Impact of fecal collection methods on sample homogenizationand extraction reproducibility in gut microbiome profiling
Mike Tayeb, Jean Macklaim, Savannah Colemeco, Christina Fragel, Christina Dillane and Laura Cunningham
DNA Genotek, Ottawa, Ontario, Canada
2020-02-15

Introduction
For an accurate analysis of the gut microbiota via
fecal sampling, it is critical to capture a snapshotof all of the microbes present in the sample
at the time of collection. The complexity anddiversity of the microbial community combinedwith the heterogeneity of the fecal matrix1 posea significant challenge when handling andprocessing samples. Ideally, multiple aliquots
extracted from the same sample should have little
or no variability and produce very similar results.
However, fecal samples can be very heterogeneousand significant microbiome profile differences
can arise when multiple aliquots of the same
sample are processed. For this reason, proper
sample homogenization at the time of collection
is critical, as it ensures that all microbes in thesample are exposed to the stabilization chemistry
and results in reproducible and consistentmicrobiome extractions.
DNA Genotek’s OMNIgene®•GUT is an all-in-one
system that enables high compliance, volumetricself-collection of fecal samples, with built-in
homogenization and stabilization of microbial
DNA from feces making it ideal for gut
microbiome profiling. In this paper we compare
the effectiveness and reproducibility ofOMNIgene•GUT extracted using QIAamp®
PowerFecal® Pro DNA Kit (QIAGEN®), and theDNA/RNA Shield™ Fecal Collection Tube (ZymoResearch) extracted using Quick-DNA™ Fecal/Soil
Microbe Miniprep Kit (Zymo Research).

Materials and methods
Sample collection
Subjects were asked to collect a bulk fecal sample intoa specimen plastic container and transport to the lab
in a Styrofoam™ box with a frozen cold pack within
three hours of collection. Upon receipt in the lab,each bulk sample was sub-sampled into one
OMNIgene•GUTPD-WP-00064 Issue 1/2020-03© 2020 DNA Genotek Inc., a subsidiary of OraSure Technologies, Inc., all rights reserved.

and one Zymo-Shield (DNA/RNA Shield Fecal
Collection Tube) according to the manufacturer’sinstructions.

Post-collection storage and DNA extraction
Samples collected in OMNIgene•GUT and Zymo-Shield
were stored at room temperature for 24 hours afterwhich triplicate extractions were performed for eachsample using PowerFecal Pro (QIAamp PowerFecal Pro
DNA Kit, 250 µL aliquot)1 and Quick-DNA (QuickDNA Fecal/Soil Microbe Miniprep Kit, 1 mL aliquot)2,respectively.

DNA analysis and metagenomic shotgun
sequencing
DNA concentration and yield were determined using
the Quant-iT™ PicoGreen™ reagent (Thermofisher
Scientific). DNA integrity and stability wereevaluated using genomic DNA screentapes on theTapeStation (Agilent Technologies).
Metagenomic shotgun sequencing libraries wereprepared using the Nextera® XT DNA LibraryPreparation Kits (Illumina®). Paired-end 150 bp
sequencing was conducted on the NextSeq™ 550(Illumina®) on a high output flow cell. Samples
were multiplexed to target a median of 2 million
reads per sample.

Metagenomic sequence processing and analysis
Demultiplexed, paired-read FASTQ files weretrimmed and quality filtered using in-house scripts.
Briefly, reads were filtered and trimmed using
kneadData v0.6.1 with Bowtie24 v.2.3.4.1 to remove
contaminating human genomic and ribosomal RNA
genes by mapping against hg37 and SILVA v128
references respectively. Timmomatic v0.38 was used
to trim low quality bases (<Q20) and to remove any
leftover sequence adapters. The resultant trimmed
and filtered reads were mapped using Kaiju5 v1.6.3
to the bacterial proGenomes database for taxonomic
assignment.

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Zymo-ShieldRTh
10 subjects

Bulk fecalsample

Transport on
ice to lab

Transfer tostabilization tubes

Hold at RT
for 24 hours

DNA extraction
in triplicate

Library prepand sequencing

Figure 1: Sample collection, transport and processing workflow.

Results
Fecal samples collected with OMNIgene•GUT
produce higher DNA yields and higher molecularweight DNA

DNA quality and quantity**2015DNA yield (µg)

The mapped read counts table was filtered to keep
reads assigned to taxonomic bins occurring in atleast 2 samples and having a total of at least 10
mapped reads. Samples had a median of 2,509,820(IQR: 1,033,238) mapped reads, post-filtering.
Using the ALDEx2 R package6, read counts weretransformed to centre-log-ratios (CLR), and theEuclidean distance between the CLR (the AitchisonDistance) was used as a measure of similarity
between samples. A lower Aitchison distance
between samples represents more similarity in therelative composition of the microbiome. Originalfigures were generated in R using the ggplot2 package
and modified for publication7. Statistical analyses
were performed in R.

