OMNIgene™•GUT provides ambient temperature stabilizationof pediatric fecal samples for microbiome profiling
Jean Macklaim, Alaya Mikalauskas, Laura Cunningham, Caroline Egloff and Evgueni Doukhanine
DNA Genotek, Ottawa, Ontario, Canada
2022-09-20

OMNIgene™•GUT (OM-200 and OMR-200) is
an all-in-one system for easy self-collection andstabilization of microbial DNA from feces for gut
microbiome profiling. In this paper, we demonstratethat OMNIgene•GUT is compatible with commonly
utilized extraction technologies, collects sufficient inputof fecal samples and stabilizes the microbiome profile
of pediatric gut microbiomes for up to 60 days atambient temperature.

Introduction
Pediatric fecal samples are often collected toinvestigate early microbial colonization of the gut
with respect to physiological and external factorsthat may influence human maturation and disease
outcomes throughout life.1,2 Accurate representationof pediatric gut microbiome relies on obtaining thein vivo representation of the richness and relative
abundance of microbes during sample collection.
Effective stabilization of fecal gut microbial profiles
by OMNIgene•GUT has been demonstrated in adults3;
however, the development of the gut during infancy
presents unique challenges, in part due to thedifference between adult and pediatric fecal
microbiome composition. Children younger than
3 years of age have a significantly lower diversity index
compared to adults, with roughly half of the microbial
taxa present in the first year of life when compared tolater life.1,2,4 In addition to differences in microbial
diversity (e.g., relative dominance of Actinobacteria
and Proteobacteria phyla) and matrix composition(dependent on factors such as diet and microbialactivity),1,2,4 these characteristics have notpreviously been addressed with regard to thesuitability of OMNIgene•GUT in preserving
pediatric gut microbiome.
The ability to generate high-quality and reliablemicrobiome sequencing data from pediatric gut
samples opens up avenues for novel research focusing
on the transient changes of gut health during early
development. In 2014, Arrieta et al. highlighted the

PD-WP-00057 Issue 1/2022-09© 2022 DNA Genotek Inc., a subsidiary of OraSure Technologies, Inc., all rights reserved.

transient and dynamic nature of pediatric gut
microbiome samples, while detecting consistentinterplay between taxonomic diversity and interindividual
variability (Figure 1).5 Accurate measurement of suchchanges and identification of key drivers require ahigh degree of confidence in the sample preservation
method and compatibility with downstream processing
and analysis of the sample. It is therefore crucial touse a method that will capture accurately, on the firstattempt, those important exposure time points thatwill form, over time, an adult-like diverse andcomplex microbiome.

Figure 1: Stages of microbial colonization of the infant and childintestine. Adapted from “The intestinal microbiome in early life:
health and disease,” by Arrieta M-C, Stiemsma LT, Amenyogbe N,
Brown EM and Finlay B, 2014, Front. Immunol. 5:427. (doi: 10.3389/
fimmu.2014.00427). Copyright © 2014 Arrieta, Stiemsma, Amenyogbe,
Brown and Finlay.

Arrieta et al. also observed that children undergo
a substantial increase in microbiota diversity during
the first few months of life. The results captured in thispaper support these conclusions and further expand
on the benefit of using OMNIgene•GUT for collecting
pediatric fecal microbiome samples. In addition,
through related and microbiome relevant measurements,
our findings are in agreement with results publishedby Williams et al., whereby “For samples transported
in ambient conditions, the limits of agreement showedwww.dnagenotek.com • support@dnagenotek.com

that the OMNIgene•GUT kit had the narrowest 95%
limits of agreement with the frozen standard as
measured by the number of operational taxonomic
units and the Shannon diversity index.6” Suchstandardization of procedures is critical for allowing
a wider number of researchers to perform impactfulstudies and compare results across experiments.
In order to evaluate OMNIgene•GUTperformance on
infant samples, we collaborated with Dr. Molly Fox at
the University of California, Los Angeles (UCLA). We
obtained access to a range of pediatric gut microbiome
samples from children who ranged in age from newborn
to 12 months. This cohort formed the core of theevaluation to determine impact of donor age, taxonomic
diversity and sample matrix on OMNIgene•GUT
performance. In addition, with the field of microbiome
research progressing into metagenomic sequencing foranalysis, we opted to include fecal samples collected in
OMNIgene•GUT from an internally conducted pediatricstudy (involving children from 4 months to 45 monthsold) utilizing metagenomic sequencing (MGS) andtaxonomic assignment following collection and storage.

