The need for stabilization: Short-term variation in microbiome profiles
Jean Macklaim, Mike Tayeb, Christina Dillane, Christina Fragel and Laura Cunningham
DNA Genotek, Ottawa, Ontario, Canada
2021-06-11

Introduction

Materials and methods

Accurate analysis of the gut microbiome requires
a complete snapshot of the microbial community
contained in a fecal sample at the time of collection.
A number of methods have been used in publishedstudies for sample handling, including the use ofice packs or dry ice for temperature-controlled
transportation or simply leaving samples at roomtemperature.1,2 However, each of these methods
has significant drawbacks. Room temperaturetransportation provides no stabilization of thesample, while ice packs maintain a cool temperaturefor only a short period of time; any delays intransport will result in temperatures incompatible
with microbiome profile stabilization.1 Transport
on dry ice can maintain a suitable stabilization
temperature for longer; however, the handling of
dry ice poses a safety risk and logistical challenge
to study participants. In addition, the sample requires
transport by hazardous materials couriers, which
substantially increases handling complexity andshipping costs.

Sample collection, storage and DNA extraction

DNA Genotek’s OMNIgene®•GUT is an all-in-one
system that enables study participants to volumetricallycollect fecal samples in the comfort of their own homes.3
The collection system is easy to use for untrained
participants and has built-in sample homogenizationand stabilization of microbial DNA from fecal samples,making it ideal for microbiome profiling by downstream
metagenomic sequencing.
In this study we demonstrate the importance of sample
stabilization by evaluating the change in microbiome
profile after simulated short-term transport undera variety of common conditions. We show thatchanges in a fecal microbiome profile occur rapidly
and progressively at room temperature (within 24hours) and that an insulated box with ice packs only
marginally mitigates the effects, with significant
changes at 48 hours after collection. In contrast,OMNIgene•GUT shows a stable profile within a 7-day
window from time of collection and has demonstrated
stabilization of the microbiome profile in human stoolfor 60 days at ambient temperature.4PD-WP-00070 Issue 1/2021-06© 2021 DNA Genotek Inc., a subsidiary of OraSure Technologies, Inc., all rights reserved.

Nineteen (19) healthy adult study participants wereasked to collect a bulk fecal sample into a plastic
specimen container and transport it to the lab in
an insulated box (sample placed between 2 frozen
ice packs) within 3 hours of collection.
Upon receipt in the lab, approximately 2 g of bulk fecal
sample was immediately sub-sampled into four 5 mL
tubes. From one of these tubes, approximately 500 mgof fecal sample was collected into an OMNIgene•GUTdevice, according to the manufacturer’s instructions.
In order to evaluate the microbial profile as close to
the time of collection as possible (T0 samples), DNA
was immediately extracted from a 50 mg portion offecal sample from each of the four tubes and from250 µL of the OMNIgene•GUT fecal sample using
the QIAamp® PowerFecal® Pro DNA Kit (QIAGEN®).
To simulate the various conditions that a sample
may encounter during transport or prior to processingin the lab, three of the 5 mL tubes were placed into
different storage conditions. One tube was left at room
temperature (20°C to 23°C), a second was placed in a-80°C freezer, and the third was placed into an insulated
box with 2 frozen ice packs and a calibrated Temp101A
ambient temperature data logger (MadgeTech). The
samples collected using OMNIgene•GUT were storedat room temperature.
At subsequent designated time points (T24h, T48h,
T72h and T7d, described in Figure 1), DNA wasextracted from 50 mg of the stored raw samples and250 µL of the OMNIgene•GUT samples. The -80°C
samples and the OMNIgene•GUT samples wereextracted in triplicate only at the 7-day time point.
After removing the portion of sample to be extracted
at each time point, the tubes were returned to theirstorage locations.
All extracted DNA was quantified using a Quant-iT™
PicoGreen™ dsDNA Assay Kit (Thermo Fisher Scientific).

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Stool sample

Roomtemperature(n = 19)

Insulated box-80°C

Extraction
at T0

Extraction
at T24h

Extraction
at T48h

Extraction
at T72h

Extraction
at T7dDiversigen BoosterShot® Sequencing

Figure 1. Schematic illustrating storage conditions and samplingtime points. Each microcentrifuge tube represents an aliquot ofsample extracted.

