In a recently published white paper, DNA Genotek researchers utilized the OMNIgene•GUT system (OM-200 and OMR-200)—an all-in-one system for fecal self-collection and stabilization of microbial DNA—with pediatric samples for gut microbiome profiling. From this study, the researchers identified three key reasons why the OMNIgene•GUT system is ideal for pediatric gut microbiome profiling:  

1. The OMNIgene•GUT system is compatible with existing, common extraction techniques.

2. The OMNIgene•GUT system maintains profile neutrality in pediatric microbiota at the point of collection.

3. The all-in-one system provides a stable microbiome profile of pediatric gut microbiomes for up to 60 days at room temperature.

Capturing the dynamic human gut microbiome

In humans, the gut microbiome — a comprehensive profile of the microorganisms living within the digestive tract and their abundance — is dynamic. The gut microbiome can change due to several factors including diet, medication, and age. Several scientific studies have shown that children under the age of three have a significantly lower microbial diversity index compared to adults.^1,2,3 Pediatric fecal samples are valuable for investigating early microbial colonization of the gut and in identifying factors that influence human maturation and disease outcomes.^1,2 

While effective stabilization of the adult fecal gut microbiome using the OMNIgene•GUT system has been demonstrated,⁴ the preservation of the pediatric gut microbiome had, until recently, not been tested. Acquiring accurate measurement of the changes happening in the pediatric gut microbiome was more challenging and required a high degree of confidence in the sample preservation method and careful capture of critical time points in the transition from infancy to adulthood. For example, one group of researchers observed that children underwent a substantial increase in microbiota diversity during the first few months of life.⁵  

To assess the performance of the OMNIgene•GUT system with pediatric fecal samples, researchers at DNA Genotek collaborated with Dr. Molly Fox from the University of California, Los Angeles. The group assessed a cohort of pediatric gut microbiome samples obtained from children ranging in ages from newborn to 12 months. In addition to this first cohort, a second cohort, which included fecal samples collected using the OMNIgene•GUT system from another pediatric study (involving children aged 4 to 45 months old), was also included. For the cohorts, microbial DNA was extracted using two different methods. From this research (detailed in the published white paper), three key observations provided good reasoning as to why the OMNIgene•GUT system is suitable for pediatric gut microbiome profiling. 

1. The OMNIgene•GUT system is compatible with common DNA extraction methods used for acquiring microbiome profiles from pediatric fecal samples

Profiling the microbiome from fecal samples can be challenging due to several sources of technical variation. Variation can be introduced in several steps on the path to obtaining a microbiome profile. One source of variation can come from the chosen extraction method used to isolate sufficient and high-quality microbial DNA. The choice of extraction method typically depends on the desired downstream application (e.g., some sequencing applications require greater amounts of DNA and thus require extraction methods that maximize DNA recovery). A second source of variation can be introduced with inconsistent preservation of DNA within the collected samples. For example, preserving in vivo DNA integrity is critical for optimal sequencing of DNA samples in applications such as long-range sequencing. Degradation of DNA can result in skewed and inaccurate microbial profiles.  

Detailed in their white paper, the researchers assessed OMNIgene•GUT system compatibility — measured by looking at microbial DNA yield and integrity — with commonly used DNA extraction methods. The microbial DNA from pediatric fecal samples (collected using the OMNIgene•GUT system) was extracted successfully using either the QIAamp PowerFecal Pro DNA Kit (QIAGEN) or the Repeated Bead Beating (RBB) DNA extraction method.⁶ In their results, the researchers were able to see sufficient DNA yield across a range of ages using the RBB method. In addition, using the RBB-extracted microbial DNA samples for various pediatric age groups, high molecular weight DNA bands were seen on an agarose gel implying that there was minimal DNA degradation in the samples.  