Materials and methods
Sample collection
Fecal samples were collected from diapers into
OMNIgene•GUT, according to the manufacturer’s
instructions. Through our collaboration with Dr. Fox,7
we used 48 OMNIgene•GUT samples to evaluate devicecompatibility for collection and extraction of pediatricfecal samples across a range of age groups (cohort 1).
OMNIgene•GUT samples collected for the internal
pediatric study were used to evaluate post-collection
stabilization performance on a WGS (shotgun
sequencing) platform (cohort 2). An aliquot of bulk
feces from each donor in cohort 2 was collected inparallel and transported in an insulated box containing
frozen ice packs.8

DNA extraction and sample storage
Samples in cohort 1 were extracted using a repeatedbead beating (RBB) method adapted from Yu andMorrison.9 Briefly, the method involved processing fecal
samples through one cycle of bead beating in lysis buffer,
followed by ammonium acetate protein and inhibitor
precipitation, DNA precipitation in isopropanol using
Eppendorf® DNA LoBind (MilliporeSigma)
microcentrifuge tubes and subsequent DNA
purification by QIAamp® spin columns (QIAGEN®).
For baseline analysis of cohort 2 samples, an aliquotof OMNIgene•GUT and an equivalent mass of fecal
sample were extracted from fresh samples using the
PD-WP-00057 Issue 1/2022-09© 2022 DNA Genotek Inc., a subsidiary of OraSure Technologies, Inc., all rights reserved.

commonly utilized and efficient lysis method QIAamp®
PowerFecal® Pro DNA Kit (QIAGEN) according to
manufacturer protocol.10 The remaining OMNIgene•GUTsamples were kept in the collection tubes for storage at
ambient temperature (23° ± 3°C) for 30 and 60 days.

DNA analysis
DNA concentration and yield were determined using
the Quant-iT™ PicoGreen® reagent (Thermo Fisher
Scientific). DNA integrity and stability over time
was evaluated on a subset of samples by runningapproximately 75 ng of purified DNA on a 0.8%
agarose gel and staining with ethidium bromide.
UltraRanger 1 kb DNA ladder (Norgen Biotek Corp.)
was used to determine the size of purified DNA.

Library preparation and metagenomic
sequencing (MGS)
Libraries were prepared from extracted genomic DNA
from cohort 2 samples using the Nextera® XT Library
Preparation Kit (Illumina®) paired with Nextera XT
Index Kit v2 (Illumina), according to the manufacturer’s
protocol for tagmentation, amplification and clean-up
steps. Libraries were normalized using DNA
concentration determined by Quant-iT™ PicoGreen™
fluorescence (Thermo Fisher Scientific) and average
fragment size determined using the D5000 ScreenTape
Assay (Agilent Technologies). The library pool was
loaded onto the Illumina NextSeq™ 550 sequencing
platform using the 300 cycles NextSeq 500/550 High
Output Kit v2.5 (Illumina).

Bioinformatics data analysis
Demultiplexed FASTQ files were trimmed and quality
filtered, and reads mapping to the human genome or
16S rRNA gene were removed. The resultant trimmed
and filtered reads were submitted to the One Codex
platform for taxonomic classification (database v. 2018).
The mapped reads counts table was filtered to keep
reads assigned to taxonomic bins occurring in at least
2 samples and having at least 10 mapped reads.
An appropriate percent abundance filter was applied
for each analysis. For compositional data analysis,counts data were transformed using the centred
log-ratio to generate relative abundance values.
Differences in the microbiome between test conditions
were evaluated using the Aitchison distance,11,12 anddifferential abundance analysis was performed on thespecies-level taxonomy using ALDEx2.13,14 All data
analysis, visual representations and statistical analyses
were performed using R15 and various package add-ons.