Shotgun metagenomic sequencing anddata analysis
All extracted samples were sequenced using
BoosterShot® methodology (Diversigen®). Briefly,
libraries were prepared using a modified procedure
adapted from the Nextera® XT DNA Library
Preparation Kit (Illumina®). Libraries were sequenced
on a NovaSeq™ 6000 (Illumina®). Sequenced read QC,
read filtering and taxonomic assignment were obtainedusing Diversigen’s Core Analysis™ pipeline. For analysis,
read count data were transformed to centred log-ratios(CLR), and changes in microbiome composition andrelative abundance were evaluated using the Aitchison
distance metric for whole microbiome comparisons,and by differential abundance analysis using ALDEx2
at aggregated levels of taxonomy (order, family, genus).
Taxa were considered significantly different with an
FDR-corrected p-value < 0.05 (Welch’s t-test) andan effect size > 1. Changes in whole microbiome
composition by Aitchison distance were statisticallyevaluated using a Kruskal-Wallis non-parametric test
between time points and collection conditions. In eachcomparison, sample replicates at T0 baseline served as
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the comparative group for statistical evaluation (e.g.,baseline versus OMNIgene•GUT at 24 hours).
Over time after sample collection, increasing changes
were observed for samples held in an insulated box
with ice packs and samples held unstabilized at room
temperature (Figure 2A and 2B). These changes
were detectable as early as 24 hours after collection(unstabilized room temperature), and by 48 hours in
an insulated box with ice packs, corresponding to anincrease in temperature over the time period (Figure 2C).
The amount of change (measured by Aitchison distance)increased over each 24-hour window of measurementwith significant differences in microbiome compositionat ≥ 24 hours at room temperature, and at ≥ 48 hours
for samples in an insulated box with ice packs. By day 7,
there was a significant change in overall microbiome
composition for these samples, while samples at thesame time point held at -80°C or at room temperaturein OMNIgene•GUT had no significant change fromT0 baseline (Figure 2D). In addition, samples storedfor 7 days in OMNIgene•GUT had a lower variability
compared to samples unstabilized at room temperature
or in an insulated box with ice packs (Median distance+/- IQR: 65.52 +/- 5.62 in OMNIgene•GUT versus
87.78 +/- 21.11 at unstabilized room temperature,
90.43 +/- 26.35 in an insulated box with ice packs).
The most striking changes in overall microbiome
composition were noted at day 7 (Figure 2D), and sotaxonomic changes at this time point were evaluated bydifferential abundance analysis compared to T0 baseline(Table 1). The largest changes were apparent at theorder level, with Lactobacillaceae (not shown) andEnterococcaceae (Figure 3) showing the largest
differences in relative abundance (twofold to ninefoldchange at room temperature and in the insulated box,
respectively). In contrast, no significant changes
were detected for samples held at -80°C nor inOMNIgene•GUT after 7 days of storage. In addition
to the changes noted in Figure 3 occurring acrossthe majority of the participants, there wereindividual-specific changes in microbiome profilefor several study participants, driven by their specificmicrobial composition. The relative abundance
profile of a representative participant is shown in
Figure 4, where there was an increase in relative
abundance of Enterobacteriaceae after the sample
had been held 7 days in an insulated box with ice
packs, whereas after 7 days of room temperaturestorage in OMNIgene•GUT, the sample composition
and taxonomic relative abundances were preserved
when compared to the T0 starting profile for thesame participant sample.

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2Results

200*

150ns100*******More similarity

Aitchison distance

****
****

50T0T24h

T48h

T72h

Microbiome profile stability at room temperature****

Aitchison distanceLess similarity

 Microbiome profile stability in insulated box with ice packs
200

**

150

100

50

T7d

T0

T24h

T48h

Time point

T72h

T7d

Insulated box
with ice packs

Roomtemperature

Time point Temperature inside insulated box with ice packs

20200Microbiome stability at 7 days

Aitchison distance

Temperature (°C)

ns1510ns
****
****

150

100
550
T0T24h

T48h

T72h

T7dTime point

T0-80°COMNIgene•GUTPreservation condition

Figure 2. Change in total microbiome composition over time.
Differences between samples are plotted as Aitchison distance (panels A, B and D), with each point being a sample pair comparison(N = 19 participants) (A: changes over 7 days in an insulated box with ice packs, B: changes over 7 days at room temperature, C: measured
temperature of the sample in an insulated box with ice packs corresponding to panel A, and D: changes at the 7-day endpoint for all
conditions). The Aitchison distance measures a change in microbiome profile (composition and relative abundance) with a lower distance
representing more similarity, and a higher distance being less similar. The distance between T0 baseline replicates (first box plot in panels A,B and D) serves as the baseline variation for every subsequent statistical comparison of condition and time point. In each box plot after thebaseline, a point is a participant sample compared to its T0 baseline. Significant differences between groups were measured with a Wilcoxon
rank-sum test (‘ns’: P > 0.05, ‘*’: P ≤ 0.05, ‘**’: P ≤ 0.01, ‘***’: P ≤ 0.001, ‘****’: P ≤ 0.0001).

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 Change in relative abundance of EnterococcaceaeChange in relative abundance of Enterococcaceae(room temperature)

2020

15

Relative abundance (CLR, log2)

Relative abundance (CLR, log2)(insulated box with ice packs)**

1051510500
T0

T7d

Time point

T0 Change in relative abundance of Enterococcaceae (OMNIgene•GUT)



2020

Relative abundance (CLR, log2)

Relative abundance (CLR, log2)

*

1510
ns

50Time point

T7d

Change in relative abundance of Enterococcaceae (-80°C)

1510ns
50T0

T7d

Time point

T0

Time point

T7d

Figure 3. Changes in Enterococcaceae relative abundance affected by storage conditions.
Relative abundance (CLR transformed, log2 scale) of Enterococcaceae plotted for samples at T0 baseline and the same samples at 7 days forthe respective conditions (A: insulated box with ice packs, B: unstabilized room temperature, C: OMNIgene•GUT, D: -80°C). Differential abundance
analysis showed a large change in relative abundance for unstabilized room temperature (twofold) and samples held in an insulated box for
7 days (ninefold) (A and B above). Significant differences by Wilcoxon rank-sum test are indicated (‘ns’: P > 0.05, ‘*’: P ≤ 0.05, ‘**’: P ≤ 0.01).

