OMNImet™•GUT enables at-home collection and ambient temperaturetransport of fecal samples for metabolomics
Olle M. De Bruin1, Elizaveta Freinkman2, Héloïse Breton1, Anne M. Evans2, Luke A. D. Miller2, UyenThao Nguyen2,
Klaus-Peter Adam2 and Evgueni Doukhanine1
1 DNA Genotek, Ottawa, Ontario, Canada
2 Metabolon Inc, Durham, North Carolina, USA

2020-06-16

Gut metabolites derived from human and/or microbial
processes, as well as via dietary intake, collectively
constitute the fecal metabolome. Metabolomics canelucidate how gut metabolites affect human healthand disease. Challenges surrounding the collection,
stability, and transport of stool samples formetabolomics have limited the scope of studies
in term of study size, accessible donor population,and geographic range available for sampling.
OMNImet™•GUT (ME-200) is a novel sampling
device that provides an all-in-one solution forambient temperature collection and transport ofhuman fecal samples for metabolomics. Evaluationof OMNImet•GUT demonstrated that the device issuitable for at-home collection of fecal samples byuntrained volunteers and is compatible with common
laboratory methodologies for both untargeted andtargeted metabolomics. Furthermore, our results
show that OMNImet•GUT captures an accurate
and precise snapshot of fecal metabolomic profiles.
Metabolomic profiles comprising >800 biochemicals
per donor, including short-chain fatty acids (SCFAs),
were stable for 7 days at room temperature (20°C to
26°C / 68°F to 79°F) and during simulated shipping
conditions (-20°C to 30°C / -4°F to 86°F). By
overcoming challenges associated with at-homecollection and transport for fecal metabolomics,
OMNImet•GUT will enable larger and bettercontrolled studies leading to key insights suchas the discovery of biomarkers and drug targets.

Introduction
Metabolomics is the comprehensive measurement of
hundreds to thousands of small-molecule biochemicals,or metabolites1. Metabolomics complements other‘omics analyses including genomics, transcriptomics
and proteomics by providing an integrative

PD-WP-00066 Issue 1/2020-06© 2020 DNA Genotek Inc., a subsidiary of OraSure Technologies, Inc., all rights reserved.

phenotypic readout. In the context of the gut
microbiome, metabolites play a well-established rolein host-microbe communication and dynamics,
making metabolomics an especially powerful tool toelucidate the mechanistic functions of gut microbes
in human health and disease2, 3, 4. To date, specific
gut microbial metabolites have been implicated in
obesity5, insulin resistance6, cardiovascular disease7,
autism-spectrum disorder8 and Parkinson’s disease9,among other human health conditions. Yet, consideringthat most analytical methods capture only asmall fraction of the biochemical diversity of gut
microbiota, host physiology and their interactions,
thousands of potentially pivotal molecules remain
to be characterized or even discovered.
A major factor limiting the large-scale application ofgut metabolomics has been the practical challengeof collecting fecal samples from human volunteers.
Many metabolites in raw fecal sample are labile torapid chemical and enzymatic degradation; therefore,
immediate freezing at -80°C is recommended forpreserving a sample’s metabolomic profile10.
However, because feces generally cannot be produced
on demand and because some donors may havedifficulty traveling to a collection site, donors strongly
prefer at-home collection, which precludes immediate
freezing at -80°C. Currently available sampling kits
for at-home collection and ambient temperaturestorage of fecal samples, such as DNA‑stabilizing
tubes and fecal immunochemical test tubes, areunsuitable for metabolomics due to high levels of
detergents, buffers, salts and other additives, which
are incompatible with liquid chromatography andmass spectrometry (LC/MS), the technique of
choice for metabolomic analysis11, 12. Thus, there
is a need for a viable collection option to enable
donor-convenient, large‑scale fecal metabolomics.

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In response to the need for a metabolomics collection
and stabilization solution, DNA Genotek™, in
collaboration with Metabolon®, has developed the
OMNImet•GUT at-home collection kit. This kit
includes a device featuring a controlled, volumetricsample input chamber, a built-in mixing ball for
rapid homogenization of the collected sample anda metabolite-stabilizing solution. In this paper, we
describe the evaluation of OMNImet•GUT as an
at-home collection device for ambient temperaturetransport of samples for fecal metabolomics analysis.
Compatibility with Metabolon’s global metabolomics
platform and quantitative SCFA assay is also
demonstrated.

These approaches have major disadvantages,
including the safety risks associated with study
participants handling dry ice, as well as risks tometabolite stability due to temperature fluctuations
during transport13. DNA Genotek already offers aunique and comprehensive solution for the collection
and stabilization of microbial DNA from feces in
the form of its OMNIgene®•GUT collection device.
Using the physical design of the OMNIgene•GUT kit,
which ensures ease-of-use, reproducibility andhomogeneity of samples14, 15, we have developed anovel complement of physical form and preservation
solution for fecal metabolites, while preserving
compatibility with metabolomics processing
platforms, including Metabolon’s global
metabolomics platform (Figure 1).

