High-throughput sequencing makes TILLING more fun

TILLING is a functional genomic method entailing efficient discovery of induced mutations in populations. Using rice and wheat, we are developing approaches that apply Illumina sequencing to TILLING. Our first approach involves resequencing target genes amplified from multi-dimensionally pooled templates representing up to 2000 individuals per experiment. Single nucleotide changes identified in aligned reads are evaluated for frequency, sequencing quality, intersection pattern in pools, and statistical relevance to produce a Bayesian score with an associated confidence threshold. The method provides robust discovery of mutations in 10 to 50 target genes per experiment at an economic cost. The second approach measures mutation density in individuals, a procedure advantageous for optimization of treatment level and choice of target genotype during development of a TILLING population. The method entails sequencing complexity-reduced DNA with restriction enzyme-phased reads coupled to algorithms that identify mutations with or without a reference genome. The third approach addresses the construction of a permanent TILLING resource providing allelic series for most genes. Mutations and their predicted effect will be stored in a database connected to a germplasm repository for public retrieval. Toward this end, we are developing exome capture and sequencing for mutation identification and anticipate that mutations in relevant exonic space for thousands of individuals can be discovered and added to a database for a reasonable cost. Together, these approaches can provide functional genomics solutions for sexually propagated economic species on a scale ranging from few genes to all genes.