OMNIgene®•GUT provides easy self-collection and stabilizationof liquid fecal samples for microbiome profiling.
Ashlee Brown, Denise Lynch, Anne Bouevitch, Evgueni Doukhanine
DNA Genotek, Ottawa, Ontario, Canada
2018-10-03

OMNIgene®•GUT is an all-in-one system for easy
self-collection and stabilization of microbial DNA fromfeces/stool for gut microbiome profiling. In this paper,
we demonstrate the performance of OMNIgene•GUTin reproducibly collecting and stabilizing gut microbiome
profiles from donors undergoing Crohn’s and chronic
colitis symptoms (Bristol type 6 and 7).

Introduction
Accurate recovery of the microbial profile has been
essential for studies linking gut microbiome
dynamics and host health. In the past, methods
such as 4°C transport or dry ice packing were utilized
as means of preservation of stool samples prior to
nucleic acid extractions. Over time, these methods
were found to be cost prohibitive or incapable ofaccurately capturing the in vivo microbiome state
without biased taxonomic changes. In addition,
as researchers are rapidly moving towards gut
microbiome collections from larger cohorts and/ordonors undergoing dysbiosis - an easy to use,
accurate and validated method of sample collection
is essential to maximize value of the generated dataand donor compliance.
In partnership with Crohn’s and Colitis Canada,
this study evaluates the compatibility andperformance of the OMNIgene•GUT collection
device for in home self-collection by donors
undergoing Crohn’s and chronic colitis symptoms.
Facets evaluated were: donor compliance, DNA
yield and quality, and sample preservation over time.
This data works to extend the performance ofOMNIgene•GUT into novel use cases, with theintention of expanding gut microbiome researchers’
toolboxes and allowing successful study executions
with unique cohorts.

PD-WP-00056 Issue 1/2018-10© 2018 DNA Genotek Inc., a subsidiary of OraSure Technologies, Inc., all rights reserved.

Materials and methods
Sample collection
OMNIgene•GUT kits were used by untrained
donors recruited through Crohn’s and Colitis Canada
to self-collect fecal samples. Each donor collected
two samples from the same bulk fecal sample, using
a different collection tool for each sample. The tools
and instructions were: standard OMNIgene•GUTspatula and the DNA Genotek spoon accessory(OM-AC2) designed for donors with dysbiosis.
The samples collected with OMNIgene•GUT, alongwith fresh fecal (bulk) sample from the same donor,
were returned to the collection coordinator the same
day as collection. The bulk sample was transported
using cold chain as per the Human Microbiome
Project standard procedure1. The collected kits
were weighed and compared to an average weightof uncollected kits to determine the amount of fecal
sample collected. The bulk samples were stored at4°C overnight and then distributed among three
OMNIgene•GUT kits, and two 5 mL tubes withoutstabilizer solution.

DNA extraction and sample storage
Baseline extractions were performed within an hourof bulk sample distribution. For all extractions,
a 0.35 mL aliquot of stabilized sample or 60 mg
of unstabilized/fresh sample was extracted using
an adapted Repeat Bead Beating protocol(Yu and Morrison, 2004)2. Briefly, the extraction
protocol modifications included reduction of lysis
buffer to 950 µL and replacement of 0.5 mm beads
with two 3 mm beads, four 2 mm beads per extraction
tube. After baseline extractions, OMNIgene•GUTsamples were stored at room temperature (23±3°C)
for 60 days, exposed to simulated transport conditions

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(50°C for 3 days) or subjected to 3 cycles of freezing
and thawing (each cycle consisted of a minimum of
3 hours at -20°C and a minimum of 3 hours at 35°C)
followed by storage at -20°C for 60 days, at which timea second set of 3 freezing and thawing cycles occurred.
Unstabilized stool aliquots were stored at room
temperature for 14 days and a separate aliquot at-80°C for 60 days. Extractions identical to baseline
protocol were performed at each time point.

DNA analysis

Results
Surveys were filled out by donors to assess easeof use of the kit and characterize their Bristol stool
type. Crohn’s and Colitis Canada facilitated donor
recruitment in order to target active sufferers ofinflammatory bowel disease (IBD). As expected,
not all donors had Bristol type 6 or 7 stool on theday of collection, with 65% of donors self-reporting
Bristol 6 or 7 (Figure 1). Results and discussion will
be primarily focusing on samples from those donors.

