OMNIgene®•GUT stabilizes the microbiome profile atambient temperature for 60 days and during transport
Evgueni Doukhanine, Anne Bouevitch, Ashlee Brown, Jessica Gage LaVecchia, Carlos Merino and Lindsay Pozza
DNA Genotek, Ottawa, Ontario, Canada
2016-05-03

OMNIgene•GUT is an all-in-one system for theeasy self-collection and stabilization of microbial
DNA from feces/stool for gut microbiome profiling.
OMNIgene•GUT is uniquely able to take asnapshot of the microbial profile at the momentof collection, and maintain it for 60 days
at ambient temperature. In this paper, wedemonstrate that profiles of gut microbiota
collected in OMNIgene•GUT are stable, highly
reproducible and represent the in vivo state.

Introduction
The study of the relationship between themicrobiome and its human host in health anddisease relies on identifying and monitoring the
microbial communities over a period of time.
Recent discoveries demonstrate the utility of thesemicrobial profiles as potential biomarkers withprognostic and diagnostic value. It is becoming
evident in the literature that due to the dynamic
nature of the gut microbiome, repeated samplingof large populations over time is essential to thedevelopment of such biomarkers. These studies,
known as Microbiome-Wide Association
Studies (MWAS) are challenged by low donor
compliance, unreliable self-collection of biologicalsamples, high cost and cumbersome shipping andhandling procedures. Current methods for stool
sampling and microbiota analysis involve thetransport of specimens at ambient temperatureor 4°C. However, these methods have the potential
to expose samples to temperatures incompatible
with microbiome stabilization. Failure to properlystabilize the microbiome during sample collection,transport and processing risk obscuring thebiological and clinical meaning of the microbiome
profile. Consequently, proper pre-analyticalprocedures should be followed to ensure thebest possible representation of the in vivo
microbiome profile.

PD-WP-00042 Issue 3/2016-05© 2016 DNA Genotek Inc., a subsidiary of OraSure Technologies, Inc., all rights reserved.

Materials and methods
Sample collection
OMNIgene•GUT kits were used by adult donors toself-collect fecal samples according to the standard
instructions provided with the kit. 15 donors wereasked to collect 3 OMNIgene•GUT samples from thesame bulk fecal sample, subsequently these samples
were split into two studies of 6 and 9 donors. First
study involved 6 donors that were used for evaluationof neutrality and sources of variability. Each of 6donors collected 3 OMNIgene•GUT samples fromthe same bulk fecal sample (n=18 total). Additionally,aliquots of fresh feces were collected from the samebulk fecal sample by each donor and transported in a
styrofoam box with frozen cold packs as per Human
Microbiome Project standard procedure1. In thesecond study collection was expanded to 9 donors
which were used to establish effectiveness ofOMNIgene•GUT in recovering high quality DNAand stabilizing the microbiome profile over time.

DNA extraction and sample storage
Baseline extractions were performed within 3 hoursof collection. For baseline analysis, a 0.25 mL aliquot
was taken from OMNIgene•GUT samples andextracted using the PowerFecal® DNA Isolation Kit(MO BIO Laboratories, Inc.)2. Each 0.25 mL sample
contains approximately 50 mg feces and 200 µL
stabilizing liquid. Equivalent amounts of feces(approximately 50 mg) were extracted fromfresh samples. Remaining OMNIgene•GUTand fresh samples were aliquoted into cryovials.
OMNIgene•GUT and unstabilized (withoutstabilization liquid) samples were stored at room
temperature (23±3°C) for up to 60 days, exposed
to simulated transport conditions (50°C for 3 days,37°C for 3 days) or subjected to 6 cycles of freezing
and thawing (each cycle consisted of a minimum of
3 hours at -20°C and a minimum of 3 hours at 30°C).

www.dnagenotek.com • support@dnagenotek.com

Additionally, an aliquot of fresh stool from eachdonor was stored at -80°C as a control. After theroom temperature holds, simulated transportconditions, freeze-thaw cycling or -80°C storage,
an aliquot was extracted from all samples.

DNA analysis
DNA concentration and yield were determinedusing the Quant-iT™ PicoGreen® reagent (LifeTechnologies). DNA integrity and stability over time
was evaluated by running approximately 50 ng ofpurified DNA on a 0.8% agarose gel and staining
with ethidium bromide. λ DNA-HindIII ladder wasused to determine the size of purified DNA.

