title: A novel process of viral vector barcoding and library preparation enables high-diversity library generation and recombination-free paired-end sequencing.
abstract: Detailed characterization and mapping of oligonucleotide function in vivo is generally a very time consuming effort that only allows for hypothesis driven subsampling of the full sequence to be analysed. Recent advances in deep sequencing together with highly efficient parallel oligonucleotide synthesis and cloning techniques have, however, opened up for entirely new ways to map genetic function in vivo. Here we present a novel, optimized protocol for the generation of universally applicable, barcode labelled, plasmid libraries. The libraries are designed to enable the production of viral vector preparations assessing coding or non-coding RNA function in vivo. When generating high diversity libraries, it is a challenge to achieve efficient cloning, unambiguous barcoding and detailed characterization using low-cost sequencing technologies. With the presented protocol, diversity of above 3 million uniquely barcoded adeno-associated viral (AAV) plasmids can be achieved in a single reaction through a process achievable in any molecular biology laboratory. This approach opens up for a multitude of in vivo assessments from the evaluation of enhancer and promoter regions to the optimization of genome editing. The generated plasmid libraries are also useful for validation of sequencing clustering algorithms and we here validate the newly presented message passing clustering process named Starcode.
authors.affiliation: Molecular Neuromodulation, Department of Experimental Medical Science, Lund University, 221 84 Lund, Sweden.
authors.forename: Marcus
authors.initials: M
authors.lastname: Davidsson
authors.affiliation: Molecular Neuromodulation, Department of Experimental Medical Science, Lund University, 221 84 Lund, Sweden.
authors.forename: Paula
authors.initials: P
authors.lastname: Diaz-Fernandez
authors.affiliation: Molecular Neuromodulation, Department of Experimental Medical Science, Lund University, 221 84 Lund, Sweden.
authors.forename: Oliver D
authors.initials: OD
authors.lastname: Schwich
authors.affiliation: Molecular Neuromodulation, Department of Experimental Medical Science, Lund University, 221 84 Lund, Sweden.
authors.forename: Marcos
authors.initials: M
authors.lastname: Torroba
authors.affiliation: Molecular Neuromodulation, Department of Experimental Medical Science, Lund University, 221 84 Lund, Sweden.
authors.forename: Gang
authors.initials: G
authors.lastname: Wang
authors.affiliation: Molecular Neuromodulation, Department of Experimental Medical Science, Lund University, 221 84 Lund, Sweden.
authors.forename: Tomas
authors.initials: T
authors.lastname: Björklund
mesh_terms: D000229:Dependovirus / Q000235:genetics*; D015723:Gene Library; D005822:Genetic Vectors / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing; D012313:RNA / Q000235:genetics*; D011995:Recombination, Genetic
chemical_list: D012313:RNA