly method, possible issues in the upstream library of DNA building blocks that feed thein vitroassembly reaction and the correct synteny of the constructs. 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 anad hocbasis. Here, we implement a systematic procedure for the comprehensive evaluation of combinatorial libraries, immediately after their creationin 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.