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.

Fragmentation of Pooled PCR Products for Highly Multiplexed TILLING

“…We conclude that fragmentation of pooled amplicons by ultrasonication provides a suitable method for producing even coverage sequencing for targeted recovery of nucleotide variation in large populations of samples. Alternative methods for amplicon fragmentation, such as use of dsDNA fragmentase have been applied for TILLING with genomic pooling of 64-fold (Burkart-Waco et al 2017). This suggests that multiple methods may be considered for experiments employing complex pools of PCR products.…”

Fragmentation of pooled PCR products for highly multiplexed TILLING

“…We conclude that fragmentation of pooled amplicons by ultrasonication provides a suitable method for producing even coverage sequencing for targeted recovery of nucleotide variation in large populations of samples. Alternative methods for amplicon fragmentation, such as use of dsDNA fragmentase have been applied for TILLING with genomic pooling of 64-fold 29 . This suggests that multiple methods may be considered for experiments employing complex pools of PCR products.…”

Deployment of Genetic and Genomic Tools Toward Gaining a Better Understanding of Rice-Xanthomonasoryzae pv. oryzae Interactions for Development of Durable Bacterial Blight Resistant Rice

“…In rice, NGS based TILLING protocols are well documented (Burkart-Waco et al, 2016) and it is a matter of time before the strategy is widely adopted to identify genes associated with resistance/susceptibility against bacterial blight pathogen in rice. Unlike NGS, where one can obtain the exact information of nucleotide base change and its position caused due to mutagen, high-resolution melting (HRM) identifies the mutation based on the differences in the melting curve of fragments of mutant and wild type.…”