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

Journal Article · Wed Oct 07 00:00:00 EDT 2015 · PLoS ONE

DOI:https://doi.org/10.1371/journal.pone.0139672· OSTI ID:1239138

Inagaki, Soichi ^[1]; Henry, Isabelle M. ^[2]; Lieberman, Meric C. ^[2]; Comai, Luca ^[2]

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1. Univ. of California, Davis, CA (United States); National Institute of Genetics, Mishima (Japan)
2. Univ. of California, Davis, CA (United States)

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.

• Research Organization: Univ. of California, Davis, CA (United States)
• Sponsoring Organization: USDOE Office of Science (SC), Biological and Environmental Research (BER)
• Grant/Contract Number: SC0007183
• OSTI ID: 1239138
• Journal Information: PLoS ONE, Vol. 10, Issue 10; Related Information: Data Availability: All short read sequence files are available under BioProject ID PRJNA287142 and SRA ID: SRP059868.; ISSN 1932-6203
• Publisher: Public Library of Science
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
sequence alignment
plant genomics
Arabidopsis thaliana
genomic library construction
polymerase chain reaction
probe hybridization
repeated sequences
sequence assembly tools