TILLING by Sequencing
Discovery of rare mutations in populations: TILLING by sequencing
Discovery of rare mutations in populations requires methods, such as TILLING, 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 and T. aestivum), we developed a  method based on Illumina sequencing of target genes amplified from multi-dimensionally 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 2D or 3D pooling schemes. The method compared  favorably to other molecular and computational approaches, providing high sensitivity and  specificity.