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Results
OMNIgene•GUT is compatible with extraction
techniques commonly used in the pediatric fecal
microbiome field
When it comes to microbial community profiling fromcomplex environments like fecal samples, there areseveral sources of technical variation. The choice of
extraction methodology for obtaining sufficient andhigh-quality microbial nucleic acids is essential formeaningful and reliable insights into these complex
communities. The potential for low microbial biomass
within pediatric samples can further affect DNA
recovery, ultimately limiting the use of downstream
analytical applications for characterizing microbial
profiles. Some sequencing applications require asubstantial amount of DNA (> 500 ng), necessitating an
extraction procedure that can maximize DNA recovery.
To this end, we utilized the QIAamp PowerFecal Pro
DNA Kit and RBB extraction method to verify thatsufficient DNA yields can be obtained when processing
fecal samples across a wide range of pediatric age
groups. An expected increase in yield across the age
range of the cohort was observed for OMNIgene•GUT
collected samples extracted with RBB (Figure 2).

integrity in the collected samples. Preservation ofhigh molecular weight DNA within the collection
device supports optimal performance of long-range
sequencing platforms, such as single-molecule real-time
sequencing (PacBio® SMRT® Sequencing) and nanopore
sequencing (Oxford Nanopore Technologies). DNA
degradation caused by nuclease activity selectively
decreases signals from lysed microbial cells, leading
to a bias in the recovered microbiome profile. In DNA
extracted from OMNIgene•GUT samples, highmolecular weight DNA bands (> 10 kb on average)
were identified across 3 donor age groups (Figure 3).

DNA yield per aliquot (µg)

Figure 3: DNA integrity of OMNIgene•GUT collected pediatricsamples. OMNIgene•GUT samples extracted by RBB at baselinefrom representative pediatric donors belonging to 3 age groups(newborn to 3 weeks (N1), 2 months (PP2), 6 months (PP6)) wererun on TapeStation (Agilent).

OMNIgene•GUT maintains profile neutrality inpediatric microbiota at the point of collection

Age group
Figure 2: DNA yield for RBB extracted OMNIgene•GUT
collected pediatric samples (N = 48) from 4 age groups: newborn
to 3 weeks (N1), 2 months (PP2), 6 months (PP6) and 12 months(PP12). For each donor, samples were extracted at baseline (T0) aftercollection in an OMNIgene•GUT device. Error bars represent the meanvalue -/+ standard deviation.

In addition to the importance of obtaining sufficient
DNA yields across post-collection sample storage
conditions, it is crucial to preserve the in vivo DNA

PD-WP-00057 Issue 1/2022-09© 2022 DNA Genotek Inc., a subsidiary of OraSure Technologies, Inc., all rights reserved.

The pediatric gut is an environment of dynamic change,
characterized by a general increase in microbial diversityand varying fecal composition throughout the child’sdevelopment, resulting in a unique and challenging
sample type. Obtaining the best in vivo representationof the gut microbiome at the exact moment of sample
collection is the goal of current stabilization technologies.
Preserving fecal microbiota at -80°C remains the gold
standard; however, transportation or immediate accessto freezers is required if collected in a clinical setting.
Chemical stabilization buffers can offer independence
from these expensive cold-chain preservation
techniques. Ideally, these buffers should keep thecollected material neutral by not promoting selective
microbial growth or accelerating DNA degradation.

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A

B

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1000

Chao1

Observed species

3

750

500

Age
(months)

750

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OM

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Figure 4: Neutrality assessment of OMNIgene•GUT stabilization chemistry by alpha diversity metrics. (A) Shannon index (B) Observed
species and (C) Chao1 metrics were used to compare donor-paired OMNIgene•GUT and fresh unstabilized sample (N = 30, cohort 2). Each dot
represents a donor-paired fresh unstabilized and OMNIgene•GUT sample where the color corresponds to age in months. No significant
differences in alpha diversity were detected between the fresh unstabilized samples and OMNIgene•GUT samples (Wilcoxon signed-rank test).

****

Additional analysis at the beta diversity level (throughthe calculation of Aitchison distance) was performedon donor-paired samples collected in OMNIgene•GUT
and fresh unstabilized samples (Figure 5). The
distance between paired samples was far lower thanthe donor-to-donor distances, demonstrating there is
no change to the sample profile at collection (sample
neutrality). Taken together with alpha diversity results,
the stabilization solution and the built-in features of theOMNIgene•GUT offer confidence in recovering an
accurate representation of the microbial community.