16S sequencing
16S rRNA sequencing library preparation,sequencing and bioinformatics were conductedby Diversigen, Microbiome Discovery Service.
V4 hypervariable region paired-end ampliconsequencing was performed using the Illumina®
MiSeq®. A subset of samples were sequenced throughGenoFIND™ Genomic Services (DNA Genotek)
using the Illumina® MiSeq® platform with V3-V4
hyperviariable region paired-end ampliconsequencing. Using QIIME and custom scripts,
sequences were quality filtered. Paired-end reads
were assembled and compared to the Greengenes
database, clustered at 96% by UCLUST. After datanormalization, sample-to-sample distance wasmeasured using weighted UniFrac and Bray-Curtis
distance on Operational Taxonomic Unit (OTU)
abundance data (utilizing taxon abundance
differences across samples, employing a pair-wisenormalization by dividing the sum of differencesby the sum of all detected OTU abundances).
In all Bray-Curtis measurements, a donor matched
T0 sample that had been extracted shortly aftercollection was used as one side of the pair-wisecomparison. Analysis of the Shannon Index (SI)for each stabilization method was performed bymeasuring the proportion of each OTU relative
to the total number of OTUs, and then multipliedby the natural logarithm of this proportion.
Summation of the resulting product acrossall OTUs produced the SI for each sample.
Sample collection methods were comparedusing the Mann-Whitney test.

PD-WP-00042 Issue 3/2016-05© 2016 DNA Genotek Inc., a subsidiary of OraSure Technologies, Inc., all rights reserved.

Results
OMNIgene•GUT stabilization liquid maintains
microbiome profile neutrality at the pointof‑collection
The study of the microbiome requires that theprofile generated represents the in vivo microbialcommunities present in the donor; thus, thecollection and stabilization method should not
introduce changes to the microbiome.
The use of chemical stabilization buffers canpotentially modify the microbial compositionof the sample by accelerating growth of somemicrobes while allowing the decay of others.
In ideal conditions, the stabilization liquid shouldbe neutral (i.e., it should not introduce any bias
to the microbiome). Comparison of the 16S rRNA
microbiome profile from fresh and OMNIgene•GUTstabilized samples showed that the liquid solution
maintains a neutral profile and does not introduce
bias (Figure 1).

Within freshWithin OMNIgene•GUTFresh vs. OMNIgene•GUT0 0.1

0.15

0.2

Bray-Curtis distance
Figure 1: Bray-Curtis dissimilarity distances within and between
fresh and OMNIgene•GUT collected samples. Mann-Whitney test
showed comparable dissimilarity in all conditions, no statisticaldifference was observed.

The study of relative OTU abundance by different
statistical methods (e.g., Weighted UniFrac) provides
a valuable description of the microbial community,
however, it can obscure the understanding of
the microbial community by minimizing thecontribution of low abundance microbes.
Proper study of the microbiome profile requires
the preservation of the “richness” of the microbialcommunities. Richness is defined as the enumerationof microbial species or OTUs present in the sample

www.dnagenotek.com • support@dnagenotek.com

2and is highly susceptible to environmental
conditions, including changes in temperature, pH,
oxygen concentration and chemical composition.
These and other factors can induce bacterial growthor decay, thereby altering the number of OTUs
detected in the sample3.

same DNA (sequencing variability) (Figure 3).
Bray Curtis dissimilarity distances were generated
within the replicate groups.
Bulk sample

Fresh and OMNIgene•GUT collected samples
from 6 donors were extracted shortly after collection.
The microbial OTUs identified in OMNIgene•GUTsamples were compared with the OTUs present incorresponding fresh samples. Shannon Index (SI)for diversity was calculated by converting OTU
abundance data into presence/absence calls. A MannWhitney test on the SI values showed no significant
difference between OMNIgene•GUT and freshsamples, indicating that OMNIgene•GUT had noimpact on the richness of the samples (Figure 2).

OMNIgene•GUTFreshDNA extraction
replicates

Sequencing
replicates

Figure 3: Sample collection scheme per donor. Each of 6 donors
collected samples from 3 different sites of the bulk sample.
Additionally, 3 DNA extraction replicates were performed from
1 of the OMNIgene•GUT samples (purple) and triplicate DNA aliquots
from 1 extraction were sent for 16S rRNA sequencing.

