The complexities involved in 16S rRNA community profiling and shotgun metagenomics methods pose significant challenges for microbiome research. Significant biases can be introduced during sample preparation, DNA extraction, PCR amplification, library preparation, sequencing, and/or data interpretation. To address these biases and provide a measure of standardization within microbiome research and applications, ATCC has developed a set of mock microbial communities comprising fully sequenced, characterized strains that can be used as universal controls for assay development and optimization.

The ATCC^® Microbiome Standards Collection

ATCC^® Microbiome Standards allow you to optimize your metagenomics research applications with confidence, and improve the consistency and reproducibility of your data run after run. From sample collection to data analysis, ATCC offers the resources you need to optimize your microbiome research and applications, including:

Mock Microbial Communities

Environmental Mixtures

Pathogen Mixtures

Site-specific Standards

Microbiome Spike-in Controls

Individual Strains and Nucleic Acids

State-of-the-art Bioinformatics

What Are ATCC^® Microbiome Standards?

ATCC^® Microbiome Standards are fully sequenced, characterized, and authenticated mock microbial communities that mimic mixed metagenomics samples. Each community is prepared from authenticated strains that have been selected for relevant phenotypic and genomic attributes such as:

What’s more, these standards are the only reference materials on the market completely manufactured from high-quality ATCC^® Genuine Cultures that are characterized by polyphasic testing, fully sequenced, and published in various databases. These standards enable the optimization of metagenomics workflows and microbiome research applications, providing reliable comparative data while improving assay consistency.

Choose Your Format and Level of Complexity

To best support your research, ATCC^® Microbiome Standards are provided in a variety of formats and levels of complexity:

The robust applicability of these controls, combine with ATCC’s commitment to authentication and characterization, make ATCC^® Microbiome Standards the ideal tool for standardizing data from a wide range of sources and generating consensus among microbiome applications and analyses.

• ■ Acinetobacter baumannii
• ■ Bacillus cereus
• ■ Bifidobacterium adolescentis
• ■ Deinococcus radiodurans
• ■ Escherichia coli
• ■ Lactobacillus gasseri
• ■ Porphyromonas gingivalis
• ■ Pseudomonas aeruginosa
• ■ Staphylococcus aureus
• ■ Streptococcus agalactiae

• ■ Actinomyces odontolyticus
• ■ Bacteroides vulgatus
• ■ Clostridium beijerinckii
• ■ Enterococcus faecalis
• ■ Helicobacter pylori
• ■ Neisseria meningitidis
• ■ Propionibacterium acnes
• ■ Rhodobacter sphaeroides
• ■ Staphylococcus epidermidis
• ■ Streptococcus mutans

Improve the Consistency of your Assays

ATCC^® Microbiome Standards support a broad array of applications ranging from method optimization to data interpretation, and they serve as superior controls for microbial community testing and assay development on any platform. By using ATCC^® Microbiome Standards combined with the One Codex data analysis module in your research, you can compare the performance and overall accuracy of your methods and ensure the validity of your results.

Figure 1. The whole cell mock microbial communities can be used to compare different DNA extraction kits. Here, we show a proof-of-concept study. Total DNA from two aliquots of the whole cell microbiome standards was extracted using two commercial extraction kits. Following extraction, the genomic DNA was analyzed using 16S rRNA and shotgun genomic sequencing analysis on the Illumina^® MiSeq^® platform, and the resulting data were analyzed using the ATCC bioinformatics tool in One Codex.

Figure 2. The genomic DNA mock microbial communities can be used to compare 16S rRNA and shotgun metagenomics assays. Here, we show a proof-of-concept study. The performance and overall accuracy of the quantitative genomic DNA mock microbial communities were tested using the Illumina^® MiSeq^® platform, and the resulting data was analyzed using the ATCC bioinformatics tool in One Codex.

• ■ Streptococcus mutans
• ■ Streptococcus agalactiae
• ■ Staphylococcus epidermidis
• ■ Staphylococcus aureus
• ■ Rhodobacter sphaeroides
• ■ Pseudomonas aeruginosa
• ■ Propionibacterium acnes

• ■ Porphyromonas gingivalis
• ■ Neisseria meningitidis
• ■ Lactobacillus gasseri
• ■ Helicobacter pylori
• ■ Escherichia coli
• ■ Enterococcus faecalis
• ■ Deinococcus radiodurans

• ■ Clostridium beijerinckii
• ■ Bifidobacterium adolescentis
• ■ Bacteroides vulgatus
• ■ Bacillus cereus
• ■ Actinomyces odontolyticus
• ■ Acinetobacter baumannii

