Time- and Cost-Efficient Identification of T-DNA Insertion Sites through Targeted Genomic Sequencing

Lepage, Étienne; Zampini, Éric; Boyle, Brian; Brisson, Normand

doi:10.1371/journal.pone.0070912

Cited by 31 publications

(34 citation statements)

References 20 publications

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“…NGS has proven to be a powerful tool for discovering genome variation including re-arrangements, gene fusions, DNA structural variations in different species (Campbell et al, 2008; Hormozdiari et al, 2011; DuBose et al, 2013). NGS coupled with bioinformatics platform applied in genomics research are widely used in the agricultural biotechnology field (Kovalic et al, 2012; Lepage et al, 2013; Park et al, 2015). Recently, several researches have focused on new approaches in molecular characterization and safety assessment of transgenic events using NGS technology (Urbanski et al, 2012; Daniela et al, 2013; Pauwels et al, 2015).…”

Section: Discussionmentioning

confidence: 99%

“…For the past few years, NGS has also provided an alternative tool in molecular characteristics of GM plants. Several NGS based methods have been developed to identify insertions of exogenous fragments in Arabidopsis thaliana (Lepage et al, 2013; Inagaki et al, 2015), rice (Daniela et al, 2013; Park et al, 2015), and maize (Rosalind et al, 2010). Compared with PCR-based methods, combination of targeted bioinformatics analysis and limited de novo assembly using WGS data has become a much simpler and more effective approach for transgenic analysis.…”

Section: Introductionmentioning

confidence: 99%

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Identification of Genomic Insertion and Flanking Sequence of G2-EPSPS and GAT Transgenes in Soybean Using Whole Genome Sequencing Method

Guo

Hong

et al. 2016

Front. Plant Sci.

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Molecular characterization of sequence flanking exogenous fragment insertion is essential for safety assessment and labeling of genetically modified organism (GMO). In this study, the T-DNA insertion sites and flanking sequences were identified in two newly developed transgenic glyphosate-tolerant soybeans GE-J16 and ZH10-6 based on whole genome sequencing (WGS) method. More than 22.4 Gb sequence data (∼21 × coverage) for each line was generated on Illumina HiSeq 2500 platform. The junction reads mapped to boundaries of T-DNA and flanking sequences in these two events were identified by comparing all sequencing reads with soybean reference genome and sequence of transgenic vector. The putative insertion loci and flanking sequences were further confirmed by PCR amplification, Sanger sequencing, and co-segregation analysis. All these analyses supported that exogenous T-DNA fragments were integrated in positions of Chr19: 50543767–50543792 and Chr17: 7980527–7980541 in these two transgenic lines. Identification of genomic insertion sites of G2-EPSPS and GAT transgenes will facilitate the utilization of their glyphosate-tolerant traits in soybean breeding program. These results also demonstrated that WGS was a cost-effective and rapid method for identifying sites of T-DNA insertions and flanking sequences in soybean.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Identification of Genomic Insertion and Flanking Sequence of G2-EPSPS and GAT Transgenes in Soybean Using Whole Genome Sequencing Method

Guo

Hong

et al. 2016

Front. Plant Sci.

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“…If the annotated T‐DNA is missing or does not cause the phenotype of interest, researchers can map other T‐DNAs in the line to try to discover genes with a role in leaf development. Here we presented a cost‐effective and simple method to map the non‐annotated T‐DNA by a workflow that is simpler than previous methods (Korbel et al ., ; Williams‐Carrier et al ., ; Polko et al ., ; Lepage et al ., ), and feasible for any plant genetics laboratory. Also, all the software tools used here have been fitted with a graphical interface and integrated in open source, cloud‐based platforms for data‐intensive research such as Galaxy (http://galaxyproject.org/; Goecks et al ., ) and iPlant (http://www.iplantcollaborative.org/; Oliver et al ., ), thus making computer equipment and command line knowledge dispensable.…”

