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Title: High-throughput analysis of T-DNA location and structure using sequence capture

Abstract

Agrobacterium-mediated transformation of plants with T-DNA is used both to introduce transgenes and for mutagenesis. Conventional approaches used to identify the genomic location and the structure of the inserted T-DNA are laborious and high-throughput methods using next-generation sequencing are being developed to address these problems. Here, we present a cost-effective approach that uses sequence capture targeted to the T-DNA borders to select genomic DNA fragments containing T-DNA—genome junctions, followed by Illumina sequencing to determine the location and junction structure of T-DNA insertions. Multiple probes can be mixed so that transgenic lines transformed with different T-DNA types can be processed simultaneously, using a simple, index-based pooling approach. We also developed a simple bioinformatic tool to find sequence read pairs that span the junction between the genome and T-DNA or any foreign DNA. We analyzed 29 transgenic lines of Arabidopsis thaliana, each containing inserts from 4 different T-DNA vectors. We determined the location of T-DNA insertions in 22 lines, 4 of which carried multiple insertion sites. Additionally, our analysis uncovered a high frequency of unconventional and complex T-DNA insertions, highlighting the needs for high-throughput methods for T-DNA localization and structural characterization. Transgene insertion events have to be fully characterized prior to usemore » as commercial products. As a result, our method greatly facilitates the first step of this characterization of transgenic plants by providing an efficient screen for the selection of promising lines.« less

Authors:
 [1];  [2];  [2];  [2]
  1. Univ. of California, Davis, CA (United States); National Institute of Genetics, Mishima (Japan)
  2. Univ. of California, Davis, CA (United States)
Publication Date:
Research Org.:
Univ. of California, Davis, CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER)
OSTI Identifier:
1239138
Grant/Contract Number:  
SC0007183
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
PLoS ONE
Additional Journal Information:
Journal Volume: 10; Journal Issue: 10; Related Information: Data Availability: All short read sequence files are available under BioProject ID PRJNA287142 and SRA ID: SRP059868.; Journal ID: ISSN 1932-6203
Publisher:
Public Library of Science
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; sequence alignment; plant genomics; Arabidopsis thaliana; genomic library construction; polymerase chain reaction; probe hybridization; repeated sequences; sequence assembly tools

Citation Formats

Inagaki, Soichi, Henry, Isabelle M., Lieberman, Meric C., and Comai, Luca. High-throughput analysis of T-DNA location and structure using sequence capture. United States: N. p., 2015. Web. doi:10.1371/journal.pone.0139672.
Inagaki, Soichi, Henry, Isabelle M., Lieberman, Meric C., & Comai, Luca. High-throughput analysis of T-DNA location and structure using sequence capture. United States. https://doi.org/10.1371/journal.pone.0139672
Inagaki, Soichi, Henry, Isabelle M., Lieberman, Meric C., and Comai, Luca. 2015. "High-throughput analysis of T-DNA location and structure using sequence capture". United States. https://doi.org/10.1371/journal.pone.0139672. https://www.osti.gov/servlets/purl/1239138.
@article{osti_1239138,
title = {High-throughput analysis of T-DNA location and structure using sequence capture},
author = {Inagaki, Soichi and Henry, Isabelle M. and Lieberman, Meric C. and Comai, Luca},
abstractNote = {Agrobacterium-mediated transformation of plants with T-DNA is used both to introduce transgenes and for mutagenesis. Conventional approaches used to identify the genomic location and the structure of the inserted T-DNA are laborious and high-throughput methods using next-generation sequencing are being developed to address these problems. Here, we present a cost-effective approach that uses sequence capture targeted to the T-DNA borders to select genomic DNA fragments containing T-DNA—genome junctions, followed by Illumina sequencing to determine the location and junction structure of T-DNA insertions. Multiple probes can be mixed so that transgenic lines transformed with different T-DNA types can be processed simultaneously, using a simple, index-based pooling approach. We also developed a simple bioinformatic tool to find sequence read pairs that span the junction between the genome and T-DNA or any foreign DNA. We analyzed 29 transgenic lines of Arabidopsis thaliana, each containing inserts from 4 different T-DNA vectors. We determined the location of T-DNA insertions in 22 lines, 4 of which carried multiple insertion sites. Additionally, our analysis uncovered a high frequency of unconventional and complex T-DNA insertions, highlighting the needs for high-throughput methods for T-DNA localization and structural characterization. Transgene insertion events have to be fully characterized prior to use as commercial products. As a result, our method greatly facilitates the first step of this characterization of transgenic plants by providing an efficient screen for the selection of promising lines.},
doi = {10.1371/journal.pone.0139672},
url = {https://www.osti.gov/biblio/1239138}, journal = {PLoS ONE},
issn = {1932-6203},
number = 10,
volume = 10,
place = {United States},
year = {Wed Oct 07 00:00:00 EDT 2015},
month = {Wed Oct 07 00:00:00 EDT 2015}
}

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Free Publicly Available Full Text
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Cited by: 20 works
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Works referenced in this record:

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Model-based Analysis of ChIP-Seq (MACS)
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A Versatile Set of Ligation-Independent Cloning Vectors for Functional Studies in Plants
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Efficient Genome-Wide Detection and Cataloging of EMS-Induced Mutations Using Exome Capture and Next-Generation Sequencing
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Generation of Backbone-Free, Low Transgene Copy Plants by Launching T-DNA from the Agrobacterium Chromosome
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Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method
journal, June 2006


A Versatile Set of Ligation-Independent Cloning Vectors for Functional Studies in Plants
journal, May 2011


Genetic applications of an inverse polymerase chain reaction.
journal, November 1988


Works referencing / citing this record:

Biofortification of rice with the essential amino acid lysine: molecular characterization, nutritional evaluation, and field performance
journal, June 2016


TDNAscan: A Software to Identify Complete and Truncated T-DNA Insertions
journal, July 2019


Genome Editing of Plants
journal, January 2017