Library Preparation for Illumina Sequencing of Microsatellite Loci v1

Through several decades, microsatellite length polymorphisms have been considered the most reliable and reproducible genetic markers for population genetic studies. As a PCR-based method using taxon-specific primers, microsatellite loci can be amplified even from low-concentration, degraded or contaminated DNA samples. However, known issues with the interpretation of microsatellite length data include band shifts, stuttering, length homoplasy, null alleles, paralogs or off-target amplicons and missing information on allele dosage in polyploids. Comparing the actual sequences at and around microsatellite loci, instead of just their length according to capillary electrophoresis, may alleviate some of these difficulties. Since multiple microsatellite loci are needed to achieve representativeness of the marker system across the genome, but multiple PCRs for large numbers of samples soon become too cost- and time-intensive, we combine multiple locus-specific PCR reactions in one or several multiplex PCRs. PCR products are subsequently prepared for Illumina sequencing, including the integration of sample-specific combinatorial sequence tags. This protocol is based on the BeGenDiv Amplicon dual indexing protocol (general structure; in turn based on Illumina's 16S Metagenomic Sequencing Library Preparation protocol), Lepais et al. 2020 for multiplex SSR amplification, and Information from the Illumina and UC Davis Genome Center websites about size selection with magnetic beads.