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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.

SUBMITTER: Davidsson M

PROVIDER: S-EPMC5118689 | biostudies-literature | 2016 Nov

REPOSITORIES: biostudies-literature

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A novel process of viral vector barcoding and library preparation enables high-diversity library generation and recombination-free paired-end sequencing.

Davidsson Marcus M Diaz-Fernandez Paula P Schwich Oliver D OD Torroba Marcos M Wang Gang G Björklund Tomas T

Scientific reports 20161122

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, ba ...[more]

PMID: 27874090

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Project description:BackgroundLarge insert paired-end sequencing technologies are important tools for assembling genomes, delineating associated breakpoints and detecting structural rearrangements. To facilitate the comprehensive detection of inter- and intra-chromosomal structural rearrangements or variants (SVs) and complex genome assembly with long repeats and segmental duplications, we developed a new method based on single-molecule real-time synthesis sequencing technology for generating long paired-end sequences of large insert DNA libraries.ResultsA Fosmid vector, pHZAUFOS3, was developed with the following new features: (1) two 18-bp non-palindromic I-SceI sites flank the cloning site, and another two sites are present in the skeleton of the vector, allowing long DNA inserts (and the long paired-ends in this paper) to be recovered as single fragments and the vector (~?8 kb) to be fragmented into 2-3 kb fragments by I-SceI digestion and therefore was effectively removed from the long paired-ends (5-10 kb); (2) the chloramphenicol (Cm) resistance gene and replicon (oriV), necessary for colony growth, are located near the two sides of the cloning site, helping to increase the proportion of the paired-end fragments to single-end fragments in the paired-end libraries. Paired-end libraries were constructed by ligating the size-selected, mechanically sheared pooled Fosmid DNA fragments to the Ampicillin (Amp) resistance gene fragment and screening the colonies with Cm and Amp. We tested this method on yeast and Setaria italica Yugu1. Fosmid-size paired-ends with an average length longer than 2 kb for each end were generated. The N50 scaffold lengths of the de novo assemblies of the yeast and S. italica Yugu1 genomes were significantly improved. Five large and five small structural rearrangements or assembly errors spanning tens of bp to tens of kb were identified in S. italica Yugu1 including deletions, inversions, duplications and translocations.ConclusionsWe developed a new method for long paired-end sequencing of large insert libraries, which can efficiently improve the quality of de novo genome assembly and identify large and small structural rearrangements or assembly errors.

Dataset Information

A novel process of viral vector barcoding and library preparation enables high-diversity library generation and recombination-free paired-end sequencing.

Publications

A novel process of viral vector barcoding and library preparation enables high-diversity library generation and recombination-free paired-end sequencing.

Similar Datasets

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