OMNIgene®•GUT accurately captures and stabilizes
the human fecal dsDNA virome
Brice Le François and Evgueni Doukhanine
DNA Genotek, Ottawa, Ontario, CanadaIntroduction
The human gut is inhabited by a tremendous varietyof microorganisms that collectively form the gut
microbiome. This diverse community is dominatedby hundreds of bacterial species, while fungal,archaeal and viral species are also present but inlower relative abundance. The human virome
remains poorly characterized and varies greatlybetween individuals. Double-stranded (ds) DNAbacteriophages of the Siphoviridae, Podoviridae
and Myoviridae families are some of the main
components of the human intestinal virome,
and they are well known to play an important rolein influencing the gut bacterial community byinfecting their host and/or promoting horizontal
gene transfer.1,2 Moreover, recent evidence suggeststhat crAssphage-like bacteriophages are also highly
abundant in the human gut.3,4 In this technical note,
we provide evidence that OMNIgene®•GUT stool
collection devices are able to accurately capture andstabilize human gut viral dsDNA. In combination
with our past data, OMNIgene•GUT collected
samples are thus suitable for microbiome studies
focusing on the characterization of both viral andbacterial profiles.

Materials and methods
Sample collection and storage
Fresh fecal samples from 6 crAssphage positive healthy
adult donors were collected in 3 OMNIgene•GUTdevices per donor. Baseline DNA extractions fromfresh samples as well as OMNIgene•GUT collected
samples were performed immediately using
QIAamp® PowerFecal® Pro DNA Kit (QIAGEN®).
OMNIgene•GUT collected samples were thenincubated at room temperature for 60 days, at 50°Cfor 3 days or subjected to 6 cycles of freeze/thaw(with room temperature holds in between) tosimulate shipping conditions. DNA extractions
were performed after each of these treatments andcompared to baseline. Alternatively, due to the lack

MK-AN-00035 Issue 1/2019-01© 2019 DNA Genotek Inc., a subsidiary of OraSure Technologies, Inc., all rights reserved.

of other viral signatures shared by a high enoughproportion of donors, fresh fecal samples from
5 healthy adult donors were spiked with purified
intact T3/T5 bacteriophages and collected in3 OMNIgene•GUT devices. A bacteriophagepermissive E. coli strain was also co-spiked into
these samples in order to ensure substantial presenceof the phages’ host. Samples were then processedusing same methodology as the crAssphage positive
fecal samples.

Real-time PCR and long-range PCR assays
Levels of crAssphage, T3 and T5 bacteriophages weremeasured in real-time PCR assays using 10 ng total
DNA as template and primer pairs specific for eachof these bacteriophages. A crAssphage-specificlong-range PCR assay was also used to assess DNA
stability in our chemistry (i.e., presence of highmolecular weight DNA). Long-range PCR
amplification of the crAssphage genome wasperformed with a different set of specific primers
using LongAmp® Taq polymerase (NEB) and 50 ng
of total DNA as template.

Results
OMNIgene•GUT captures the human virome at
the point of collection without introducing a bias
We tested the ability of OMNIgene•GUT devices
to capture and stabilize an endogenous virus suchas crAssphage (sensu stricto). The relative levels ofcrAssphage were compared by real-time PCR infresh and OMNIgene•GUT stabilized stool samples
at baseline (T0) and showed no significant differences(Figure 1). Similar results were seen with stoolsamples spiked with either T5 (Siphoviridae family)or T3 (Podoviridae family) bacteriophages (Figure 1and data not shown). Taken together this demonstrates
the ability of OMNIgene•GUT devices to captureviral profiles with no bias from the sample
stabilization technology.

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T5baseline(T0)

OMNIgene•GUTcrAssphagebaseline(T0)

OMNIgene•GUTAFreshcrAssphage

Unstabilized- 80°C - 14 days
RT - 14 days
T06 Freeze/thaw

FreshOMNIgene•GUT05101520

RT - 60 days
50°C - 3 days
T0250

Relative level (Ct value)

61218

Relative level (Ct value)

Figure 1: Relative levels of the crAssphage and T5 bacteriophages
in fresh stool compared to OMNIgene•GUT collected samples atbaseline (T0). Total DNA was extracted from each sample using
the QIAamp PowerFecal Pro DNA kit. Real-time PCR analysis wasperformed with primers specific for each bacteriophage using
10 ng total DNA as a template. No significant differences wereseen using paired one-way ANOVA. n=5 for spiking experiments(T5 bacteriophage) and n=6 for crAssphage-positive samples.