Section: Discussionmentioning

confidence: 99%

Leaf phenomics: a systematic reverse genetic screen for Arabidopsis leaf mutants

et al. 2014

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SUMMARYThe study and eventual manipulation of leaf development in plants requires a thorough understanding of the genetic basis of leaf organogenesis. Forward genetic screens have identified hundreds of Arabidopsis mutants with altered leaf development, but the genome has not yet been saturated. To identify genes required for leaf development we are screening the Arabidopsis Salk Unimutant collection. We have identified 608 lines that exhibit a leaf phenotype with full penetrance and almost constant expressivity and 98 additional lines with segregating mutant phenotypes. To allow indexing and integration with other mutants, the mutant phenotypes were described using a custom leaf phenotype ontology. We found that the indexed mutation is present in the annotated locus for 78% of the 553 mutants genotyped, and that in half of these the annotated T-DNA is responsible for the phenotype. To quickly map non-annotated T-DNA insertions, we developed a reliable, cost-effective and easy method based on whole-genome sequencing. To enable comprehensive access to our data, we implemented a public web application named PhenoLeaf (http://genetics.umh.es/phenoleaf) that allows researchers to query the results of our screen, including text and visual phenotype information. We demonstrated how this new resource can facilitate gene function discovery by identifying and characterizing At1g77600, which we found to be required for proximal-distal cell cycle-driven leaf growth, and At3g62870, which encodes a ribosomal protein needed for cell proliferation and chloroplast function. This collection provides a valuable tool for the study of leaf development, characterization of biomass feedstocks and examination of other traits in this fundamental photosynthetic organ.

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“…Southern‐by‐Sequencing, described here using maize as an example, offers a high‐throughput and targeted NGS‐based alternative to traditional molecular characterization analyses for screening and selection of transformation events. Previously, insertion site analysis via targeted sequencing has been described for Arabidopsis thaliana (L.) Heynh., ecotype Columbia‐4, (Lapage et al, 2013) by targeting a small portion of the T‐DNA ends, which leaves most of the inserted T‐DNA uncharacterized. Whole‐transgene characterization by array hybridization and targeted sequencing has been described in mice (DuBose et al, 2013) but is not as applicable to a high‐throughput format as an in‐solution based capture method.…”

Section: Resultsmentioning

confidence: 99%

Southern‐by‐Sequencing: A Robust Screening Approach for Molecular Characterization of Genetically Modified Crops

et al. 2015

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Molecular characterization of events is an integral part of the advancement process during genetically modified (GM) crop product development. Assessment of these events is traditionally accomplished by polymerase chain reaction (PCR) and Southern blot analyses. Southern blot analysis can be time-consuming and comparatively expensive and does not provide sequence-level detail. We have developed a sequence-based application, Southernby-Sequencing (SbS), utilizing sequence capture coupled with nextgeneration sequencing (NGS) technology to replace Southern blot analysis for event selection in a high-throughput molecular characterization environment. SbS is accomplished by hybridizing indexed and pooled whole-genome DNA libraries from GM plants to biotinylated probes designed to target the sequence of transformation plasmids used to generate events within the pool. This sequence capture process enriches the sequence data obtained for targeted regions of interest (transformation plasmid DNA). Taking advantage of the DNA adjacent to the targeted bases (referred to as nextto-target sequence) that accompanies the targeted transformation plasmid sequence, the data analysis detects plasmid-to-genome and plasmid-to-plasmid junctions introduced during insertion into the plant genome. Analysis of these junction sequences provides sequence-level information as to the following: the number of insertion loci including detection of unlinked, independently segregating, small DNA fragments; copy number; rearrangements, truncations, or deletions of the intended insertion DNA; and the presence of transformation plasmid backbone sequences. This molecular evidence from SbS analysis is used to characterize and select GM plants meeting optimal molecular characterization criteria. SbS technology has proven to be a robust event screening tool for use in a highthroughput molecular characterization environment.

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Time- and Cost-Efficient Identification of T-DNA Insertion Sites through Targeted Genomic Sequencing

Cited by 31 publications

References 20 publications

Identification of Genomic Insertion and Flanking Sequence of G2-EPSPS and GAT Transgenes in Soybean Using Whole Genome Sequencing Method

Identification of Genomic Insertion and Flanking Sequence of G2-EPSPS and GAT Transgenes in Soybean Using Whole Genome Sequencing Method

Leaf phenomics: a systematic reverse genetic screen for Arabidopsis leaf mutants

Southern‐by‐Sequencing: A Robust Screening Approach for Molecular Characterization of Genetically Modified Crops

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