title: 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.
authors: Laboratoire d'Ecologie Alpine (LECA), Centre National de la Recherche Scientifique, F-38000, Grenoble, France.|Laboratoire d'Ecologie Alpine (LECA), Université Grenoble-Alpes, F-38000, Grenoble, France.
authors: M
authors: M
authors: De Barba
authors: Laboratoire d'Ecologie Alpine (LECA), Centre National de la Recherche Scientifique, F-38000, Grenoble, France.|Laboratoire d'Ecologie Alpine (LECA), Université Grenoble-Alpes, F-38000, Grenoble, France.
authors: C
authors: C
authors: Miquel
authors: Laboratoire d'Ecologie Alpine (LECA), Centre National de la Recherche Scientifique, F-38000, Grenoble, France.|Laboratoire d'Ecologie Alpine (LECA), Université Grenoble-Alpes, F-38000, Grenoble, France.
authors: S
authors: S
authors: Lobréaux
authors: ONCFS, Equipe Ours, RN 117, F-31800, Villeneuve de Rivière, France.
authors: P Y
authors: PY
authors: Quenette
authors: Department of Ecology and Natural Resource Management, Norwegian University of Life Sciences, PO Box 5003, NO-1432, Ås, Norway.|Norwegian Institute for Nature Research, NO-7485, Trondheim, Norway.
authors: J E
authors: JE
authors: Swenson
authors: Laboratoire d'Ecologie Alpine (LECA), Centre National de la Recherche Scientifique, F-38000, Grenoble, France.|Laboratoire d'Ecologie Alpine (LECA), Université Grenoble-Alpes, F-38000, Grenoble, France.
authors: P
authors: P
authors: Taberlet
mesh_terms: D000483:Alleles; D000818:Animals; D004247:DNA / Q000032:analysis*; D004463:Ecology; D005819:Genetic Markers; D005828:Genetics, Population; D005838:Genotype; D060005:Genotyping Techniques; D059014:High-Throughput Nucleotide Sequencing; D018895:Microsatellite Repeats; D060885:Multiplex Polymerase Chain Reaction; D001503:Ursidae / Q000235:genetics
chemical_list: D005819:Genetic Markers; D004247:DNA
keywords: 
  Ursus arctos
  ; faecal DNA; high-throughput sequencing; individual identification; parentage analysis; short tandem repeat (STR)