Time- and cost-efficient identification of T-DNA insertion sites through targeted genomic sequencing.
Forward genetic screens enable the unbiased identification of genes involved in biological processes. In Arabidopsis, several mutant collections are publicly available, which greatly facilitates such practice. Most of these collections were generated by agrotransformation of a T-DNA at random sites in the plant genome. However, precise mapping of T-DNA insertion sites in mutants isolated from such screens is a laborious and time-consuming task. Here we report a simple, low-cost and time efficient approach to precisely map T-DNA insertions simultaneously in many different mutants. By combining sequence capture, next-generation sequencing and 2D-PCR pooling, we developed a new method that allowed the rapid localization of T-DNA insertion sites in 55 out of 64 mutant plants isolated in a screen for gyrase inhibition hypersensitivity.
Department of Biochemistry, Université de Montréal, Montréal, Quebec, Canada.
Étienne
É
Lepage
Éric
É
Zampini
Brian
B
Boyle
Normand
N
Brisson
D017360:Arabidopsis / Q000187:drug effects / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics*; D023061:Gene Order; D018745:Genome, Plant; D023281:Genomics; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods; D016254:Mutagenesis, Insertional; D009154:Mutation; D030821:Plants, Genetically Modified; D059005:Topoisomerase II Inhibitors / Q000494:pharmacology
D004269:DNA, Bacterial; C036483:T-DNA; D059005:Topoisomerase II Inhibitors