Results and discussion

Metabolite coverage is maintained aftercollection into OMNImet•GUT

OMNImet•GUT enables at-home collectionof fecal samples and integration with
Metabolon’s global metabolomics platform

As the gold standard of fecal sample collection,
immediate freezing at -80˚C was used as thereference condition in this study. Sample collection
and storage in OMNImet•GUT was comparedto immediate freezing at -80˚C for sensitivity as
reflected in the number of metabolites detected

Without the means to stabilize metabolites atambient temperature after at-home collection,
researchers typically resort to transporting raw fecal
samples on dry ice or, alternatively, freezing them ina home freezer followed by shipping on wet ice.

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Figure 1: Integration with Metabolon’s global metabolomics platform. DNA Genotek’s OMNImet•GUT
device and Metabolon’s global metabolomics platform provide an integrated solution for collection, storage,transport and analysis of human fecal sample in metabolomics studies (figure provided by Metabolon).

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2

by Metabolon’s global metabolomics platform.
Overall, the number of metabolites detected insamples from 7 donors was similar between thetwo collection methods (Figure 2), demonstratingthat collection with OMNImet•GUT does not
introduce a systematic bias or experimental artefacts
and maintains a gold standard level of detection
sensitivity for metabolomics analysis.
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stabilization within the device. Further analysis of
Spearman coefficients after grouping metabolites
by their biochemical classification (superpathway)
revealed that peptides were likely a contributing
factor of discrepancy between flash-frozen and
OMNImet•GUT samples at all timepoints (Figure 3).
The 37 peptide superpathway biochemicals detectedin this study were primarily dipeptides, which may
originate from food sources, gut tissue and/or
microbial activity and whose importance in fecal
metabolomics is currently unclear. By contrast, theamino acid and xenobiotic superpathways, whichcontain most of the known bioactive microbial
metabolites, displayed median Spearman coefficientsin the 0.9 range at all timepoints (T0 to T7) in
OMNImet•GUT relative to matched flash-frozen
samples (Figure 3). These results indicate thatstorage in OMNImet•GUT preserves the vast
majority of metabolites in raw fecal sample withboth fidelity and stability.

PD-WP-00066 Issue 1/2020-06© 2020 DNA Genotek Inc., a subsidiary of OraSure Technologies, Inc., all rights reserved.

T1

T4

T7

Freeze-thaw

Median R

0.89

0.88

0.86

0.86

0.89

Table 1: Correlation analysis across n=7 biological replicates (after
averaging each donor’s n=3 technical replicates) in each OMNImet•GUT
storage condition (baseline (T0), room temperature for 1, 4, or 7 days
(T1, T4, T7), or freeze-thaw) vs. flash-frozen. The median Spearmancoefficient across all detected metabolites is shown.

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To be suitable for at-home collection and storageof fecal samples, a device must not distort the
metabolomic profile of the raw fecal sample andmust adequately stabilize metabolites over time.
To assess the fidelity and stability of metabolomic
profiles from OMNImet•GUT samples,
Spearman correlations were calculated for all
detected metabolites between flash-frozen samples
and matched OMNImet•GUT samples held for
0, 1, 4 and 7 days at room temperature (table 1).
The median Spearman coefficient between flash-frozen
and OMNImet•GUT-collected samples held at room
temperature for 0 days (i.e., samples immediately
frozen in OMNImet•GUT devices) was 0.89, indicatingthat OMNImet•GUT preserves the metabolomic
profile of the raw sample with a high degree offidelity. Furthermore, median Spearman correlations
to flash-frozen remained in the 0.86-0.88 range
at 1, 4 and 7 days of room temperature storage in
OMNImet•GUT, indicating sustained metabolite

T0

Metabolite stability and analytical precision are
maintained after collection into OMNImet•GUT

Condition

Figure 2: Preservation of metabolomic coverage. Technicalreplicate samples (n=3) from each of 7 donors were collected intoeither a flash-frozen or an OMNImet•GUT device (ME-200), followed
by immediate freezing at -80°C. The number of metabolites detected
in all technical replicates (n=3) of each donor’s samples is plotted.

Figure 3: Accurate measurement of individual metabolites andmetabolic pathways. Correlation analysis across n=7 biologicalreplicates (after averaging each donor’s n=3 technical replicates) ineach OMNImet•GUT storage condition (baseline (T0), room temperaturefor 1, 4, or 7 days (T1, T4, T7), or freeze-thaw) vs. flash-frozen (FF).
The median Spearman coefficient across each major class of biochemicals(metabolic superpathway) is plotted and the number of biochemicals
in each superpathway (detected in flash-frozen samples) is labeled.