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200

100

More similarity

To evaluate the preservation of pediatric microbial
profiles in OMNIgene•GUT samples, alpha diversity
metrics (Shannon index, number of observed species
and Chao1) were calculated at the species taxonomic
level to assess species richness and evenness.
OMNIgene•GUT samples and corresponding fresh
samples were extracted from 30 donors in cohort 2.
A Wilcoxon signed-rank test of values from each alphadiversity metric showed no significant difference between
donor-paired OMNIgene•GUT samples and fresh
samples, indicating that the richness and the microbial
composition of the samples were not affected by thechemical stabilization of the OMNIgene•GUT (Figure 4).

Less similarity

To assess the neutrality of OMNIgene•GUT with this
distinctive sample type, we collected fecal samples fromdonors ranging in age from 4 months to 45 months.

Aitchison distance

Shannon index

1000
4

Unstabilized vs.
OMNIgene•GUT

Unstabilized
between donors

Figure 5: Beta diversity assessment of pediatric samples.
Aitchison distance (beta diversity) for donor-paired samples collectedin OMNIgene•GUT compared to fresh unstabilized samples at baseline(N = 30, cohort 2) shows significantly lower difference (Wilcoxon
rank-sum test) compared to donor-to-donor differences betweenfresh
unstabilized samples at baseline (‘****’: P ≤ 0.0001). These significantly
low differences demonstrate that no change is occurring to the sample
profile after collection in OMNIgene•GUT.

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A

B

1000

3

1100
1000

900

Chao1

Observed species

4

800

Age
(months)

900

40
30

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T6
0

T3
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T0

T6
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T3
0

T0

T6
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T3
0

600
T0

Time point
Figure 6: Alpha diversity metrics over 30 and 60 days compared to baseline. (A) Shannon index (B) Observed species and (C) Chao1 metrics
were used to compare pediatric samples (N = 30, cohort 2) collected in OMNIgene•GUT at baseline (T0), 30 days (T30) and 60 days (T60) aftercollection. Each dot represents a donor sample collected in OMNIgene•GUT with color corresponding to age in months. No significant differencesin alpha diversity were detected between time points (Kruskal-Wallis).

Kruskal-Wallis, p < 2.2e−16

OMNIgene•GUT stabilizes pediatric microbiome
profiles beyond the point of collection
To access the stability of samples collected in
OMNIgene•GUT, we evaluated the changes in the
microbiome at 30 and 60 days using alpha (Figure 6)
and beta (Figure 7) analyses. Statistical measurement
through Kruskal-Wallis did not show any significant
difference in 30- and 60-day stored samples compared
to baseline, suggesting a lack of time-dependent
shifts during storage. In contrast, distances between
pediatric donors showed a large magnitude of
microbiome compositional differences between
donors within the same cohort. This result is
indicative of how effectively the OMNIgene•GUT
device’s stabilization chemistry prevents microbial
profile changes during storage conditions, including
a 60-day hold at ambient temperature.
Conversely, a highly significant difference (Figure 7)
was observed when comparing beta diversity (Aitchison
distance) between donors, highlighting the ability ofOMNIgene•GUT devices to conserve the uniquedonor microbial profile even within samples held
60 days post-collection at ambient temperature.

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300

****
****
ns

Aitchison distanc e

Shannon index

C

1100

200

100

T0 vs. T30

T0 vs. T60

T0 between
donorsFigure 7: Beta diversity assessment of pediatric samples over timeat ambient temperature. Each point is a donor sample at baseline (T0)compared to the same sample after storage at ambient temperaturefor 30 days (T30) or 60 days (T60) in OMNIgene•GUT. For statisticalcomparison, the final box shows the between-donor distance ofbaseline (T0) samples across all donor-to-donor permutations. The
magnitude of change (distance) and the variability for each time-point
group is far lower than the between-donor change, suggesting the
microbiome profiles are stable over time in the OMNIgene•GUT device.
Kruskal-Wallis non-parametric test was applied between groups(‘****’: P ≤ 0.0001) and a Wilcoxon rank-sum test was applied forthe two-group comparison (‘ns’: P > 0.05).

