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TILLING (Targeting Induced Local Lesions IN Genomes) is a powerful reverse genetics method in plant functional genomics and breeding to identify mutagenized individuals with improved behavior for a trait of interest. Pooled high throughput sequencing (HTS) of the targeted genes allows efficient identification and sample assignment of variants within genes of interest in hundreds of individuals. Although TILLING has been used successfully in different crops and even applied to natural populations, one of the main issues for a successful TILLING experiment is that most currently available bioinformatics tools for variant detection are not designed to identify mutations with low frequencies in pooled samples or to perform sample identification from variants identified in overlapping pools. Our research group maintains the Next Generation Sequencing Experience Platform (NGSEP), an open source solution for analysis of HTS data. In this manuscript, we present three novel components within NGSEP to facilitate the design and analysis of TILLING experiments: a pooled variants detector, a sample identifier from variants detected in overlapping pools and a simulator of TILLING experiments. A new implementation of the NGSEP calling model for variant detection allows accurate detection of low frequency mutations within pools. The samples identifier implements the process to triangulate the mutations called within overlapping pools in order to assign mutations to single individuals whenever possible. Finally, we developed a complete simulator of TILLING experiments to enable benchmarking of different tools and to facilitate the design of experimental alternatives varying the number of pools and individuals per pool. Simulation experiments based on genes from the common bean genome indicate that NGSEP provides similar accuracy and better efficiency than other tools to perform pooled variants detection. To the best of our knowledge, NGSEP is currently the only tool that generates individual assignments of the mutations discovered from the pooled data. We expect that this development will be of great use for different groups implementing TILLING as an alternative for plant breeding and even to research groups performing pooled sequencing for other applications.

Accurate, Efficient and User-Friendly Mutation Calling and Sample Identification for TILLING Experiments