BBacteriophage T5Unstabilized- 80°C - 14 days
RT - 14 days**T0
6 Freeze/thaw

OMNIgene•GUT stabilizes viral DNA duringtransport and up to 60 days at room temperature
The ability of the OMNIgene•GUT kits to stabilize
viral DNA during simulated transport and storage
at room temperature for extended periods of time
was tested by subjecting fecal samples collected inOMNIgene•GUT to elevated temperatures (50°C for3 days), freezing (6 cycles of freeze/thaw interspersedwith holds at room temperature) or long-term storage
at room temperature (up to 60 days). For each ofthese conditions, total DNA was extracted andrelative levels of the bacteriophages were measured
by real-time PCR and compared to baseline levels(T0). All of the simulated shipping and storage
conditions tested had no impact on the relative
levels of crAssphage or T5/T3 bacteriophages inOMNIgene•GUT collected samples (Figure 2 anddata not shown), highlighting the ability of ourchemistry to stabilize viral profiles over time.
Importantly, extended incubation of the unstabilized
samples at room temperature (14 days) led to alarge increase in T5 bacteriophage levels following
infection/lysis of the bacterial host. This demonstrates
the need for proper stabilization to accurately
measure gut viral profiles (Figure 2). No changes
in crAssphage levels were observed in unstabilized
samples; this is likely due to the fact that its host ispredicted to be a member of the obligate anaerobicgenus Bacteroides, and would not be expected togrow and/or survive in aerobic conditions.

MK-AN-00035 Issue 1/2019-01© 2019 DNA Genotek Inc., a subsidiary of OraSure Technologies, Inc., all rights reserved.

OMNIgene•GUT50°C - 3 days
RT - 60 days
T00

6

12

1824

Relative level (Ct value)

Figure 2: OMNIgene•GUT stabilizes crAssphage (A) andT5 bacteriophage (B) during simulated shipping conditions andstorage at room temperature for 60 days. Total DNA was extracted
from each sample using the QIAamp PowerFecal Pro DNA kit andreal-time PCR analysis was performed with primers specific for
crAssphage or T5 phage using 10 ng total DNA as a template.
No significant differences were seen using paired one-way
ANOVA for crAssphage (n=6) or T5 bacteriophage (n=5).
However, a significant change was seen for samples left
unstabilized, **p<0.01 paired one-way ANOVA.

The presence of high molecular weight viral DNA
was also assessed in these samples using a long-range
PCR assay specific for a 9.5 kb fragment of thecrAssphage genome. A high molecular weight PCRband was observed in OMNIgene•GUT stabilized
samples at baseline, but also following incubation
at room temperature for extended periods of time(60 days), subjected to high temperatures (50°Cfor 3 days) or 6 cycles of freeze/thaw (Figure 3).
This indicates that our chemistry not only stabilizes
viral profiles but also maintains the integrity of highmolecular weight viral DNA.

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Room temperature hold
10 –50°C holdFreeze/thaw cycles

NegativecontrolConclusions
Using representative viruses from some the mainbacteriophage families of the human gut virome(crAssphage-like, Siphoviridae-T5 and Podoviridae-T3),
we demonstrate that OMNIgene•GUT devices captureand stabilize the viral profiles:•Figure 3: OMNIgene•GUT protects high molecular weight viral DNA
during simulated shipping conditions and storage at room
temperature for 60 days. Total DNA was extracted from each sample
using the QIAamp PowerFecal Pro DNA kit and a crAssphage specificlong-range PCR assay was performed using 50 ng total DNA as atemplate. Presence of a 9.5 kb PCR band indicates the presence of highmolecular weight viral DNA in the sample. Data from a representative
donor is shown here.

•No detectable bias in viral levels are introduced atthe point-of-collection.
High molecular weight double-stranded viral
DNA is stabilized in our chemistry for up to60 days at room temperature or when subjected
to harsher simulated shipping conditions(high temperature or multiple freeze/thaw).

OMNIgene•GUT devices are therefore highly suitablefor gut virome studies focusing on dsDNA viruses.

References123
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Waller AS, Yamada T, Kristensen DM, Kultima JR, Sunagawa S, Koonin EV, Bork P. Classification and quantification of bacteriophagetaxa in human gut metagenomes. ISME J. 8;7:1391-402. (2014).
Carding SR1, Davis N, Hoyles L. The human intestinal virome in health and disease. Aliment Pharmacol Ther. Nov; 46;9:800–815. (2017).
Dulith BE et al. A highly abundant bacteriophage discovered in the unknown sequences of human faecal metagenomes. Nat Commun.
24;5:4498. (2014)
Yutin N. et al. Discovery of an expansive bacteriophage family that includes the most abundant viruses from the human gut.
Nat. Microbiol. 3:38–46. (2018)

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