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Title:   Author  item
                                            BURKART-WACO, DIANA - University Of Californiaitem
                                            TSAI, HELEN - University Of Californiaitem
                                            NGO, KATHIE - University Of Californiaitem
                                            HENRY, ISABELLE - University Of Californiaitem
                                            COMAI, LUCA - University Of Californiaitem
                                            Tai, Thomas
  Submitted to: Book Chapter
                                    Publication Type: Book / Chapter
                                    Publication Acceptance Date: 3/16/2016
                                    Publication  Date: N/A
                                    Citation: N/AInterpretive Summary: Targeting of Induced Local Lesions in Genomes (TILLING) is a reverse genetics method that combines generation of chemically-induced mutants and high throughput mutation discovery platforms. This book chapter describes a detailed protocol for the generation and sequencing of pooled mutant DNAs amplified from specific gene targets. A brief description of a computational analysis pipeline for calling real mutations versus changes resulting from sequencing-based errors is also provided. The example presented involves rice although the method has been successfully applied to several species.
                                Technical Abstract: Advances in DNA sequencing (i.e., next-generation sequencing, NGS) have greatly increased the power and efficiency of detecting rare mutations in large mutant populations. Targeting Induced Local Lesions in Genomes (TILLING) is a reverse genetics approach for identifying gene mutations resulting from chemical mutagenesis. In traditional TILLING, mutation discovery is accomplished through mismatch cleavage of mutant and wild-type DNA heteroduplexes using endonucleases. This is followed by Sanger sequencing to determine the specific sequence changes. TILLING by sequencing (TBS) uses NGS to facilitate the concurrent detection and sequence characterization of mutations, which allows researchers to prioritize mutants for further analyses. NGS increases the sensitivity of mutation detection and thus improves screening efficiency by allowing the pooling of more DNAs. Here we describe a protocol for TBS using rice as an example. First, target genes are amplified from mutant population of interest. Then, amplicons are combined in overlapping pool designs to maximize throughput and increase likelihood of mutation detection during sequencing. Once sequence data is obtained, mutations are called using statistical approaches that weigh likelihood of rare mutations versus the probability of sequencing error.


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