Do-it-Yourself Mock Community Standard for Multi-Step Assessment of Microbiome Protocols

Area of Science:

• Microbiology
• Molecular Biology
• Bioinformatics

Background:

• Microbiome research relies on high-throughput sequencing (HTS) for analyzing diverse environments.
• Effective controls, like mock microbial communities, are crucial for validating HTS methods.
• Commercially available mock communities can be expensive and may not represent non-human microbiomes.

Purpose of the Study:

• To provide protocols for designing and constructing a do-it-yourself (DIY) mock microbial community.
• To establish a cost-effective and customizable positive control for microbiome studies.
• To ensure the accuracy and reliability of cultivation-independent microbiome analyses.

Main Methods:

• Strain identification and verification using Sanger sequencing.
• Creation of glycerol stocks for long-term cryostorage of microbial strains.
• Development of standard curves to quantify microbial cell concentrations (CFU/ml).
• Assembly of the mock community using calculated concentrations and standard curves.
• Assessment of strain viability and cryostorage methods.

Main Results:

• Detailed protocols for DIY mock community construction are presented.
• Methods for verifying strain identity and quantifying cell density are described.
• Guidance on assembling, storing, and interpreting results from DIY mock communities is provided.

Conclusions:

• DIY mock communities offer a customizable and economical alternative to commercial controls.
• These communities serve as essential positive controls for validating microbiome analysis workflows.
• DIY mock communities aid in assessing contamination and identifying protocol biases in HTS microbiome studies.