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In addition to alpha diversity and microbiome
composition distance measurements, differential
abundance analysis was applied to compare
OMNIgene•GUT collected samples at baseline
to 30- and 60-day extractions to determine if there
were any taxonomic species that consistently andsignificantly changed across the cohort. Our previous
work highlighted rapid and progressive onset of
microbiome profile change when gut samples arecollected without a -80°C cold-chain transport orchemical stabilizer, even at time holds of 24 hours
with ice packs.16 Owing to the effectiveness of thestabilization chemistry, we did not find any significantly
different species relative abundances for any of thetime points (Figure 8). These results confirm that
the OMNIgene•GUT device can effectively stabilize
pediatric gut microbiome samples post-collection
without introducing any storage-induced bias intaxonomic recovery.
As a final assessment of performance, a taxonomic
stacked bar plot at species-level resolution wasconstructed for a subset of cohort 2 OMNIgene•GUTstabilized samples following metagenomic sequencing
results (Figure 9). The subset of donor-paired samples
for representation was selected based on donor age
range and represented from youngest to oldest
within the study cohort. With our current scientificunderstanding of pediatric gut microbiome development,
as depicted in Figure 1 from Arrieta et al., there is an
expectation of increase in diversity and complexity oftaxonomic presence as infants develop. The taxonomic
profiles from pediatric donors from 3 age groups(Figure 9) demonstrate some of these expected changes
in microbiome composition as environmental factors
and age influence the microbiome. One of the firstcolonizers of the pediatric gut, Bifidobacteriaceae,
becomes less abundant over time, and while microbiome
diversity increases (more rare taxa represented in“Other”), interindividual variability decreases.

PD-WP-00057 Issue 1/2022-09© 2022 DNA Genotek Inc., a subsidiary of OraSure Technologies, Inc., all rights reserved.

Figure 8: Effect plots for differential abundance analysis
of pediatric baseline samples collected in OMNIgene•GUT
versus the same samples stored at ambient temperature in
OMNIgene•GUT for 30 or 60 days. Each dot is a taxonomic species,
with the y-axis showing fold change (log2) between time points scaledby the within group variance (x-axis). An effect size > 1, represented
by the dashed lines, would indicate a significant change in relative
abundance due to the fold change between samples exceeding thewithin group variance. No significant changes in species relative
abundance were detected.

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Bacterial relative abundances< 10 months

10-18 months

35-45 months

Family level

100
Other

Relative abundance (%)

Rikenellaceae

75

Clostridiales (order)
Streptococcaceae
Prevotellaceae

50

Clostridiaceae
Akkermansiaceae
Lachnospiraceae

25

Bacteroidaceae
Ruminococcaceae

0

Bifidobacteriaceae

A

B

C

D

E

F

G

H

I

Donor
Figure 9: Relative abundance stacked bar plot showing the taxonomic profile for representative donors from cohort 2, grouped into
3 age ranges. The top 10 family-level taxonomies (by average relative abundance) are shown, with the remaining reads grouped as “Other.”Conclusions
Consideration of sample collection and stabilization
chemistry methods is critical for obtaining accurate
representation of the pediatric gut microbiome during
various stages of life. As shown in Figure 9, distinct
differences between pediatric gut samples fromdifferent age groups can be recovered when collectedusing OMNIgene•GUT. In-depth assessment of thecollection device is required to confirm effectivenessand compatibility with:•

Extraction methods of processing a wide rangeof microbial bioload to recover sufficient nucleic
acid for sequencing applications.

•

A wide range of Bristol stool scale sample types.

•

Matrix uniqueness across donors, particularly
in early development stages.

•

A wide range of microbial cell types.

PD-WP-00057 Issue 1/2022-09© 2022 DNA Genotek Inc., a subsidiary of OraSure Technologies, Inc., all rights reserved.

We have demonstrated that OMNIgene•GUTstabilizes the gut microbiome of pediatric samples,collected from infants ranging in age from newborn to
45 months, without compromising sample integrity
or microbial richness. Showcasing the utility ofOMNIgene•GUT for assessing and quantifying
complex microbial structures within pediatricstudies, and powering scalable studies throughan accessible at-home collection device.

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References
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temperature for 60 days and during transport. PD-WP-00042. DNA Genotek Inc.
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OMNIgene•GUT® microbial DNA purification protocol using QIAGEN® QIAamp® PowerFecal® DNA Kit. PD-PR-00434. DNA Genotek Inc.
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