High-Throughput Analysis of T-DNA Location and Structure Using Sequence Capture.
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 be...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Public Library of Science (PLoS)
2015-01-01
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| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC4596565?pdf=render |
| _version_ | 1818506596169285632 |
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| author | Soichi Inagaki Isabelle M Henry Meric C Lieberman Luca Comai |
| author_facet | Soichi Inagaki Isabelle M Henry Meric C Lieberman Luca Comai |
| author_sort | Soichi Inagaki |
| collection | DOAJ |
| description | 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. Our method greatly facilitates the first step of this characterization of transgenic plants by providing an efficient screen for the selection of promising lines. |
| first_indexed | 2024-12-10T22:06:42Z |
| format | Article |
| id | doaj.art-3f8cbb8eb40548e8b63e0d821b238d73 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-10T22:06:42Z |
| publishDate | 2015-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-3f8cbb8eb40548e8b63e0d821b238d732022-12-22T01:31:44ZengPublic Library of Science (PLoS)PLoS ONE1932-62032015-01-011010e013967210.1371/journal.pone.0139672High-Throughput Analysis of T-DNA Location and Structure Using Sequence Capture.Soichi InagakiIsabelle M HenryMeric C LiebermanLuca ComaiAgrobacterium-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. Our method greatly facilitates the first step of this characterization of transgenic plants by providing an efficient screen for the selection of promising lines.http://europepmc.org/articles/PMC4596565?pdf=render |
| spellingShingle | Soichi Inagaki Isabelle M Henry Meric C Lieberman Luca Comai High-Throughput Analysis of T-DNA Location and Structure Using Sequence Capture. PLoS ONE |
| title | High-Throughput Analysis of T-DNA Location and Structure Using Sequence Capture. |
| title_full | High-Throughput Analysis of T-DNA Location and Structure Using Sequence Capture. |
| title_fullStr | High-Throughput Analysis of T-DNA Location and Structure Using Sequence Capture. |
| title_full_unstemmed | High-Throughput Analysis of T-DNA Location and Structure Using Sequence Capture. |
| title_short | High-Throughput Analysis of T-DNA Location and Structure Using Sequence Capture. |
| title_sort | high throughput analysis of t dna location and structure using sequence capture |
| url | http://europepmc.org/articles/PMC4596565?pdf=render |
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