Mapping multiple co-sequenced T-DNA integration sites within the Arabidopsis genome.
BACKGROUND
Insertion mutagenesis, using transgenes or endogenous transposons, is a popular method for generating null mutations (knockouts) in model organisms. Insertions are mapped to specific genes by amplifying (via TAIL-PCR) and sequencing genomic regions flanking the inserted DNA. The presence of multiple TAIL-PCR templates in one sequencing reaction results in chimeric sequence of intermittently low quality. Standard processing of this sequence by applying Phred quality requirements results in loss of informative sequence, whereas not trimming low-quality sequence causes inclusion of low-complexity homopolymers from the ends of sequence runs. Accurate mapping of the flanking sequences is complicated by the presence of gene families.


RESULTS
Methods for extracting informative regions from sequence traces obtained by sequencing multiple TAIL-PCR fragments in a single reaction are described. The completely sequenced Arabidopsis genome was used to identify informative TAIL-PCR sequence regions. Methods were devised to define and select high quality matches and precisely map each insert to the correct genome location. These methods were used to analyze sequence of TAIL-PCR-amplified flanking regions of the inserts from individual plants in a T-DNA-mutagenized population of Arabidopsis thaliana, and are applicable to similar situations where a reference genome can be used to extract information from poor-quality sequence.
Presting
Gernot G
GG
Torrey Mesa Research Institute, 3115 Merryfield Row, San Diego, CA 92121, USA. gernot@genome.clemson.edu
D017360:Arabidopsis / Q000737:chemistry / Q000235:genetics*; D001483:Base Sequence; D004252:DNA Mutational Analysis / Q000379:methods; D004251:DNA Transposable Elements / Q000235:genetics*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics*; D020869:Gene Expression Profiling / Q000379:methods; D018745:Genome, Plant; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D016415:Sequence Alignment / Q000379:methods*; D017422:Sequence Analysis, DNA / Q000379:methods*; D017385:Sequence Homology
D004251:DNA Transposable Elements; D004269:DNA, Bacterial; C036483:T-DNA