Fast sequence-based microsatellite genotyping development workflow for any non-model species

Application of high-throughput sequencing technologies to microsatellite genotyping (SSRseq) has been shown to remove many of the limitations of electrophoresis-based methods and to refine inference of population genetic diversity and structure. However, early proof of concept and species specific development studies resulted in dispersed information making it cumbersome for prospective users to identify a clear path to SSRseq approach set up in species of new interest. To overcome these difficulties, we present here a streamlined SSRseq development workflow that includes microsatellite development, multiplexed marker amplification and sequencing, and automated bioinformatics data analysis. We demonstrate its application to five groups of species across kingdoms (fungi, plant, insect and fish) with different levels of polymorphism and genomic resource availability. We found that relying on previously developed microsatellite assay is not optimal and leads to a resulting low number of reliable locus being genotyped. In contrast, de novo ad hoc primer designs gives highly multiplexed microsatellite assays that can be sequenced to produce high quality genotypes for 20 to 40 loci. We highlight critical upfront development factors to consider for effective SSRseq setup in a wide range of situations. The automated sequence analysis pipeline, which accounts for all linked polymorphisms along the sequence, quickly generates a powerful multi-allelic haplotype-based genotypic dataset. Cost and time effective application of SSRseq approaches are within reach for any species, calling to new theoretical and analytical frameworks to extract more information from multi-nucleotide polymorphism marker systems.