How to Choose a Microbiome Standard

The advancement of NGS-based technologies has led to rapid growth in microbiome research, enabling scientists to decipher microbial community composition, function, and interactions. Many studies conclude that technical variability in microbiome processing methods leads to significant differences in results[1–3]. Most discrepancies can be explained by differences in nucleic acid extraction, NGS library preparation, bioinformatic data processing, and the choice of reference databases.

Despite the complexity introduced by protocol variation, data is being generated at an unprecedented pace. However, without appropriate microbiome standards or quality control materials, many important conclusions cannot be reliably reproduced or compared across datasets.

Commonly used controls and reference reagents are often referred to as ‘standards’ because they enable comparisons across methods, equipment, and protocols. Microbiome standards are essential for microbial community profiling and quality control. While the microbial composition of experimental samples is often unknown and variable, standards provide a defined and consistent baseline for comparison. They help reveal potential biases, validate protocols, and enable inter-laboratory comparisons, supporting reproducibility across experiments.

The concept of a microbiome standard is straightforward: use a well-characterised and quantified microbial input to assess the consistency of an experimental workflow. These standards are typically run in parallel with test samples to monitor workflow performance. The resulting profile serves as a reference to calibrate and, when necessary, troubleshoot the pipeline.

Several types of microbiome quality control materials are available, each designed to assess different stages of the NGS-based microbiome analysis workflow. This article provides guidance on selecting the most appropriate microbiome standard for your application.

Microbiome standards fall into several categories, including mock microbial communities, true diversity reference materials, and spike-in controls. Each category can serve as a positive control and detect specific sources of bias throughout the workflow.

Mock communities made from whole cells are the most widely used microbiome standards because they assess the full workflow. They are also essential for evaluating lysis efficiency. For instance, the ZymoBIOMICS Microbial Community Standard contains equal abundances of microbes with varying cell wall properties. If Gram-negative species are overrepresented in results compared to Gram-positive species, this may indicate lysis inefficiencies.

Site-specific standards like the ZymoBIOMICS Gut Microbiome Standard are designed for specific sample types, supporting method validation in gut microbiome studies[6–7].

Log-distributed cellular standards such as ZymoBIOMICS Microbial Community Standard II (Log Distribution) contain species at a wide range of concentrations (10²–10⁸ cells per prep), supporting detection limit assessment[8].

DNA-based microbiome standards are used downstream of DNA extraction and are primarily tools for evaluating library preparation and bioinformatics pipelines. For example, the ZymoBIOMICS Microbial Community DNA Standard helps identify preparation and analysis biases[9–10].

Log-distributed versions, such as ZymoBIOMICS Microbial Community DNA Standard II, support detection limit assessment at the DNA level.

The ZymoBIOMICS HMW DNA Standard, designed for long-read sequencing applications, is the only high molecular weight mock community standard currently available. It has been used to evaluate long-read sequencing chemistries and genome assembly tools[11–12].

A true diversity reference is a natural material with a stable and complex microbial profile. Unlike synthetic mock communities, these references reflect the diversity and abundance of real samples. The ZymoBIOMICS Fecal Reference with TruMatrix™ Technology is the first commercially available true diversity microbiome standard. It is stabilised for long-term storage and consistency between lots.

These references can be used to evaluate method performance and inter-run variability. They are especially useful for testing bioinformatic pipelines under real-world conditions due to their natural complexity and fixed composition.

*TruMatrix™ is a trademark of The BioCollective.

Spike-in controls are added directly to experimental samples and provide absolute quantification as well as in situ microbiome quality control. The species used are not typically found in the human microbiome, ensuring no interference with native microbial communities.

By adding these unique microbes in defined amounts, users can determine the absolute microbial load in each sample. In situ spike-ins are particularly useful for pathogen detection using NGS workflows.

Two formats are available:

• ZymoBIOMICS Spike-in Control I: for high biomass samples (e.g. stool)

• ZymoBIOMICS Spike-in Control II: for low biomass samples (e.g. sputum, BAL fluid)

Recent years have seen rising demand for microbiome standards, references, and controls designed to meet different experimental needs. The ZymoBIOMICS product range offers a wide variety of options to help researchers improve reproducibility and data accuracy in microbiome NGS studies.

The table below summarises the key applications of each standard: