The report of my death was an exaggeration: A review for researchers using microsatellites in the 21st century

Microsatellites (or simple sequence repeats, SSR) are widely used markers in population genetics. Traditionally, genotyping was and still is carried out through recording fragment length. Now, next‐generation sequencing (NGS) makes it easy to obtain also sequence information for the loci of interest. This avoids misinterpretations that otherwise could arise due to size homoplasy. Here, an NGS strategy is described that allows to genotype hundreds of individuals at many custom‐designed SSR loci simultaneously, combining multiplex PCR, barcoding, and Illumina sequencing. We created three different datasets for which alleles were coded according to (a) length of the repetitive region, (b) total fragment length, and (c) sequence identity, in order to evaluate the eventual benefits from having sequence data at hand, not only fragment length data. For each dataset, genetic diversity statistics, as well as F ST and R ST values, were calculated. The number of alleles per locus, as well as observed and expected heterozygosity, was highest in the sequence identity dataset, because of single‐nucleotide polymorphisms and insertions/deletions in the flanking regions of the SSR motif. Size homoplasy was found to be very common, amounting to 44.7%–63.5% (mean over all loci) in the three study species. Thus, the information obtained by next‐generation sequencing offers a better resolution than the traditional way of SSR genotyping and allows for more accurate evolutionary interpretations.

Section: Discussionmentioning

confidence: 99%

SSR‐seq: Genotyping of microsatellites using next‐generation sequencing reveals higher level of polymorphism as compared to traditional fragment size scoring

Šarhanová

Pfanzelt

Brandt

et al. 2018

“…Additionally, NGS (next‐generation sequencing) technologies have a large potential for traditional microsatellite (simple sequence repeat, SSR) analysis (de Barba et al, ). Although RAD‐sequencing methods are becoming more widely adopted, they still require relatively high coverage per locus and thus high‐throughput sequencing (Hodel et al, ). With lower coverage, the amount of missing data increases, compromising population genetic analyses of the subsequent datasets (Arnold, Corbett‐Detig, Hartl, & Bomblies, ; Curto, Schachtler, Puppo, & Meimberg, ).…”

Section: Introductionmentioning

confidence: 99%

Application of a SSR‐GBS marker system on investigation of European Hedgehog species and their hybrid zone dynamics

Curto

Winter

Seiter

et al. 2019

By applying second‐generation sequencing technologies to microsatellite genotyping, sequence information is produced which can result in high‐resolution population genetics analysis populations and increased replicability between runs and laboratories. In the present study, we establish an approach to study the genetic structure patterns of two European hedgehog species Erinaceaus europaeus and E. roumanicus . These species are usually associated with human settlements and are good models to study anthropogenic impacts on the genetic diversity of wild populations. The short sequence repeats genotyping by sequence (SSR‐GBS) method presented uses amplicon sequences to determine genotypes for which allelic variants can be defined according to both length and single nucleotide polymorphisms (SNPs). To evaluate whether complete sequence information improved genetic structure definition, we compared this information with datasets based solely on length information. We identified a total of 42 markers which were successfully amplified in both species. Overall, genotyping based on complete sequence information resulted in a higher number of alleles, as well as greater genetic diversity and differentiation between species. Additionally, the structure patterns were slightly clearer with a division between both species and some potential hybrids. There was some degree of genetic structure within species, although only in E. roumanicus was this related to geographical distance. The statistically significant results obtained by SSR‐GBS demonstrate that it is superior to electrophoresis‐based methods for SSR genotyping. Moreover, the greater reproducibility and throughput with lower effort which can be obtained with SSR‐GBS and the possibility to include degraded DNA into the analysis, allow for continued relevance of SSR markers during the genomic era.

“…Patterns of connectivity and gene flow can be successfully investigated using both microsatellite markers (Hodel et al., ; Morin, Luikart, & Wayne, ) and SNP (single‐nucleotide polymorphism) markers (Van Inghelandt, Melchinger, Lebreton, & Stich, ). However, measuring inbreeding coefficients and effective population sizes is more suited to genome‐wide markers, for which SNPs are an increasingly popular choice (e.g., Bjelland, Weigel, Vukasinovic, & Nkrumah, ; Luikart, Ryman, Tallmon, Schwartz, & Allendorf, ; Saura et al., ).…”

Section: Introductionmentioning

confidence: 99%

Fresh is best: Accurate SNP genotyping from koala scats

Schultz

Cristescu

Littleford‐Colquhoun

et al. 2018

Maintaining genetic diversity is a crucial component in conserving threatened species. For the iconic Australian koala, there is little genetic information on wild populations that is not either skewed by biased sampling methods (e.g., sampling effort skewed toward urban areas) or of limited usefulness due to low numbers of microsatellites used. The ability to genotype DNA extracted from koala scats using next‐generation sequencing technology will not only help resolve location sample bias but also improve the accuracy and scope of genetic analyses (e.g., neutral vs. adaptive genetic diversity, inbreeding, and effective population size). Here, we present the successful SNP genotyping (1272 SNP loci) of koala DNA extracted from scat, using a proprietary DArTseq™ protocol. We compare genotype results from two‐day‐old scat DNA and 14‐day‐old scat DNA to a blood DNA template, to test accuracy of scat genotyping. We find that DNA from fresher scat results in fewer loci with missing information than DNA from older scat; however, 14‐day‐old scat can still provide useful genetic information, depending on the research question. We also find that a subset of 209 conserved loci can accurately identify individual koalas, even from older scat samples. In addition, we find that DNA sequences identified from scat samples through the DArTseq™ process can provide genetic identification of koala diet species, bacterial and viral pathogens, and parasitic organisms.