OMNIgene®•GUT enables reliable collection of high quality fecal samples
for gut microbiome studies
Evgueni Doukhanine, Anne Bouevitch, Lindsay Pozza and Carlos Merino
DNA Genotek, Ottawa, Ontario, Canada
2014-09-19

OMNIgene®•GUT is an all-in-one system for easy
self-collection and stabilization of microbial DNA fromfeces for gut microbiome profiling. In this paper wedemonstrate that OMNIgene•GUT allows for reliableand reproducible self-collection of fecal sample. The
design of the kit ensures consistent sample input, while
the stabilization liquid and mixing properties of the kit
ensure that the sample is homogenized and liquefied at
the point of collection to simplify handling in thelaboratory. DNA extraction from OMNIgene•GUTdelivers consistent amounts of high quality DNA
sufficient for multiple high-throughput metagenomic
analyses. Importantly, the chemical properties of theOMNIgene•GUT stabilization liquid are compatible
with commercial processing methods, maintain aneutral profile and do not introduce any bias into
downstream applications. These properties simplify
workflows in the laboratory, increase efficiency andreduce overhead.

Introduction
For any analysis of the gut microbiota, it is essential
to capture an accurate microbiome snapshot of therichness and relative abundance of microbes present
at the moment of collection. Current protocols
described in the literature suggest a variety of fecal
sample transport methods including ambient
temperature, 4°C or frozen. Each of these methods
has the potential to expose samples to conditions
incompatible with microbiome stabilization.
Improvements to self-collection, transport andprocessing are urgently needed in order to address
the diversity in form and microbial content of humanstool, as well as the range of pre-analytical factors
which can introduce variability. OMNIgene•GUT
is designed to overcome these challenges anduniquely enable the scalability and reproducibilityof metagenomic research and biomarker discovery.

PD-WP-00040 Issue 2/2015-06© 2015 DNA Genotek Inc., a subsidiary of OraSure Technologies, Inc., all rights reserved.

This paper evaluates the suitability of OMNIgene•GUTfor use in home self-collection and laboratory
processing of fecal samples for microbiomeassociation studies with a focus on: a) consistency offecal sample input, b) DNA yield and concentration,c) compatibility with commonly used DNA isolation
techniques and d) performance in downstream
applications.

Materials and methods
Sample collection
OMNIgene•GUT kits were used by untrained donors
to self-collect fecal samples according to the standard
instructions provided with the kit. Each of six donors
Bulksamples
sample from the same bulk
collected threefecal sample (n=18 total) using pre-weighed
OMNIgene•GUT collection devices. After collection,
the kits were weighed, establishing the amount of fecal
sample collected. Additionally, an aliquot of fresh
feces was collected from the same bulk fecal sample
OMR-200
Fresh
and transported in a styrofoam box with frozen
cold packs (as per Human Microbiome Projectstandard procedure)1.

Bulk sample

OMNIgene•GUT
FreshMultiple
samples
Extraction
replicates

Figure 1: Sample collection scheme per donor. Each of six donors
collected samples from three different sites of the bulk sample.
DNA extraction from all OMNIgene•GUT and fresh samples wasperformed. Additionally, three DNA extraction replicates were done
from one of the OMNIgene•GUT samples.

www.dnagenotek.com • support@dnagenotek.com

Baseline extractions were performed within threehours of collection. For baseline analysis, a 0.25 mL
aliquot was extracted from OMNIgene•GUTsamples using the PowerFecal® DNA Isolation Kit(MO BIO Laboratories, Inc.)2. Each 0.25 mL sample
contained approximately 50 mg of feces and 200 µL
stabilizing liquid. Equivalent amounts of fecal
sample (approximately 50 mg) were extracted
from fresh samples.

DNA analysis
DNA concentration and yield were determined using
the Quant‑iT™ PicoGreen® reagent (Life Technologies).
DNA integrity was evaluated by runningapproximately 50 ng of purified DNA on a 0.8%
agarose gel and staining with ethidium bromide.
A Lambda Hind III ladder was used to determine
the size of purified DNA.