2. The OMNIgene•GUT system maintains profile neutrality in pediatric microbiota at the point of collection

As mentioned earlier, the gut microbiome is dynamic (i.e., microorganisms can appear/disappear or increase/decrease). In particular, the pediatric gut is well known to change with age. The current goal of stabilization technologies is to capture and preserve the in vivo gut microbiome in its exact state at the moment of sample collection. To preserve the representative snapshot of the gut microbiome, freezing samples (-80°C) is the gold standard approach, though chemical stabilization buffers offer a more cost-effective and transportation-friendly alternative. Similar to cold temperatures, stabilization buffers keep the collected microbial sample “neutral” by:  

• Discouraging the growth of certain microbes within the sample.

• Preventing DNA degradation. 

In their white paper, the researchers found that the stabilization buffer from the OMNIgene•GUT system could maintain a so-called neutral environment for pediatric fecal samples at the exact moment they were collected. The researchers assessed the preservation of the pediatric microbial profile by looking at several microbial diversity metrics for species richness and evenness. For some of the participants, fecal samples collected using the OMNIgene•GUT system were compared to fresh, unstabilized fecal samples. This comparison demonstrated that the stabilization buffer within the OMNIgene•GUT system did not alter the microbial composition of the samples and confirmed that the sample microbial profile was an accurate snapshot of the point of collection.  

3. The OMNIgene•GUT system stabilizes pediatric microbiome profiles beyond the point of collection

Having established that the OMNIgene•GUT system could accurately preserve and capture the pediatric microbiome profile at the point of collection, the researcher sought to determine whether the pediatric microbiome profiles were stable beyond the point of collection. Two time points, 30 and 60 days, were selected to evaluate whether the pediatric microbiome changed within the same fecal sample.  

The researchers did not see any significant difference in 30-day and 60-day stored samples compared to baseline, implying that pediatric microbial profiles remained unchanged up to 60 days. In support of this, none of the taxonomic microbial species relative abundances changed across the cohort between baseline and 30 or 60 days, suggesting the system does not introduce storage-induced bias in the recovery of taxonomic microbial species for up to 60 days post-collection. 

Key takeaways 

Standardization of any research workflow is a critical and necessary step for consistency, accurate repetition, and comparison of research results. In this sense, the dynamic pediatric gut microbiome is a particularly challenging system to study. The research conducted in this recent white paper highlights three key reasons as to why the OMNIgene•GUT system is ideal for pediatric gut microbiome profiling from fecal samples.  

The results demonstrate that the OMNIgene•GUT system is not only compatible with two well-established DNA extraction techniques, but that this system is also capable of effectively stabilizing and preserving the pediatric gut microbiome at both the initial sample collection point and beyond. Using the OMNIgene•GUT system reduces variation introduced in steps on the path to acquiring an accurate pediatric gut microbiome. 

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References 

1. Yang I, Corwin EJ, Brennan PA, Jordan S, Murphy JR, Dunlop A. (2016). The infant microbiome: implications for infant health and neurocognitive development. Nurs Res. doi: 10.1097/NNR.0000000000000133

2. Rodríguez JM, Murphy K, Stanton C, Ross RP, Kober OI, Juge N, Avershina E, Rudi K, Narbad A, Jenmalm MC, Marchesi JR, Collado MC. (2015). The composition of the gut microbiota throughout life, with an emphasis on early life. Microb Ecol Health Dis. doi: 10.3402/mehd.v26.26050

3. Tanaka M, Nakayama J. (2017). Development of the gut microbiota in infancy and its impact on health in later life. Allergol Int. doi: 10.1016/j.alit.2017.07.010

4. Doukhanine E, Bouevitch A, Brown A, LaVecchia JG, Merino C, Pozza (2016). OMNIgene®•GUT stabilizes the microbiome profile at ambient temperature for 60 days and during transport. PD-WP-00042. DNA Genotek Inc.

5. Arrieta, M-C., Stiemsma, L. T., Amenyogbe, N., Brown, E. M., & Finlay, B. (2014). The intestinal microbiome in early life: health and disease. Front Immunol. doi: 10.3389/fimmu.2014.00427

6. Yu Z, Morrison M. (2004). Improved extraction of PCR-quality community DNA from digesta and fecal samples. BioTechniques. doi: 2144/04365ST04