High‐throughput microsatellite genotyping in ecology: improved accuracy, efficiency, standardization and success with low‐quantity and degraded DNA

Abstract

Microsatellite markers have played a major role in ecological, evolutionary and conservation research during the past 20 years. However, technical constrains related to the use of capillary electrophoresis and a recent technological revolution that has impacted other marker types have brought to question the continued use of microsatellites for certain applications. We present a study for improving microsatellite genotyping in ecology using high-throughput sequencing (HTS). This approach entails selection of short markers suitable for HTS, sequencing PCR-amplified microsatellites on an Illumina platform and bioinformatic treatment of the sequence data to obtain multilocus genotypes. It takes advantage of the fact that HTS gives direct access to microsatellite sequences, allowing unambiguous allele identification and enabling automation of the genotyping process through bioinformatics. In addition, the massive parallel sequencing abilities expand the information content of single experimental runs far beyond capillary electrophoresis. We illustrated the method by genotyping brown bear samples amplified with a multiplex PCR of 13 new microsatellite markers and a sex marker. HTS of microsatellites provided accurate individual identification and parentage assignment and resulted in a significant improvement of genotyping success (84%) of faecal degraded DNA and costs reduction compared to capillary electrophoresis. The HTS approach holds vast potential for improving success, accuracy, efficiency and standardization of microsatellite genotyping in ecological and conservation applications, especially those that rely on profiling of low-quantity/quality DNA and on the construction of genetic databases. We discuss and give perspectives for the implementation of the method in the light of the challenges encountered in wildlife studies.

CHIIMP: An automated high‐throughput microsatellite genotyping platform reveals greater allelic diversity in wild chimpanzees

“…In addition, genotyping‐by‐sequencing (GBS) distinguishes alleles of the same size that contain substitutions or differ in length by a single nucleotide (Adams, Brown, & Hamilton, ). Although initially developed for human forensics (Fordyce et al., ; Van Neste, Van Nieuwerburgh, Van Hoofstat, & Deforce, ), GBS technologies have recently been used to genotype wild animals, including Atlantic cod (Vartia et al., ), brown bear (De Barba et al., ), boarfish (Farrell, Carlsson, & Carlsson, ), and muskrat (Darby, Erickson, Hervey, & Ellis‐Felege, ). These studies demonstrated the utility of GBS for molecular ecology applications (Darby et al., ; Farrell et al., ) and showed that even samples containing small quantities of host DNA, such as dung and hair, can be used for these analyses (De Barba et al., ).…”

“…Although initially developed for human forensics (Fordyce et al., ; Van Neste, Van Nieuwerburgh, Van Hoofstat, & Deforce, ), GBS technologies have recently been used to genotype wild animals, including Atlantic cod (Vartia et al., ), brown bear (De Barba et al., ), boarfish (Farrell, Carlsson, & Carlsson, ), and muskrat (Darby, Erickson, Hervey, & Ellis‐Felege, ). These studies demonstrated the utility of GBS for molecular ecology applications (Darby et al., ; Farrell et al., ) and showed that even samples containing small quantities of host DNA, such as dung and hair, can be used for these analyses (De Barba et al., ). However, alleles were primarily called manually by visual inspection of read length histograms (Darby et al., ; Farrell et al., ; Vartia et al., ), and none of these studies have compared the performance of capillary electrophoresis and high‐throughput sequencing side‐by‐side to validate and improve the genotyping approach.…”

A cost‐and‐time effective procedure to develop SNP markers for multiple species: A support for community genetics

“…Therefore, any multispecies data obtained from a common and standardized procedure could help resolve this issue. Here, SNP markers have been isolated using a standardized protocol and are easily transferable to any other laboratory, in an easier way than microsatellites as genotyping does not rely on an estimation of allele size (but see De Barba et al., ). Due to relatively small amplicon sizes, markers are also usable for degraded DNA, for example, with samples from noninvasive protocols or from museum collections (Mendisco et al., ; Morin & Mccarthy, ).…”

Microsatellite diversity of a critically endangered sturgeon, Acipenser sturio L. 1758, assessed from museum and archaeological tissue remains

“…The difficulty in genotyping the degraded DNA has considerably increased the time and cost of the analysis, limiting the amount of data produced in 2008–2009. Since then, progress has been made in the field of molecular population analysis which would allow for more efficient processing today (De Barba et al., ).…”

“…A reasonable explanation for this finding could be a lack of genetic information, linked to the reduced number of microsatellites analysed here or insufficient sampling. However, promising methodological developments, in conjunction with high‐throughput sequencing, should help in the future to find new markers to increase the number of microsatellites suitable for studying the nuclear diversity of the sturgeons in greater details and using highly degraded DNA samples (De Barba et al., ).…”