High-Throughput Microsatellite Amplicon Genotyping

In order to obtain affordable, high-throughput genotyping for parentage and sibship analyses in large numbers of individuals, we expanded on a recent high throughput SNP genotyping protocol to perform high throughput microsatellite amplicon genotyping (MAG) in catfish populations. We co-amplified up to 37 loci as 400-500bp dual barcoded amplicons in 96-well format reactions. Amplicons were pooled within each plate, then equimolar plate pools were combined and purified with magnetic beads. This super-pooled library was loaded onto the Illumina MiSeq or HiSeq platforms to produce single 300bp reads plus dual index reads. Sequences from individual samples were demultiplexed using bcl2fastq software. A bash script extracted locus-specific reads from each sample, defined the microsatellite repeat in each read and determined its length (allele), then collated allele counts for each locus within each sample. Genotypes from six loci were compared between MAG and fluorescent fragment analysis. Only 34 of 2,953 total genotypes were divergent at one allele and 16 were divergent at both alleles. These loci contained di-, tri-, tetra-, or pentanucleotide repeats. Relative quantities of allelic reads reflected relative fluorescent peak heights produced using traditional microsatellite analysis platforms. MAG permits a higher level of multiplexing than electrophoretic fragment analysis. MAG also provides flexibility in locus selection/substitution in the multiplex and flexibility in the type of polymorphism (microsatellite, SNP, simple indel), and could be used to transition from established microsatellite genotyping standards to SNP-based standards.