DNA concentration and yield were determined using
the Quant-iT™ PicoGreen® dsDNA Reagent (Invitrogen).
DNA integrity was evaluated by running approximately
70 ng of purified DNA on a 0.8% agarose gel andstaining with ethidium bromide. The UltraRanger
1 kb DNA Ladder™ (Norgen) was used to determine
the size of purified DNA.

16S sequencing
16S rRNA library preparation, sequencing andbioinformatics were conducted in-house throughDNA Genotek’s GenoFIND™ service. V3-V4
hypervariable region paired-end amplicon sequencing
was performed with a PE-300 V3 kit on an Illumina®
MiSeq® platform. Paired-end sequencing reads weremerged, screened for length, and filtered for quality
using proprietary DNA Genotek scripts. Filteredsequences were aligned to the GreenGenes reference
database at 97% identity using the NINJA-OPSalgorithm, v1.5.1. Samples were rarefied to 20,000
or 25,000 reads per sample depending on the study.
Operational Taxonomic Units (OTUs) that were notpresent with more than 10 counts per sample in any
sample were removed, and all remaining OTUs werecollapsed at the species-level (L7) when possible,
otherwise were assigned highest available taxonomic
resolution. Shannon alpha diversity and Bray-Curtis
dissimilarity, were calculated using QIIME v1.9.1 on
the collapsed OTU tables. LEfSe (Linear discriminant
analysis Effect Size) was used to determine if anyorganisms were differentially abundant between
time points (Segata et al., 2011).3

PD-WP-00056 Issue 1/2018-10© 2018 DNA Genotek Inc., a subsidiary of OraSure Technologies, Inc., all rights reserved.

Figure 1. Distribution of donor-reported Bristol stool type.
65% of donors identified as Bristol 6 or 7. ‘Other’ encompasses
donors that identified more than a single type on the survey.

Donors were asked to fill the volumetric cavity in
the OMNIgene•GUT kit with the spatula or thesampling spoon. The sampling spoon was preferredby all Bristol 7 donors, and collected 580 ± 120 mg(mean ± SD) with a minimum of 430 mg. The spatula
was generally preferred for Bristol ≤6 and collected
560 ± 160 mg (mean ± SD) with a minimum of 330 mg.
Therefore, based on a 0.35 mL extraction input,OMNIgene•GUT samples contained at least 50 mg
of feces per extraction (Figure 2). Both sampling tools
were successfully used to collect the required amountof stool into the OMNIgene•GUT device.

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2

A m o u n t c o lle c te d (g )1 .00 .80 .6

400 m g0 .4

The average DNA yield per 0.35 mL OMNIgene•GUTextraction aliquot was 4.49 ± 3.67 µg (mean ± SD).
Considering current minimum DNA input
requirements for sequencing assays, a single
OMNIgene•GUT sample collected by donors
undergoing dysbiosis has more than sufficient capacity
to provide sufficient DNA for these assays (Table 1).
16S rRNA
sequencing

0 .2

0 .0
S p a t u laSpoon

B r is t o l ≤ 6

B r is t o l 7

Figure 2. Amount of fecal sample collected into OMNIgene•GUT byeach of the donor preferred tools, spatula for Bristol ≤6 and spoon forBristol 7. For both sets of donor groups, the preferred tools collected
suﬃcient sample to facilitate at least 50 mg of stool per 350 µL extraction.

Samples collected from dysbiosis donors withOMNIgene•GUT provide consistent DNA yieldand quality, sufficient for downstream
sequencing applications
The total DNA yield from Bristol 6 and 7 samples
collected into OMNIgene•GUT kits was 31.43 ± 25.67 µg(mean ± SD), with 90% of samples having > 4.05 µg.
DNA yield from multiple extractions from the sameOMNIgene•GUT sample over time was reproducible(see “intra-variability” in Figure 3), demonstrating
the ability of OMNIgene•GUT to fully homogenize
samples and provide consistent sample aliquots.

Figure 3. Total DNA yield from representative Bristol 6 and 7OMNIgene•GUT collected samples. The figure represents theinter-variability, OMNIgene•GUT samples from the same bulk
stool, and intra-variability, extraction aliquots from the sameOMNIgene•GUT sample (as outlined by the box).