Real-time PCR
To assess compatibility with third party DNA
isolation kits, changes in PCR efficiency (as measuredby Cq values) was used to detect potential carryover of inhibitors. Fecal microbial DNA from threedonors’ samples stabilized in OMNIgene•GUT andfresh samples was amplified using 16S rRNA
real-time PCR. PCR primers were chosen from aregion of the 16S rRNA gene that is known to beconserved across a wide variety of microorganisms,
and is not found in eukaryotes. The primers wereBacrRNA173-F 5’ ATTACCGCGGCTGCTGG 3’ andBacrRNA173-R 5’ CCTACGGGAGGCAGCAG 3’.
20 ng of total DNA was taken from each sample tobe tested; standard curves were generated using
2-fold serial dilutions of E. coli DNA, from 2.5 ng
to 80 ng. All real-time PCR runs were performedon the Corbett Life Sciences Rotor-Gene™. Cq values
from the OMNIgene•GUT samples and unstabilized
samples were calculated using the default
quantitation analysis with the built-in software,
Rotor-Gene™ 6000 ver. 1.7. The difference inamplification was quantified using ∆∆Cq method3.

16S rRNA sequencing
16S rRNA library preparation, sequencing andbioinformatics were conducted by Metanome,
Microbiome Discovery Service, with additional
analytics provided by Resphera Biosciences.
V4 hypervariable region paired-end amplicon
PD-WP-00040 Issue 2/2015-06© 2015 DNA Genotek Inc., a subsidiary of OraSure Technologies, Inc., all rights reserved.

sequencing was performed using the Illumina®
MiSeq®. Using an amplicon length of up-to 500 nt,
an average coverage of 25,000 reads per sample
was achieved. Using QIIME and custom scripts,
sequences were quality filtered. Paired-end reads
were assembled and searched against the Greengenes
reference database, clustered at 96% by UCLUST.
After data normalization, sample-to-sample distance
was measured using Bray-Curtis distance on OTUabundance data (performs a pair-wise normalization
by dividing the sum of differences by the sum of allabundances). Sample collection methods werecompared using the Mann-Whitney test.

Results and discussion
OMNIgene•GUT allows collection ofa consistent amount of fecal sample
The volumetric properties of OMNIgene•GUT weretested in a population of 18 samples as per thecollection methods described in the materials andmethods section. The standard OMNIgene•GUT user
collection protocol resulted in samples containing 2mL stabilizing solution and an average (± SD) of 613± 144 mg of feces, with a minimum of 400 mg offeces (Figure 2). Therefore, based on a 0.25 mL input
volume for extraction (i.e., PowerFecal DNA IsolationKit), OMNIgene•GUT samples consisted of at least
50 mg feces per extraction aliquot.
1.00.8Sample weight (g)DNA extraction and sample storage

0.6
0.4
0.2
0.0

Figure 2: Fecal sample amount (g) obtained in 18 OMNIgene•GUTsamples (6 donors × 3 samples each). The box plot represents, frombottom to top, minimum, 25th percentile, median, 75th percentile.

Fecal samples collected with OMNIgene•GUTrender reliable DNA yield and concentration
The total DNA yield from OMNIgene•GUT collectedsamples was 19.18 ± 8.89 µg (mean ± SD), with 90%of samples having ≥ 7.6 µg. DNA yield from multiple

www.dnagenotek.com • support@dnagenotek.com

2

extractions from the same OMNIgene•GUT sample
was highly reproducible (see “intra-variability”
in Figure 3), demonstrating the value ofOMNIgene•GUT’s ability to fully homogenize
fecal samples and elicit consistent sample aliquots.

μg of DNA per device

403020100High molecular weight DNA is consistently
extracted from samples collected withOMNIgene•GUTDue to the competitive nature of PCR, DNAdegradation in microbiome samples can lead to theintroduction of bias since smaller DNA fragments
may be preferentially amplified. Therefore, minimizing
DNA degradation in fecal samples is critical toensuring accurate and valid microbiome profiling
results. As assessed by agarose gel, the DNA extracted
from OMNIgene•GUT samples had a molecularweight greater than 10 kb (Figure 4), thereby
reducing a potential source of bias in PCR-based
downstream applications.

Inter Intra Inter Intra Inter Intra Inter Intra
Donor 1

Donor 2

Donor 3

Donor 4

Figure 3: Total DNA yield (µg) for OMNIgene•GUT samples. The figure
represents the inter-variability (three sites from the same bulk sample)
and intra-variability (three aliquots from the same OMNIgene•GUTsample, as outlined by the green box).

