title: High-throughput discovery of rare human nucleotide polymorphisms by Ecotilling.
abstract: Human individuals differ from one another at only approximately 0.1% of nucleotide positions, but these single nucleotide differences account for most heritable phenotypic variation. Large-scale efforts to discover and genotype human variation have been limited to common polymorphisms. However, these efforts overlook rare nucleotide changes that may contribute to phenotypic diversity and genetic disorders, including cancer. Thus, there is an increasing need for high-throughput methods to robustly detect rare nucleotide differences. Toward this end, we have adapted the mismatch discovery method known as Ecotilling for the discovery of human single nucleotide polymorphisms. To increase throughput and reduce costs, we developed a universal primer strategy and implemented algorithms for automated band detection. Ecotilling was validated by screening 90 human DNA samples for nucleotide changes in 5 gene targets and by comparing results to public resequencing data. To increase throughput for discovery of rare alleles, we pooled samples 8-fold and found Ecotilling to be efficient relative to resequencing, with a false negative rate of 5% and a false discovery rate of 4%. We identified 28 new rare alleles, including some that are predicted to damage protein function. The detection of rare damaging mutations has implications for models of human disease.
authors.affiliation: Basic Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA. btill@fhcrc.org
authors.forename: Bradley J
authors.initials: BJ
authors.lastname: Till
authors.forename: Troy
authors.initials: T
authors.lastname: Zerr
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Bowers
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Greene
authors.forename: Luca
authors.initials: L
authors.lastname: Comai
authors.forename: Steven
authors.initials: S
authors.lastname: Henikoff
mesh_terms: D000465:Algorithms; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D023281:Genomics / Q000191:economics / Q000379:methods*; D006801:Humans; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide
chemical_list: D017931:DNA Primers