Targeted Genomic Sequencing for Marker Assisted/Genomic Selection in Crops

The development of next generation sequencing (NGS) has greatly increased the amount of information generated and has the potential to improve the way researchers perform genomic and marker assisted selection (MAS). Using NGS genotyping-by-sequencing (GBS) has provided a quick and low cost method with the potential to generate thousands of markers for analysis. The application of GBS data for hexaploid wheat (Triticum aestivum L.) has been problematic primarily due to the complex genome structure and absence of a well annotated, sequenced genome. Lack of in-house bioinformatic capabilities compounds the difficulty of analysis of big data generated by GBS. An alternate approach that addresses these issues and allows for genomic selection and MAS is to use a targeted amplicon sequencing method. This protocol multiplexes 1,000’s of mapped 90K SNPs and known informative genes that saturate the wheat genome at ~ 2cM intervals. NGS libraries were constructed with two PCR programs that use >1,000 oligo pairs and can be barcoded to run up to 864 samples on the Ion Proton system. With this grouping of samples and oligo pairs a read depth of ~100 sequences per sample per amplicon can be achieved. Samples were run on relevant Pacific Northwest (PNW) germplasm and validated through a set of custom designed bioinformatic scripts. This pipeline clusters and aligns sequences based on sequence homology, and generates a consensus to call the alleles at the polymorphic site. This low cost, high output targeted sequencing approach can be easily adapted to use in any species.