Next-generation sequencing for targeted discovery of rare mutations in rice

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, DNA from a mutant population is quantified and combined in an overlapping pool design. Then, target genes are amplified from DNA pools and amplicons are combined 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 PCR and sequencing error.