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 »
- Authors:
-
- Univ. of California, Davis, CA (United States); National Institute of Genetics, Mishima (Japan)
- 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}
}
Web of Science
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