10FreshOMNIgene•GUT

Figure 2: OMNIgene•GUT preserves sample richness. Richness wasassessed by assigning presence/absence to individual OTUs andcompared using Shannon-Index. Mann-Whitney test showed nosignificant differences between fresh and OMNIgene•GUT samples.

Sources of variability in fecal sample collection
Bray-Curtis analysis showed systematic dissimilarity
within replicates of fresh and OMNIgene•GUTsamples. To understand the sources of suchdissimilarity, we evaluated the variability
introduced during collection and processing offecal samples. Biological variability was assessedby generating microbiome profiles from 3 freshand 3 OMNIgene•GUT samples collected fromdifferent sites within the same bulk sample.
Technical variability was assessed using
OMNIgene•GUT collected samples because thiscollection system provides homogenized liquidsamples, reducing experimental errors during
processing. We compared the profiles of replicate
DNA extractions from the same tube (extractionvariability) and replicate library preps from thePD-WP-00042 Issue 3/2016-05© 2016 DNA Genotek Inc., a subsidiary of OraSure Technologies, Inc., all rights reserved.

Similar variability was observed in biologicalreplicates of fresh and OMNIgene•GUT collected
samples (Bray-Curtis distances 0.14± 0.01 and0.11± 0.01 respectively) (Figure 4). Our analysis
showed that technical and biological variability
introduce some dissimilarity to the 16S rRNA
microbiome profile (Bray-Curtis distances biologicalvariability 0.11; extraction variability 0.09 andsequencing variability 0.08). We concluded thatthe source of dissimilarity observed can be explainedby the technical or biological variability, and thatOMNIgene•GUT does not introduce any bias.
Fresh2OMNIgene•GUT3
Shannon Index value

Biologicalreplicates

Biological reps
Biological reps
Extraction reps

Sequencing reps
00.05

0.10

0.15

Bray-Curtis distance

Figure 4: OMNIgene•GUT samples render highly reproducible
microbiome profiles. Mann-Whitney tests on Bray-Curtis distances
showed comparable dissimilarity in triplicate samples.

www.dnagenotek.com • support@dnagenotek.com

3

OMNIgene•GUT preserves microbiome profiles
for up to 60 days
In addition to maintaining profile neutrality, theaccurate preservation of microbial community
structure over time is a requirement for MWAS.
We evaluated the capability of OMNIgene•GUT tostabilize samples during storage at 23°C for 60 days
and exposure to simulated transport conditions(see next section).
OMNIgene•GUT stabilized samples wereextracted at baseline (T0) and again after storagefor 14 and 60 days at room temperature (23°C).
Paired unstabilized samples were extracted atbaseline (T0, fresh) and after 14 days at room
temperature (23°C) or 14 days at -80°C (Figure 5).
The similarity of the samples at baseline and after
storage was evaluated using Bray-Curtis distances.
Mann-Whitney analysis showed no significant
difference in the Bray-Curtis distance for theOMNIgene•GUT samples stored at 23°C for 60 days
when compared to unstabilized samples stored at-80°C for 14 days. In contrast, the Bray-Curtis
distances for the unstabilized samples kept at 23°C
for 14 days were significantly different whencompared either to the OMNIgene•GUT(60 days)or -80°C storage.

-80°C
OMNIgene•GUT**

Unstabilized
00.20.4
Bray-Curtis distancebiological replicates (~63% similarity). Proper
stabilization therefore has a large effect on profile
clustering over time.
60%
63.5%

95%96%

FreshOMNIgene•GUTT0

Fresh OMNIgene•GUTT14

Unstabilized

T14

Figure 6: Dendrogram of microbiome weighted UniFrac % similarityof a representative donor. Extractions from 3 biological replicateswere evaluated for each condition. Low % similarity to fresh sample
indicates changes in the microbiome profile over time.

OMNIgene•GUT effectively preserves themicrobiome profile during transport
Samples are commonly exposed to undesirable
conditions during transport from the point-ofcollection to the processing laboratory. To simulate
standard shipping conditions, unstabilized andOMNIgene•GUT stabilized samples were exposed
to 50°C for 3 days, 37°C for 3 days or multiple
freeze-thaw cycles. OMNIgene•GUT preserved highmolecular weight DNA bands whereas unstabilized
samples showed various degrees of degradation,
particularly when exposed to 50°C or freeze-thaw
cycles (Figure 7).