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3Given the relatively minor discrepancies between flashfrozen samples and samples stored in OMNImet•GUT
for up to 7 days, we sought to assess the impact ofstoring samples in OMNImet•GUT on the ability toderive biological insights from human subject data. To
do this, we performed Hierarchical Clustering Analysis(HCA) and Principal Component Analysis (PCA) on
matched samples from 7 donors that had been flashfrozen or stored in OMNImet•GUT for varying lengthsof time. In both analyses, samples clustered clearly bydonor, while differences among storage conditions andtimepoints were minor relative to inter-donor

differences (Figures 4 and 5). In particular, no timedependent trends were consistently observed in either
the PCA or HCA. These results indicate that storage in
OMNImet•GUT for up to 7 days at room temperaturepreserves the unique metabolomic fingerprint of
human fecal samples. Taken together, the Spearman,
ICC, PCA and HCA analyses demonstrate thatOMNImet•GUT samples continue to accurately reflect
the metabolomic profile of raw fecal samples after
storage for up to 7 days at room temperature.

Storage Condition
Flash-frozen

Color
Color by
by PARAM_GROUP_DESCRIPTIONPARAM_GROUP_DESCRIPTION

Freeze-thaw
Fresh
Fresh frozen
frozenP186
T0 F/T
P186
F/T
P186
T1 T0
P186
T0
P186
P186 T1
T1

T4

ME-200Because metabolite stabilization is such a critical
performance metric for an at-home fecal collection
and storage device, we sought to obtain anotherquantitative measure of metabolite stability over time in
OMNImet•GUT. In this case, T0 samples (i.e., samples
immediately frozen in OMNImet•GUT devices) wereused as the reference condition in order to isolate thetime effect. Intraclass Correlation Coefficients (ICC)
between T0 and subsequent timepoints were calculated
as described12, using samples from 7 individual donors.
The median ICC across all detected metabolites
after 4 and 7 days of room temperature storage in
OMNImet•GUT was 0.93 and 0.90, respectively(maximum = 1.0). ICC values above 0.9 reflect excellent
agreement between datasets16, 17, again indicating
successful stabilization of metabolites over 7 daysof storage at room temperature.

P186
P186 T4
T4

T7

P186
P186 T7
T7
Shape
Shape by
by PARAM_SUBJECT_OR_ANIMAL_ID
PARAM_SUBJECT_OR_ANIMAL_ID

Donors
1
1

1
2
2
2

3
3

3
4

4
4
5

5
6
6
7
7

Figure 4: PCA. Principal component analysis of metabolomic datacollected using fecal samples from 7 donors (n=3 technical replicatesper donor) that were flash-frozen or stored in OMNImet•GUT as
indicated: baseline (T0), room temperature for 1, 4, or 7 days (T1, T4,
T7), or freeze-thaw.
Superpathway:

Relative Abundance:
-4.00

4.00

PARAM_GROUP_DESCRIPTION
Storage condition:

Flash-frozen
T0
T1
T4

ME-200F/T
Freeze-thaw

T7SUPER_PATHWAY
Superpathway:

Amino Acid
Carbohydrate
Cofactors and Vitamins
EnergyLipid
Nucleotide
Partially Characterized
Molecules Peptide
Xenobiotics

Donor:
Donor 1
Donor 2
Donor 3
Donor 4
Donor 5
Donor 6Storage Condition:
Donor ID:

Donor 7

Figure 5: HCA. Hierarchical clustering analysis of metabolomics data collected using fecal samples from 7 donors (n=3 technical replicates per donor)
that were flash-frozen or stored in OMNImet.GUT as indicated: baseline (T0), room temperature for 1, 4, or 7 days (T1, T4, T7), or freeze-thaw. (The samedataset as Figure 4).
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In addition to stability, precision is a criticalconsideration in metabolomic analysis. Therefore, wesought to benchmark the precision of OMNImet•GUT
samples against that of flash-frozen samples.
PCA (Figure 4) showed that within-donor variability(n=3 technical replicates per condition fromeach of 7 donors) was comparable for samples in
OMNImet•GUT and flash-frozen samples. In a
separate analysis of n=6 technical replicates percondition collected from each of three donors, nostatistically significant differences were observed
between the median relative standard deviation(RSD) across all biochemicals in the OMNImet•GUT
samples relative to matched flash-frozen replicates(data not shown). Together, these data indicate thatcollection into OMNImet•GUT device does not
introduce additional technical variability into
metabolomic analyses relative to the gold standard
collection method.

