Colovas Joanna, Bintarti Ari Fina, Mechan Llontop Marco E, Grady Keara L, Shade Ashley
Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan.
Department of Plant, Soil, and Microbial Sciences, Michigan State University, East Lansing, Michigan.
Curr Protoc. 2022 Sep;2(9):e533. doi: 10.1002/cpz1.533.
Microbiomes provide critical functions that support animals, plants, and ecosystems. High-throughput sequencing (HTS) has become an essential tool for the cultivation-independent study of microbiomes found in diverse environments, but requires effective and meaningful controls. One such critical control is a mock microbial community, which is used as a positive control for nucleic acid extraction, marker gene amplification, and sequencing. While mock community standards can be purchased, they can be costly and often include only medically relevant microbial strains that are not expected to be major players in non-human microbiomes. As an alternative, it is possible to design and construct a do-it-yourself (DIY) mock community, which can then be used as a positive control that is specifically customized to the protocol needs of a particular study system. In this article, we describe protocols to select appropriate microbial strains for the construction of a mock community. We first describe the steps to verify the identity of community members via Sanger sequencing. Then, we provide guidance on assembling and storing the DIY mock community as viable whole cells. This includes steps to create standard growth curves referenced to plate counts for each member, so that the community members can be quantified and later compared in terms of their "expected versus returned" relative contributions after sequencing. We also describe appropriate methods for the cryostorage of the fully assembled mock community as viable whole cells, so that they can be used as a unit in a microbiome analysis, from the lysis and nucleic acid extraction steps onwards. Finally, we provide an example of returned data and interpretation of DIY mock community sequences, discussing how to assess possible contamination and identify protocol biases for particular members. Overall, DIY mock communities serve to determine success and possible bias in a cultivation-independent microbiome analysis. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Strain identification and verification using Sanger sequencing Basic Protocol 2: Creation of glycerol stocks of each mock community strain for long-term cryostorage Basic Protocol 3: Assessment of strain freezer viability without cryoprotectant Basic Protocol 4: Creation of standard curve to determine CFU/ml of a liquid culture as a function of optical density Basic Protocol 5: Full mock community assembly using community concentration calculations and standard curves.
微生物群落提供支持动物、植物和生态系统的关键功能。高通量测序（HTS）已成为对不同环境中发现的微生物群落进行非培养研究的重要工具，但需要有效且有意义的对照。其中一个关键对照是模拟微生物群落，它被用作核酸提取、标记基因扩增和测序的阳性对照。虽然可以购买模拟群落标准品，但它们成本高昂，且通常只包含医学相关的微生物菌株，而这些菌株在非人类微生物群落中预计并非主要成分。作为替代方案，可以设计和构建一个自制（DIY）模拟群落，然后将其用作专门针对特定研究系统的实验方案需求定制的阳性对照。在本文中，我们描述了为构建模拟群落选择合适微生物菌株的实验方案。我们首先描述通过桑格测序验证群落成员身份的步骤。然后，我们提供关于将自制模拟群落组装并储存为活的全细胞的指导。这包括创建以每个成员的平板计数为参考的标准生长曲线的步骤，以便对群落成员进行定量，并在测序后根据其“预期与返回”的相对贡献进行比较。我们还描述了将完全组装好的模拟群落作为活的全细胞进行冷冻保存的合适方法，以便从裂解和核酸提取步骤开始，它们可以作为一个整体用于微生物群落分析。最后，我们提供了返回数据的示例以及对自制模拟群落序列的解释，讨论了如何评估可能的污染并识别特定成员的实验方案偏差。总体而言，自制模拟群落有助于确定非培养微生物群落分析的成功与否以及可能存在的偏差。© 2022作者。由Wiley Periodicals LLC出版的《当前实验方案》。基本方案1：使用桑格测序进行菌株鉴定和验证 基本方案2：为长期冷冻保存创建每个模拟群落菌株的甘油菌液 基本方案3：评估无冷冻保护剂时菌株在冷冻库中的活力 基本方案4：创建标准曲线以确定液体培养物中每毫升菌落形成单位（CFU/ml）与光密度的函数关系 基本方案5：使用群落浓度计算和标准曲线进行完整模拟群落组装