0.6

Figure 5: Bray-Curtis distance dissimilarity between unstabilized(14 days at 23°C), OMNIgene•GUT (60 days at 23°C) and -80°C (14 daysat -80°C) samples when compared with fresh samples. Significant
dissimilarity was assessed using Mann-Whitney (**P≤0.001).

In order to understand the reproducibility among
replicates, a cluster analysis of weighted UniFrac wasperformed using biological replicates of fresh (T0),unstabilized samples (T14 days at 23°C) andOMNIgene•GUT stabilized samples (T0 and T14days at 23°C) (Figure 6). The fresh biologicalreplicates and T0 and T14 OMNIgene•GUT
biological replicates clustered tightly (96% similarity).
The unstabilized biological replicates clustered
together with a high separation from the fresh

PD-WP-00042 Issue 3/2016-05© 2016 DNA Genotek Inc., a subsidiary of OraSure Technologies, Inc., all rights reserved.

Figure 7: DNA integrity of OMNIgene•GUT samples subjected
to simulated transport conditions. OMNIgene•GUT samples froma representative donor were stored at 23°C for 60 days (T60),
50°C for 3 days (50°C), 37°C for 3 days (37°C) or exposed to6 freeze‑thaw cycles (F/T). Unstabilized samples from the same
donor were stored at 23°C for 14 days (T14), 1 day 50°C (50°C),
3 days at 37°C (37°C) and at -80°C for 14 days (-80°C).

www.dnagenotek.com • support@dnagenotek.com

4

16S rRNA analysis confirmed that OMNIgene•GUT
preserves the microbial community structure evenat extreme temperatures. A Mann-Whitney testcomparing Bray-Curtis distances of OMNIgene•GUTsamples subjected to common shipping temperatureand paired samples held at -80°C showed nosignificant differences. Conversely, unstabilized
samples held at 37°C or subjected to freeze-thaw
cycles showed significant differences when comparedwith samples held at -80°C (Figure 8).
F/T
Unstabilized 37°C
50°C
F/T
OMNIgene•GUT 37°C
50°C-80°C

*

0.0

0.2
0.4
0.6
Bray-Curtis distance**0.8

Conclusions
Stabilization, in the context of metagenomics, is amulti-dimensional attribute that encompasses:•••

Neutrality (ability to capture unbiased profiles)
Reproducibility (homogenous sample material
from which, highly concordant aliquots can betaken), andDNA integrity (molecular weight), as measured
over time.

Based on tightly controlled experiments and rigorousanalysis, OMNIgene•GUT is the first and only
device of its kind proven to effectively stabilize gut
microbiota in human stool for 60 days at room
temperature in addition to during real-life shippingand handling conditions and freeze-thaw cycling.
This is of utmost importance to cost-effective scalingof MWAS, as well as for ensuring effective biomarker
discovery and research translation.

Figure 8: Bray-Curtis distance dissimilarity of OMNIgene•GUTsamples exposed to simulated shipping conditions (as in Figure 7).
Mann-Whitney test showed no differences between OMNIgene•GUTsamples stored at various temperatures and unstabilized
samples stored at -80°C. Significant dissimilarity was observed in
unstabilized samples held at 37°C or subjected to freeze-thaw (F/T)
conditions when compared to paired -80°C samples (*P≤0.05 and**P≤0.01, respectively).

References
123

Manual of Procedures - Human Microbiome Project (2010).
OMNIgene•GUT gut microbial DNA purification using MoBio PowerFecal DNA Isolation Kit. DNA Genotek. PD-PR-00434.
The Open Microbiology Journal, 3, 40-46 (2009).

OMNIgene®•GUT (OM-200) is not available for sale in the United States.
OMNIgene®•GUT (OMR-200) is for research use only, not for use in diagnostic procedures.
Some DNA Genotek products may not be available in all geographic regions.
®OMNIgene is a registered trademark of DNA Genotek Inc. All other brands and names contained herein are the property of their respective owners.
All DNA Genotek protocols, white papers and application notes, are available in the support section of our website at www.dnagenotek.com.

PD-WP-00042 Issue 3/2016-05© 2016 DNA Genotek Inc., a subsidiary of OraSure Technologies, Inc., all rights reserved.

www.dnagenotek.com • support@dnagenotek.com

5