A Bacterial relative abundances

B
Family level

100

Eubacteriaceae
Bacteroidales (order)
Tannerellaceae

50

Lactobacillaceae
Clostridiales (order)

Aitchison distanceEnterobacteriaceae% Abundance

110

Other75

Ruminococcaceae

25

Lachnospiraceae
Bacteroidaceae

0Change in microbiome profile over 7 days

Bifidobacteriaceae

T0

T7d

Insulated box
with ice packs

T0

T7d100Insulated box with ice packs

90

OMNIgene•GUT80

7060
T0

T24h

T48h

T72h

T7d

Time pointOMNIgene•GUTFigure 4. Representative participant: changes over 7 days in an insulated box with ice packs. A: Relative abundance stacked bar plot
showing the taxonomic profile for top 10 family-level taxa over 7 days in an insulated box with ice packs compared to OMNIgene•GUT, and
B: the same samples showing change in microbial profile measured in Aitchison distance over time held in an insulated box with ice packs
compared to OMNIgene•GUT samples held at room temperature.

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Table 1. Significantly changing taxa for samples stored
unstabilized in an insulated box with ice packs or unstabilized
at room temperature. List of taxonomic features at the order, familyand genus level that were significantly different at 7 days comparedto T0 baseline (Welch’s t-test, p-value < 0.05) between samples stored
in an insulated box and at room temperature. There were no taxa
found to be significantly different in either the -80°C or OMNIgene•GUTstabilized samples.

Order

Insulated box with ice packs

Room temperature

LactobacillalesLactobacillales

Erysipelotrichales

Burkholderiales

Conclusions•

An insulated foam shipping container with ice packs(cold-chain method) is a common form of sample
transport for fecal samples and has been used inprotocols such as the Human Microbiome Project.
Results from this study using the cold-chain method
show a similar amount of change as those samples
held unstabilized at room temperature (the forcedfailure condition).

•Changes to the fecal microbiome occur rapidly(within 24 hours) and progressively in the 1-6 days
following, resulting in negative impacts on downstream
discovery and the power to detect biological effects
in the microbiome.

•Overall, these results suggest that an insulated
shipping box with ice packs is not an effective method
for stabilization of the taxonomic profile and thatstabilization is essential if you are unable to process
fecal microbiome samples within 24 hours.

•In contrast, samples stored at -80°C and those stored
at room temperature in OMNIgene•GUT show no
significant change in taxonomic profile over the
7-day period. However, when compared with dryice, OMNIgene•GUT does not incur the expenseand logistical challenges associated with shippinga hazardous material.

•In summary, at-home sample collection using
OMNIgene•GUT removes many of the logisticalchallenges related to cold-chain transport, improvesuser experience and increases compliance. Additionally,
the unique design of the OMNIgene•GUT collection
kit ensures that the sample is collected volumetrically,
properly homogenized and stabilized at the time ofcollection, resulting in a more consistent, reliable
and reproducible microbiome profile.5

Burkholderiales
Enterobacterales

FamilyBacteroidaceae

Coriobacteriaceae

Barnesiellaceae

EnterococcaceaeOdoribacteraceae

Lactobacillaceae

Tannerellaceae

Oscillospiraceae

Enterococcaceae

Ruminococcaceae

Lactobacillaceae

Sutterellaceae

Streptococcaceae
Oscillospiraceae
Ruminococcaceae
Erysipelotrichaceae
Sutterellaceae
Genus

Bacteroides

Bacteroides

Barnesiella

Parabacteroides

References
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Nel Van Zyl, K., Whitelaw, A. C., & Newton-Foot, M. (2020). The effect of storage conditions on microbial communities in stool. PLoS ONE, 15(1),e0227486. DOI: 10.1371/journal.pone.0227486
Gorzelak, M. A., Gill, S. K., Tasnim, N., Ahmadi-Vand, Z., Jay, M., & Gibson, D. L. (2015). Methods for improving human gut microbiome data
by reducing variability through sample processing and storage of stool. PLoS ONE, 10(8), e0134802. DOI: 10.1371/journal.pone.0134802
DNA Genotek Inc. Collection of fecal sample using OMNIgene®•GUT device. PD-PR-00442.
Doukhanine, E., Bouevitch, A., Brown, A., LaVecchia, J. G, Merino, C., & Pozza, L. (2016). OMNIgene®•GUT stabilizes the microbiome profileat ambient temperature for 60 days and during transport. PD-WP-00042, DNA Genotek Inc.
Tayeb, M., Macklaim, J., Colemeco, S., Fragel, C., Dillane, C., & Cunningham, L. (2020). Impact of fecal collection methods on sample homogenizationand extraction reproducibility in gut microbiome profiling. PD-WP-00064, DNA Genotek Inc.

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