Using NEXTflex Amplicon Studio for SNPs Detection in the Flax (Linum usitatissimum) Genome by Illumina-based Targeted Sequencing

Various mutagens are used to induce mutations in plant genomes in order to introduce selective phenotypical features. However, the methods traditionally used to identify these desired mutations, such as map-based cloning, TILLING, PCR-RFLP, and CAPS, are very expensive. The introduction of NGS targeted sequencing of specific genes opens a frontier for large scale screening of samples from populations in which mutations were forcibly induced. Primer design is a critical issue which must be optimized to facilitate efficient large scale targeted sequencing projects. Development of robust, highly multiplexed amplicon panels offering consistent, high quality results requires the design of primers with the same amplification efficiencies.

With well-defined primer selection criteria, and a robust, interactive and flexible design, the NEXTflex™ Amplicon Studio™ allows production of the best candidate primers. A novel scoring system has been incorporated into the primer design process, ensuring candidate primers are always represented numerically. This scoring system allows for formal quality comparison between all primers in a set, and also allows many sets to be compared. Using the NEXTflex Amplicon Studio,  a custom amplicon panel was designed spanning the coding regions of the pectin methylesterase (PME) genes (LuPME10, LuPME73, LuPME79, LuPME105) for discovery of variations in SNPs after using ethyl methanesulfonate (EMS) to induce point mutations in the flax (Linum usitatissimum) genome.