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.
Mol Ecol Resour. 2017 May;17(3):492-507. doi: 10.1111/1755-0998.12594. Epub 2016 Aug 29.
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.
微卫星标记在过去 20 年的生态、进化和保护研究中发挥了重要作用。然而，与毛细管电泳相关的技术限制以及影响其他标记类型的技术革命，使得某些应用中继续使用微卫星标记受到质疑。我们提出了一项使用高通量测序（HTS）改进生态学中微卫星基因分型的研究。这种方法需要选择适合 HTS 的短标记，在 Illumina 平台上对 PCR 扩增的微卫星进行测序，并对序列数据进行生物信息学处理以获得多位点基因型。它利用了 HTS 可以直接访问微卫星序列这一事实，从而可以明确地识别等位基因，并通过生物信息学实现基因分型过程的自动化。此外，大规模平行测序能力使单个实验运行的信息量大大超过毛细管电泳。我们通过对 13 个新微卫星标记和一个性别标记的多重 PCR 扩增的棕熊样本进行基因分型来说明该方法。HTS 微卫星提供了准确的个体识别和亲子关系鉴定，并导致粪便降解 DNA 的基因分型成功率（84%）显著提高，同时与毛细管电泳相比降低了成本。HTS 方法在生态和保护应用中，特别是在依赖于低量/质量 DNA 分析和遗传数据库构建的应用中，具有提高微卫星基因分型成功率、准确性、效率和标准化的巨大潜力。我们根据野生动物研究中遇到的挑战讨论并展望了该方法的实施。