PD-WP-00056 Issue 1/2018-10© 2018 DNA Genotek Inc., a subsidiary of OraSure Technologies, Inc., all rights reserved.

Metagenomic PCR-freesequencing sequencing

Assay input
requirement(per sample)~50 ng~100 ng

1-2 µg# assays possible inone OMNIgene•GUTextraction aliquot†>800>404# assays possible withone OMNIgene•GUTsample†

>6000

>300

31

Table 1. Number of sequencing assays per OMNIgene•GUT sample.
†Based on mean DNA yield and an assumption of 7 extraction
aliquots per sample.

DNA extracted from OMNIgene•GUT samples
had a molecular weight greater than 10 kb, thereby
reducing a potential source of bias in PCR-based
downstream applications that could preferentially
amplify smaller DNA fragments (Figure 4).

Figure 4. Agarose gel of OMNIgene•GUT and unstabilized stool
samples from a representative Bristol 7 donor. Stabilized samples
were extracted at baseline and post storage at room temperaturefor 60 days, control fresh sample from matched donor wasextracted at baseline.

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Microbiome profile neutrality of donors withdysbiosis is maintained in OMNIgene•GUT
A crucial aspect of microbiome studies is that any
sample taken represents the in vivo microbialcommunity present in the donor. Storage at -80°C
remains the standard for preservation of themicrobiota, but is not feasible or cost effective inmany situations – particularly for in homecollections. Chemical stabilization buffers offer thebenefits of being free from additional expense andinconvenience of cold chain preservation, but needto demonstrate neutrality where-in the microbial
profile is not altered by DNA degradation or growthof specific microbes. Neutrality was established bycomparing OMNIgene•GUT baseline to fresh sample
extractions, this dissimilarity was then statisticallytested against dissimilarity between replicate
extractions of baseline fresh samples (Figure 5).
Finding no statistical significance confirmed thatstabilization within OMNIgene•GUT did notintroduce microbiome profile changes beyond what
is typically observed through biological sampling.

of samples at baseline and after storage was evaluatedusing Bray-Curtis dissimilarity analysis (Figure 6).
As expected, unstabilized samples stored for 14 days
at room temperature showed a large increase in
Bray-Curtis dissimilarity from baseline. MannWhitney analysis showed significant differences
between OMNIgene•GUT samples and unstabilized
samples, both held at -80°C and room temperature.
This confirms that stool samples collected withOMNIgene•GUT from donors undergoing dysbiosis
show significantly less profile change over time thanunstabilized samples held at -80°C.
Unstabilized

RT for14 daysOMNIgene•GUT-80°C for
60 days*****-20°C, 6x F/Tfor 60 days
50°C for3 daysRT for60 daysA0.0

Fresh vs. OMNIgene•GUT

0.2

0.4

0.6

Bray-Curtis dissimilarity
nsWithin fresh

0.00

0.05

0.10

0.15

0.20

Bray-Curtis dissimilarity

Figure 5. Bray-Curtis dissimilarity of OMNIgene•GUT baselinecompared to fresh (marked in purple), as well as, baseline variability
within fresh aliquots (marked in blue); no statistical difference wasfound between the two sets of dissimilarity values. Bray-Curtis
dissimilarity results for fresh aliquots (marked in blue) were usedfrom the OMNIgene•GUT validation study4, with triplicate aliquots
from 28 donors.

Gut microbiome profiles of donors with dysbiosis
are preserved for up to 60 days in OMNIgene•GUTOMNIgene•GUT samples were kept at room
temperature for 60 days and exposed to simulatedtransport conditions: 50°C for 3 days and freeze-thaw
cycling with interim storage at -20°C for 60 days.
Additionally, control unstabilized sample aliquots
were kept at room temperature for 14 days and-80°C for 60 days. The microbiome profile changes

PD-WP-00056 Issue 1/2018-10© 2018 DNA Genotek Inc., a subsidiary of OraSure Technologies, Inc., all rights reserved.

Figure 6. Bray-Curtis dissimilarity of stool samples collected withOMNIgene•GUT from donors undergoing dysbiosis and then exposed
to storage and simulated shipping conditions. Dissimilarity wasgenerated by comparing sample’s microbial profile at baseline to itspaired time held sample. Significant difference in microbiome profilechange was observed in unstabilized (marked in blue) samples heldat RT or -80°C when compared to paired OMNIgene•GUT (markedin purple) stabilized samples (*P≤0.05 and ****P≤0.0005, respectively).

