Quality Control and Statistical Evaluation of Combinatorial DNA Libraries using Nanopore Sequencing

BioTechniques, vol. 69, pp. 379–383

Abstract

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.

Authors 5

1. Cédric Lood

   KU Leuven

   Affiliation as printed

   Department of Biosystems,Laboratory of Gene Technology, KU Leuven, Leuven, Belgium

   Department of Microbial & Molecular Systems,Centre of Microbial & Plant Genetics,Laboratory of Computational Systems Biology, KU Leuven, Leuven, Belgium

   Department of Biosystems, Laboratory of Gene Technology, KU Leuven, Leuven, Belgium

2. Hans Gerstmans

   Ghent University · KU Leuven

   Affiliation as printed

   Department of Biosystems,Laboratory of Gene Technology, KU Leuven, Leuven, Belgium

   Department of Biosystems,MeBioS-Biosensors Group, KU Leuven, Leuven, Belgium

   Department of Biotechnology,Laboratory of Applied Biotechnology, Ghent University, Ghent, Belgium

   Department of Biosystems, Laboratory of Gene Technology, KU Leuven, Leuven, Belgium

   Department of Biosystems, MeBioS-Biosensors Group, KU Leuven, Leuven, Belgium

   Department of Biotechnology, Laboratory of Applied Biotechnology, Ghent University, Ghent, Belgium

3. Yves Briers

   Ghent University

   Affiliation as printed

   Department of Biotechnology,Laboratory of Applied Biotechnology, Ghent University, Ghent, Belgium

   Department of Biotechnology, Laboratory of Applied Biotechnology, Ghent University, Ghent, Belgium

4. Vera van Noort Aachen Institute of Biology, University of Leiden, Leiden

   Leiden University · KU Leuven

   Affiliation as printed

   Department of Microbial & Molecular Systems,Centre of Microbial & Plant Genetics,Laboratory of Computational Systems Biology, KU Leuven, Leuven, Belgium

   Institute of Biology,University of Leiden, Leiden, The Netherlands

5. Rob Lavigne corresponding

   KU Leuven

   Affiliation as printed

   Department of Biosystems,Laboratory of Gene Technology, KU Leuven, Leuven, Belgium

   Department of Biosystems, Laboratory of Gene Technology, KU Leuven, Leuven, Belgium