Source Standards Based on Specific Applications

Pathogen Detection

ATCC has developed a mock microbial community for clinically relevant pathogen detection in partnership with the LGC Group, an international leader in genomics, measurement standards, and reference materials. With the ATCC Metagenomic Control Material for Pathogen Detection (ATCC^® MSA-4000™), you can expect:

• Genomic DNA prepared from fully sequenced ATCC^® Genuine Cultures
• A combination of strains observed in clinical infections, including antimicrobial-resistant organisms
• Absolute quantification of genomic DNA and assignment of genome copy number using Droplet Digital™ PCR to improve analytical precision

• ■ Staphylococcus aureus subsp. aureus (MRSA)
• ■ Staphylococcus aureus subsp. aureus (MSSA)
• ■ Streptococcus pneumoniae
• ■ Streptococcus pyogenes
• ■ Streptococcus agalactiae
• ■ Enterococcus faecalis

• ■ Pseudomonas aeruginosa
• ■Klebsiella pneumoniae subsp. pneumoniae
• ■Acinetobacter baumannii
• ■Escherichia coli
• ■Neisseria meningitidis

Figure 3. The ATCC Metagenomic Control Material for Pathogen Detection (ATCC^® MSA-4000™) can be used to compare various molecular diagnostic methods. Here, we used the (A) control material to evaluate (B) digital PCR and compare (C) 16S rRNA and shotgun metagenomics analysis methods. *The percent relative abundance of Staphylococcus aureus includes both MRSA and MSSA.

Environmental Testing

The ABRF-MGRG metagenomics reference standards were developed and packaged in partnership with the Association of Biomolecular Resource Facilities Metagenomics Research Group (ABRF-MGRG) as part of the Extreme Microbiome Project (XMP). With the ABRF-MGRG metagenomics reference standards (ATCC^® MSA-3000™, MSA-3001™, MSA-3002™), you can expect:

• Genomic DNA prepared from fully sequenced ATCC^® Genuine Cultures representing bacterial and archaeal species found in extreme environments
• Mock communities that are human DNA-free, RNA-free, and tested to ensure purity via sequencing
• Access to One Codex, the leading bioinformatics platform for microbial genomics

• ■ Enterococcus faecalis
• ■ Staphylococcus epidermidis
• ■ Micrococcus luteus
• ■ Haloferax volcanii
• ■ Halobacillus halophilus
• ■ Bacillus subtilis
• ■ Pseudoalteromonas haloplanktis
• ■ Escherichia coli
• ■ Chromobacterium violaceum
• ■ Pseudomonas fluorescens

Figure 4. The ABRF-MGRG metagenomics reference standards can be used to compare different sequencing platforms. As a proof-of-concept, ATCC^® MSA-3001™ was used a control material to compare the performance of the Illumina^® MiniSeq™, MiSeq^®, NextSeq^®, and HiSeq^® platforms. Data were analyzed using the ATCC bioinformatics tool in One Codex. ABRF-MGRG metagenomics reference standards can also be used to evaluate run-to-run reproducibility (data not shown).

Source Standards Based on Body Site

ATCC has developed several site-specific mock microbial communities for research on the human microbiome. With these standards, you can expect:

• Mock microbial communities representing the oral, skin, gut, and vaginal microbiomes
• Comprising normal and atypical flora
• Genomic DNA or whole cells prepared from fully sequenced ATCC^® Genuine Cultures
• Anaerobic and aerobic microbial strains
• A combination of Gram-positive and -negative bacterial cultures

These site-specific microbiome standards are ideal for use as controls in microbial profiling of mixed populations or for research on the effects of dysbiosis on human health.

Calibrate your Experiments with Spike-in Controls

Microbiome spike-in controls from ATCC comprise exogenous bacteria and archaea that do not exist in the human microbiome under physiological conditions, making them easy to distinguish from human bacteria via standard sequencing methods. These microbiome standards offer a valuable way to evaluate the accuracy and reproducibility of library preparation, sequencing, and data analysis in both 16S rRNA community profiling and shotgun metagenomics. Further, they enable inter-sample comparison, thereby providing a way to standardize microbiome research across different laboratories.

Figure 5. Microbiome spike-in controls are ideal for evaluating the accuracy and reproducibility of different sequencing methods. As a proof-of-concept, 1 μL of the spike-in control (1 x 10⁶ genomes/strain) was added to 20 μL of the 10 strain even mix genomic material standard (ATCC^® MSA-1000™). Samples were then evaluated using 16S rRNA community profiling. The percentage of reads recovered were determined using the One Codex database. Strains were evaluated at the genus level.