Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Av. do Café s/n, Monte Alegre, Ribeirão Preto, São Paulo, SP, 14040-903, Brazil.
Appl Microbiol Biotechnol. 2019 Jan;103(1):69-82. doi: 10.1007/s00253-018-9464-9. Epub 2018 Oct 25.
The reduction of the price of DNA sequencing has resulted in the emergence of large data sets to handle and analyze, especially in microbial ecosystems, which are characterized by high taxonomic and functional diversities. To assess the properties of these complex ecosystems, a conceptual background of the application of NGS technology and bioinformatics analysis to metagenomics is required. Accordingly, this article presents an overview of the evolution of knowledge of microbial ecology from traditional culture-dependent methods to culture-independent methods and the last frontier in knowledge, metagenomics. Topics that will be covered include sample preparation for NGS, starting with total DNA extraction and library preparation, followed by a brief discussion of the chemistry of NGS to help provide an understanding of which bioinformatics pipeline approach may be helpful for achieving a researcher's goals. The importance of selecting appropriate sequencing coverage and depth parameters to obtain a suitable measure of microbial diversity is discussed. As all DNA sequencing processes produce base-calling errors that compromise data analysis, including genome assembly and microbial functional analysis, dedicated software is presented and conceptually discussed with regard to potential applications in the general microbial ecology field.
DNA 测序价格的降低导致了大量数据集的出现，需要对这些数据集进行处理和分析，特别是在微生物生态系统中，这些生态系统具有高度的分类和功能多样性。为了评估这些复杂生态系统的特性，需要了解 NGS 技术和生物信息学分析在宏基因组学中的应用的概念背景。因此，本文概述了从传统的依赖培养方法到非依赖培养方法以及宏基因组学的知识前沿，微生物生态学知识的演变。涵盖的主题包括用于 NGS 的样品制备，从总 DNA 提取和文库制备开始，然后简要讨论 NGS 的化学，以帮助理解哪种生物信息学管道方法可能有助于实现研究人员的目标。讨论了选择适当的测序覆盖度和深度参数以获得合适的微生物多样性衡量标准的重要性。由于所有的 DNA 测序过程都会产生碱基调用错误，从而影响数据分析，包括基因组组装和微生物功能分析，因此专门介绍并从概念上讨论了专用软件在一般微生物生态学领域的潜在应用。
