Microsatellite development from genome skimming and transcriptome sequencing: comparison of strategies and lessons...

Even though microsatellite loci frequently have been isolated using recently developed next-generation sequencing (NGS) techniques, this task is still difficult because of the subsequent polymorphism screening requires a substantial amount of time. Selecting appropriate polymorphic microsatellites is a critical issue for ecological and evolutionary studies. However, the extent to which assembly strategy, read length, sequencing depth, and library layout produce a measurable effect on microsatellite marker development remains unclear. Here, we use six frog species for genome skimming and two frog species for transcriptome sequencing to develop microsatellite markers, and investigate the effect of different isolation strategies on the yield of microsatellites.

The results revealed that the number of isolated microsatellites increases with increased data quantity and read length. Assembly strategy could influence the yield and the polymorphism of microsatellite development. Larger k-mer sizes produced fewer total number of microsatellite loci, but these loci had a longer repeat length, suggesting greater polymorphism. However, the proportion of each type of nucleotide repeats was not affected; dinucleotide repeats were always the dominant type. Finally, the transcriptomic microsatellites displayed lower levels of polymorphisms and were less abundant than genomic microsatellites, but more likely to be functionally linked loci.

These observations provide deep insight into the evolution and distribution of microsatellites and how different isolation strategies affect microsatellite development using NGS.