Abstract
Mutations tagged by transposon insertions can be readily mapped and identified in organisms with sequenced genomes. Collections of such mutants allow a systematic analysis of gene function, and can be sequence-indexed to build invaluable resources. Here we present Mu-seq (Mutant-seq), a high-throughput NextGen sequencing method for harnessing high-copy transposons. We illustrate the efficacy of Mu-seq by applying it to the Robertson’s Mutator system in a large population of maize plants. A single Mu-seq library, for example, constructed from 576 different families (2304 plants), enabled 4, 723 novel, germinal, transposon insertions to be detected, identified, and mapped with single base-pair resolution. In addition to the specificity, efficiency, and reproducibility of Mu-seq, a key feature of this method is its adjustable scale that can accomodate simultaneous profiling of transposons in thousands of individuals. We also describe a Mu-seq bioinformatics framework tailored to high-throughput, genome-wide, and population-wide analysis of transposon insertions.
Highlights
Insertional mutagenesis has provided a vital foundation for development of large-scale collections of sequence-indexed mutants for model organisms
We initially used conventional and 454-based sequencing of MuTAIL PCR amplicons to identify novel Mu insertion sites in the UniformMu maize population [11,12,13]. These data revealed seven terminal inverted repeat (TIR) variants that accounted for 95% of the new Mutransposon insertions captured by this approach in the UniformMu population (Figure S1)
Key features of Mu-seq that enable efficient creation and mining of transposon mutagenesis resources depend on maximizing the information obtained from every sequence read
Summary
Insertional mutagenesis has provided a vital foundation for development of large-scale collections of sequence-indexed mutants for model organisms. Advances in building and mining these resources have had a major impact on functional genomics research across a range of species from Drosophila [1,2] to plants. The latter include a nearly comprehensive collection of T-DNA insertion lines for Arabidopsis [3,4], transposon-based resources for rice [5,6,7], and in maize, the Robertson’s Mutator transposon has been widely used for both forward and reverse genetics [8,9,10,11,12,13,14]. Efforts to create a searchable, sequence-index of transposons in the resource [11,12,13] highlighted the need for an efficient, reproducible, sequence-based method that could concurrently handle genome-wide discovery and mapping of Mu transposons in thousands of plants
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