OMNImet•GUT is compatible with freezethawing and simulated shipping conditions
Simulating transport of samples through different
temperature conditions was performed by subjecting
samples collected into OMNImet•GUT to freezethaw at -20˚C followed by 30˚C. This treatment
simulates a scenario of air transport followed byground transport without temperature control,or, alternatively, samples that have been frozen(e.g., in a household freezer) and then delivered
to a study site without temperature control.

Short-chain fatty acids are stable in
OMNImet•GUT for at least 7 days at room
temperature
Short-chain fatty acids (SCFAs) are key mediatorsof gut microbial activity and play important roles
in regulating the immune system, maintaining the
gut epithelial barrier and preventing disorders suchas metabolic syndrome, inflammatory bowel diseaseand certain types of cancer18. Therefore, we assessed
the performance of OMNImet•GUT in stabilizing
SCFAs at room temperature. To this end, a pooled
fecal sample derived from 7 donors was stored atroom temperature for varying lengths of time in
OMNImet•GUT and in an unstabilized condition(cryovial), followed by targeted, quantitative analysis
of eight SCFAs. While concentrations of SCFAs
in raw samples increased approximately 2- to
10-fold after 96h (4 days) at room temperature,
concentrations in OMNImet•GUT samples
remained unchanged between 0 and 13 days atroom temperature (Figure 6). These results indicatethat OMNImet•GUT quenches bacterial metabolism
and that SCFAs are stable in the device for at least

Acetate stability

2-methylbutyrate stability
140.005000.00

3500.00

300.003000.00

250.00

80.00
60.00

ng/ml butyrate

ng/ml acetate

4000.00
3000.00
2000.00

2000.00
1500.00
1000.00

40.00
1000.00

500.00

0.00

0.00

0.00
T1

T4

T7

T0

T13

T1

T4

T7

160.00120.00140.00ng/ml isovalerate

80.00
60.00
40.00

ng/ml propionate

120.00100.00100.00
80.00
60.00

20.00

0.00

0.00
T0

T1

T4

T7

T13

T1

T4

T7

2000.00

250.001500.00

1000.00

T4

T7

T0

T13

T4

T7

T13

T7

T13

200.00150.00100.00
50.00

0.00
T1

T1

Valerate stability
300.00500.00

T0

T0

T13

2500.00

40.00

20.00

100.00

Propionate stability

Isovalerate stability

Isobutyrate stability
140.00150.00

0.00
T0

T13

200.0050.00

ng/ml valerate

T0

ng/ml hexanoate

2500.00

100.0020.00

ng/ml isobutyrate

Hexanoatestability

Butyrate stability

6000.00

120.00ng/ml 2 -methylbutyrate

As seen through PCA, HCA and Spearman
correlation (Figures 4, 5 and table 1), freeze-thaw
cycling had minimal effect on the metabolomic
profile of samples from all donors, indicating thatOMNImet•GUT-collected samples are not sensitive
to freeze-thawing and can be shipped without
temperature-controlled conditions.

T1

T4

T7

T13

0.00
T0

T1

T4

Unstabilized:
ME-200: [

A/

B/

C/

D]

Figure 6: Stability of SCFA in OMNImet•GUT. Aliquots of a 7-donor pooled raw fecal sample were either left unstabilized (n=4 replicates) or
stored in OMNImet•GUT (ME-200) (n=4 replicates, A/B/C/D) at room temperature for the indicated number of days (T0 to T13), followed byquantification of eight short-chain fatty acids (SCFAs) using targeted LC-MS/MS.

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7 days at room temperature. In a similar experimentwith samples from 7 individual donors, all SCFAs
from all donors remained stable in OMNImet•GUT
for 7 days at room temperature (data not shown).
These results demonstrate that OMNImet•GUT
is compatible with Metabolon’s short-chain fatty
acid assay and stabilizes SCFAs for at least 7 days
at room temperature.

Conclusions
In the context of most fecal metabolomics studies,
at-home collection is necessary to recruit a
representative donor population and achieveadequate statistical power, which are prerequisites
for identification of meaningful and translatablemetabolite-disease associations. To date, nostandardized, validated at-home collection kit
for fecal metabolomics has been available.

Measurement of >800 individual biochemicals perhuman donor on Metabolon’s global metabolomics
platform, as well as quantitative analysis of 8 SCFAs,
showed that samples collected in OMNImet•GUT
devices remained stable at room temperature for up
to 7 days and recapitulated the metabolomic profilesof matched flash-frozen samples while maintaining
similar technical precision. These results show thatsample collection and storage in OMNImet•GUT
compares favorably with the gold standard flashfreezing methodology. OMNImet•GUT will enable
the collection of high-quality fecal metabolomics
data at a scale previously not possible and drive a
new wave of discoveries in the field.
For Materials and methods, consult Methods appendix:
OMNImet•GUT (ME-200) enables at-home collection
and ambient temperature transport of fecal samples formetabolomics (PD-WP-00068) at www.dnagenotek.com

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