title: Discovery of rare mutations in populations: TILLING by sequencing.
abstract: Discovery of rare mutations in populations requires methods, such as TILLING (for Targeting Induced Local Lesions in Genomes), for processing and analyzing many individuals in parallel. Previous TILLING protocols employed enzymatic or physical discrimination of heteroduplexed from homoduplexed target DNA. Using mutant populations of rice (Oryza sativa) and wheat (Triticum durum), we developed a method based on Illumina sequencing of target genes amplified from multidimensionally pooled templates representing 768 individuals per experiment. Parallel processing of sequencing libraries was aided by unique tracer sequences and barcodes allowing flexibility in the number and pooling arrangement of targeted genes, species, and pooling scheme. Sequencing reads were processed and aligned to the reference to identify possible single-nucleotide changes, which were then evaluated for frequency, sequencing quality, intersection pattern in pools, and statistical relevance to produce a Bayesian score with an associated confidence threshold. Discovery was robust both in rice and wheat using either bidimensional or tridimensional pooling schemes. The method compared favorably with other molecular and computational approaches, providing high sensitivity and specificity.
authors.affiliation: Department of Plant Biology and Genome Center, University of California, Davis, California 95616, USA.
authors.forename: Helen
authors.initials: H
authors.lastname: Tsai
authors.forename: Tyson
authors.initials: T
authors.lastname: Howell
authors.forename: Rebecca
authors.initials: R
authors.lastname: Nitcher
authors.forename: Victor
authors.initials: V
authors.lastname: Missirian
authors.forename: Brian
authors.initials: B
authors.lastname: Watson
authors.forename: Kathie J
authors.initials: KJ
authors.lastname: Ngo
authors.forename: Meric
authors.initials: M
authors.lastname: Lieberman
authors.forename: Joseph
authors.initials: J
authors.lastname: Fass
authors.forename: Cristobal
authors.initials: C
authors.lastname: Uauy
authors.forename: Robert K
authors.initials: RK
authors.lastname: Tran
authors.forename: Asif Ali
authors.initials: AA
authors.lastname: Khan
authors.forename: Vladimir
authors.initials: V
authors.lastname: Filkov
authors.forename: Thomas H
authors.initials: TH
authors.lastname: Tai
authors.forename: Jorge
authors.initials: J
authors.lastname: Dubcovsky
authors.forename: Luca
authors.initials: L
authors.lastname: Comai
mesh_terms: D017343:Genes, Plant / Q000235:genetics; D005828:Genetics, Population; D018745:Genome, Plant / Q000235:genetics*; D016296:Mutagenesis / Q000235:genetics*; D009154:Mutation / Q000235:genetics*; D012275:Oryza / Q000235:genetics*; D010865:Pilot Projects; D011336:Probability; D017422:Sequence Analysis, DNA / Q000379:methods*; D013698:Templates, Genetic; D014908:Triticum / Q000235:genetics*