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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 LU ; Diaz-Fernandez, Paula; Schwich, Oliver D.; Torroba, Marcos; Wang, Gang LU and Björklund, Tomas LU (2016) In Scientific Reports 6.
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... (More)

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.

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author
organization
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type
Contribution to journal
publication status
published
subject
in
Scientific Reports
volume
6
publisher
Nature Publishing Group
external identifiers
  • scopus:84996497597
  • wos:000388241600001
ISSN
2045-2322
DOI
10.1038/srep37563
language
English
LU publication?
yes
id
25692e96-faec-434d-bdd6-e287d255a525
date added to LUP
2016-12-12 08:00:24
date last changed
2017-10-22 05:23:36
@article{25692e96-faec-434d-bdd6-e287d255a525,
  abstract     = {<p>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.</p>},
  articleno    = {37563},
  author       = {Davidsson, Marcus and Diaz-Fernandez, Paula and Schwich, Oliver D. and Torroba, Marcos and Wang, Gang and Björklund, Tomas},
  issn         = {2045-2322},
  language     = {eng},
  month        = {11},
  publisher    = {Nature Publishing Group},
  series       = {Scientific Reports},
  title        = {A novel process of viral vector barcoding and library preparation enables high-diversity library generation and recombination-free paired-end sequencing},
  url          = {http://dx.doi.org/10.1038/srep37563},
  volume       = {6},
  year         = {2016},
}