Additionally, LEfSe analysis of unstabilized samples
stored for 14 days at room temperature and compared
to baseline found 13 significant taxonomic
differences, ranging from family to species level.
Contrary, no significant differences were found afterstorage of stool samples in OMNIgene•GUT kits for
60 days at room temperature or exposed to simulatedtransport conditions. As a control, LEfSe analysis
was carried out on stool samples that wereimmediately stored at -80°C after collection and heldfor 60 days until extraction, no significant differenceswere identified. Together this evidence shows that
the OMNIgene•GUT device can effectively stabilize
microbiome signals in stool collected by donors
undergoing dysbiosis, allowing extended storage
conditions without a need for cold chain.

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4Enrichment and depletion of microbes linked
to IBD are observable in gut microbiome profilesof donors with dysbiosis
We observe a total of 17 microbiota markers forColonic Crohn’s disease (CCD) in our cohort and53% of those markers follow published enrichment
and depletion trends (Walters, Xu and Knight, 2014)5
when compared to control donors. For example,Parabacteroides distasonis is depleted in donors
with dysbiosis by ~14 fold when compared tocontrols. We also observe a total of 23 microbiota
markers for Ileal Crohn’s disease (ICD) and 65%of those markers follow published trends (Walters,Xu and Knight, 2014)5. As an example, we observed
a ~35 fold enrichment of Veillonella dispar in ourdysbiosis cohort. These CCD and ICD findings are
preliminary and the remaining data not following
published trends may be a result of the particularcohort or study size. Broad taxonomic shifts that havebeen published regarding IBD-linked microbes
include a depletion of Firmicutes and Bacteroidetes
and enrichment of Proteobacteria and Actinobacteria(Sartor and Mazmanian, 2012)6, similar trends arealso observable in our cohort (Figure 7).

Conclusions
The OMNIgene•GUT kit provides the means
to effectively collect and stabilize microbiome profiles
from donors undergoing dysbiosis. These stool
samples are soft or liquid in consistency andcommon for people suffering from inflammatory
bowel disease, Crohn’s or chronic colitis. The intuitive
design of the spoon accessory combined withOMNIgene•GUT allows for easier collection process
and in home use for donors. Samples collectedin OMNIgene•GUT recover DNA yields and quality
sufficient for multiple downstream applications.
In addition, these samples maintain their microbial
profile neutrality without any introduced bias;
therefore, ensuring the in vivo dysbiosis profileof the donor can be accurately interrogated.
Profile preservation is achieved for up to 60 days
at room temperature and various transportconditions, allowing for banking of samples
and repeat analysis.

Figure 7. Phylum-level abundance charts of representative donors
illustrating previously published depletion and enrichment trends
for IBD-linked microbes.

References
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Manual of Procedures – Human Microbiome Project (2010).
Yu, Z. and Morrison, M. (2004). Improved extraction of PCR-quality community DNA from digesta and fecal samples. BioTechniques, 36(5): 808–812.
Segata, N., Izard, J., Waldron, L., Gevers, D., Miropolsky, L., Garrett, W.S. and Huttenhower, C. (2011). Metagenomic biomarker discovery and explanation. Genome Biology, 12(6),R60. http://doi.org/10.1186/gb-2011-12-6-r60
PD-WP-00040: OMNIgene•GUT enables reliable collection of high quality fecal samples for gut microbiome studies (2014).
Walters, W.A., Xu, Z. and Knight, R. (2014). Meta-analyses of human gut microbes associated with obesity and IBD. FEBS Letters, 588. http://doi.org/10.1016/j.febslet.2014.09.039
Sartor, R.B. and Mazmanian, S.K. (2012). Intestinal microbes in inflammatory bowel diseases. Am. J. Gastroenterol. Suppl., 1: 15-21. http://doi.org/10.1038/ajgsup.2012.4

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All DNA Genotek protocols, white papers and application notes, are available in the support section of our website at www.dnagenotek.com.

PD-WP-00056 Issue 1/2018-10© 2018 DNA Genotek Inc., a subsidiary of OraSure Technologies, Inc., all rights reserved.

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