DNA yield from OMNIgene•GUT is sufficientfor downstream sequencing applications
The average DNA yield per 0.25 mL extraction
aliquot (approximately 50 mg feces) was 2.40 ±1.11 µg (mean ± SD). Given that 16S rRNA library
prep typically requires a minimum of 5 ng purified
DNA per assay and metagenomic sequencing
requires a minimum of 100 ng per library prep, asingle OMNIgene•GUT sample has the capacity toprovide sufficient DNA for >3500 16S sequencing
assays and 190 metagenomic sequencing assays(Table 1).
16S rRNAsequencing

Metagenomic
sequencing

PCR-freesequencing

Input requirement(per sample)~5 ng~100 ng

1–2 µg# assays possible inone OMNIgene•GUT
extraction aliquot†

>475

>20

2

# total assays
possible with one
OMNIgene•GUTsample†>3,500>190

9Table 1: Number of sequencing assays per OMNIgene•GUT sample.
† Based on mean total DNA yield.
PD-WP-00040 Issue 2/2015-06© 2015 DNA Genotek Inc., a subsidiary of OraSure Technologies, Inc., all rights reserved.

Figure 4: Agarose gel of fresh and OMNIgene•GUT fecal samples
collected from a representative donor. Site replicates refer to multiple
collections from a single fecal sample of one donor. Extraction
replicates were performed on a single collection replicate.

DNA extracted from OMNIgene•GUTstabilized samples demonstrates
compatibility and optimal performanceon downstream applications
Fecal samples contain several PCR inhibitorsincluding humic acids, polyphenols, polysaccharides
and heme, among others. Commercial extraction kits
include effective inhibitor-removal systems, ensuring
proper performance in downstream applications.
The compatibility of samples collected withOMNIgene•GUT with the PowerFecal DNA Isolation
Kit was assessed. Three donors collected one fresh
sample and one OMNIgene•GUT sample from thesame bulk fecal sample. DNA was extracted froman aliquot of each sample. DNA performance wasdetermined by 16S rRNA real-time PCR using
equal amounts of DNA. No difference in Cq values
were observed between DNA extracted from thecontrol or OMNIgene•GUT samples, indicatingwww.dnagenotek.com • support@dnagenotek.com

3

that OMNIgene•GUT method of stabilization
did not impact inhibitor removal or real‑time
PCR performance (Figure 5).

Within freshWithin OMNIgene•GUT

Fresh vs. OMNIgene•GUT0.1

0.15

0.2

Bray-Curtis distanceOMNIgene•GUTFresh
NTC

Figure 5: Real-time 16S rRNA PCR of fecal DNA extracted
from 3 donors using OMNIgene•GUT or fresh samples (∆∆Cq = 0.54).
NTC-no template control.

OMNIgene•GUT stabilization liquid maintains
a neutral microbiome profile and does not
introduce bias
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 tothe microbiome profile). To test neutrality, fresh
samples and OMNIgene•GUT collected samples
were compared using 16S rRNA sequencing.
Bray‑Curtis analysis of microbiome profiles
obtained using 16S rRNA sequencing showedthat OMNIgene•GUT does not introduce anybias to the microbiome profile.

Figure 6: Dissimilarity within and between donor matched freshand OMNIgene•GUT collected fecal samples. Bray-Curtis analysis
showed no statistical difference in dissimilarity between freshand OMNIgene•GUT.

Conclusions
OMNIgene•GUT provides a means to collect samples
containing sufficient material for microbiome-wide
association studies to bank and share for repeat
analyses. The OMNIgene•GUT stabilization liquid iscompatible with a commercially available extraction
process, and DNA quality is suitable for downstream
applications including qPCR and sequencing.
Furthermore, the preservative function ofOMNIgene•GUT is capable of maintaining DNA
integrity, thereby minimizing bias in commonly usedmicrobial profiling applications. Reproducible andreliable self-collection with OMNIgene•GUT
uniquely enables access to study participants,
facilitating population-based microbiome-wide
association studies.

References
1
2
3

Manual of Procedures - Human Microbiome Project (2010).
OMNIgene•GUT gut microbial DNA purification using MoBio PowerFecal DNA Isolation Kit. DNA Genotek. PD-PR-00434.
Pfaffl, M. A new mathematical model for relative quantification in real-time RT–PCR, Nucleic Acids Res. 29(9): E45 (2001).

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-00040 Issue 2/2015-06© 2015 DNA Genotek Inc., a subsidiary of OraSure Technologies, Inc., all rights reserved.

www.dnagenotek.com • support@dnagenotek.com

4