10

50OM+PFPZS+QD

Collection + extraction

Figure 2: Total DNA yield (µg) from a 250 µL aliquot OMNIgene•GUT(purple) and Zymo-Shield (green) fecal sample extracted with
PowerFecal Pro and Quick-DNA respectively.

The mean DNA yield per 250 µL aliquot ofOMNIgene•GUT-stabilized feces (approximately50 mg feces) was significantly higher than fromthe same volume aliquot of feces stabilized inZymo-Shield (11.85 +/- 3.52 µg vs 1.14 +/- 0.7 µg;paired t-test p < 0.01) as shown in Figure 2.
The average molecular weight of DNA extracted
from OMNIgene•GUT was significantly higher
than the Zymo-Shield (16.7 kb vs 3.2 kb; pairedt-test P < 0.01). DNA extraction using the QuickDNA resulted in significant shearing of the DNA
compared to DNA extracted using PowerFecal Pro
as seen in Figure 3.

Figure 3: Pseudo-gel image generated by Agilent TapeStation gDNA
Assay showing DNA extracted from OMNIgene•GUT (OM) andmatching Zymo-Shield (ZS) samples extracted with PowerFecal Pro
and Quick-DNA respectively.

Metagenomic sequencing analysis
Samples collected in OMNIgene•GUT
have higher subsampling reproducibility
and introduce less variability in themicrobiome profile
The magnitude of change in the total microbiome(Aitchison distance) is lower and the amount ofvariance between replicate samples is smaller forsamples collected using OMNIgene•GUT andPD-WP-00064 Issue 1/2020-03© 2020 DNA Genotek Inc., a subsidiary of OraSure Technologies, Inc., all rights reserved.

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2

Less similarity

OM+PFP
ZS+QD

140

A

120

100
More similarity

160

160

Aitchison distance

extracted with PowerFecal Pro (mean Aitchison
distance 89.74 and coefficient of variance 3.22 asshown in Figure 4 and Figure 5). In contrast, samples
collected using Zymo-Shield and extracted with
Quick-DNA protocol showed the most variability
between replicates and replicates were less similar in
their microbiome profiles (mean Aitchison distance
104.63 and coefficient of variance 7.09).

80

Aitchison distance

140

1120

80

OM+PFPZS+QD
Collection + extraction

456
Subject

78910

BConclusions•10.0

Coefficient of variance

3Figure 5: The between-replicate Aitchison distances are plotted
per-donor (x-axis) and colored according to collection+extraction
methodology: OMNIgene•GUT samples extracted with PowerFecal
Pro (left side-purple dots) and Zymo-Shield samples extracted with
Quick-DNA (right side-green dots). A lower Aitchison distance
represents microbiota profiles that are more similar to each other.
In addition, the spread between triplicate points represents thevariability in the measured microbiota in replicated extractions.

100

12.5

27.5

•

5.0

2.5

OM+PFPZS+QD

Collection + extraction

Figure 4: Groupwise A) Difference in Aitchison distance andB) Coefficient of variance of the Aitchison distance between replicate
samples collected with OMNIgene•GUT extracted with PowerFecal Pro(purple) and replicate samples collected with Zymo-Shield extractedwith Quick-DNA (green).

••

PD-WP-00064 Issue 1/2020-03© 2020 DNA Genotek Inc., a subsidiary of OraSure Technologies, Inc., all rights reserved.

DNA yield and molecular weight fromOMNIgene•GUT samples was significantly
higher than samples collected using Zymo-Shield.
In addition, OMNIgene•GUT samples extractedwith PowerFecal Pro showed less DNA
fragmentation.
Samples collected using OMNIgene•GUTshowed reduced intra-sample replicate variability
resulting in higher sampling reproducibility
compared to samples collected using ZymoShield. This reduced technical variability gives
confidence that a single sample aliquot isrepresentative of the entire sample, and thatmultiple extractions from the same sample
will produce the same results.
The unique design of the OMNIgene•GUTcollection kit ensures that the sample is collectedvolumetrically and is properly homogenized,
resulting in more consistent and reproducible
microbiome profile.
Collection device form factor is a criticalconsideration when it comes to choosing a
reliable and reproducible collection andstabilization device.

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3References
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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-00064 Issue 1/2020-03© 2020 DNA Genotek Inc., a subsidiary of OraSure Technologies, Inc., all rights reserved.

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