Evaluation of DNA extraction methods to obtain accurate
and reliable results from gut microbiome samples
A guide to the optimal extraction method for OMNIgene®•GUT
B. LeFrançois and L. Cunningham
DNA Genotek, Ottawa, Ontario, Canada 2019-01-10

Introduction

Methods

When it comes to microbial community profilingfrom complex environments like stool, sources oftechnical variation such as collection procedures,storage conditions and sample extraction can greatly
impact the accuracy of the data. In conjunction withsample stabilization buffer offering post-collection
protection from microbial profile changes, the choiceof extraction methodology for obtaining sufficientand high quality microbial nucleic acids is essential
for meaningful and reliable insights into thesecomplex communities.

Stool samples from eight adult human donors werecollected in OMNIgene•GUT under IRB guidance andDNA was extracted from a 250 µL aliquot using either
the QIAamp® PowerFecal® DNA Kit (QIAGEN®), theQIAamp PowerFecal Pro DNA Kit (QIAGEN),the RBB+C method3 or the ZymoBIOMICS™ DNA
Miniprep Kit (Zymo Research). Extractions were
performed within 72 hours of sample collection intoOMNIgene•GUT. DNA yields, lysis efficacy as wellas the quality and integrity of the recovered DNA
was compared for these four methods.

Despite the lack of a standardized or optimized
extraction method for efficiently recovering DNA
from all stool sample types, most methods developed
over the years rely on mechanical and/or enzymatic
lysis of microbial cells to release DNA. The lysis
component of the extraction method plays a criticalrole in the efficiency of DNA recovery, especiallyfor challenging sample matrices (presence of fibers
or undigested particles) or low biomass samples(ex., infant or donors undergoing dysbiosis).
A recent comparison of extraction protocols
showed that mechanical lysis (i.e., bead beating)is paramount for efficient DNA release from toughto lyse gram-positive bacteria and is positively
correlated with increased sample diversity.1

For the PowerFecal (QIAamp PowerFecal DNA Kit)
and PowerFecal Pro (QIAamp PowerFecal ProDNA Kit) extractions, bead beating was performedfor 10 minutes using a 2 mL tube vortex adapter(QIAGEN). For the RBB+C extractions, bead beating
was performed on a Bead Ruptor Elite (OMNIInternational) at 5.5 m/s for 3 minutes, conditions
that were found to be optimal for lysis while
maintaining good DNA integrity. For ZymoBIOMICS(ZymoBIOMICS DNA Miniprep Kit) extractions,bead beating was also performed on the Bead Ruptor
Elite using conditions recommended by Zymo
Research technical support (5 cycles consisting of
1 minute bead beating at 6.5 m/s with 5 minutes
incubation on ice between each cycle).

DNA Genotek’s OMNIgene•GUT collection andstabilization devices constitute a reliable method
to easily and accurately capture the microbialcommunity present in stool samples at the timeof collection.2 In this application note, we tested
and compared the extraction performance offour commonly used bead beating based DNA
extraction methods on stool samples collectedwith the OMNIgene•GUT device.

MK-AN-00034 Issue 1/2019-01© 2019 DNA Genotek Inc., a subsidiary of OraSure Technologies, Inc., all rights reserved.

Results
Our extraction data demonstrate that PowerFecal Pro
produced the highest average DNA yield acrosssamples collected from multiple donors, followed byZymoBIOMICS, RBB+C and PowerFecal (Figure 1).
Interestingly, RBB+C showed the least variability
from donor to donor while the performance ofZymoBIOMICS appears to be highly donor dependent.

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RBB+CZymoBIOMICS

To assess lysis efficiency of the various extractions,
we quantified the relative levels of two hard to lyse
gram-positive bacteria (Bifidobacterium longum andFaecalibacterium prausnitzii) and gram-negative
bacteria (Bacteroides genus) in 10 ng total DNA
using real-time PCR.
Lysis of the gram-positive bacteria was suboptimalin PowerFecal extracted samples (Figure 2), asdemonstrated by the significantly higher Ct values
for Bifidobacterium longum and Faecalibacteriumprausnitzii. A higher Ct value is correlated withlower levels of the bacterial DNA in the sample
and indicates suboptimal lysis of the assayed species.
In contrast, PowerFecal Pro, RBB+C andZymoBIOMICS extractions all gave lower Ct values,
indicative of a more efficient lysis. As expected,levels of the easy to lyse gram-negative bacteria
genus Bacteroides were similar across extractions.
30

PowerFecalCt values

PowerFecal ProRBB+C20

ZymoBIOMICS

100B. longum

F. prausnitzii

Bacteroides

Figure 2: Relative levels of gram-positive or negative bacteria in stool
DNA samples extracted using PowerFecal, PowerFecal Pro, RBB+C
or ZymoBIOMICS. Levels of Bifidobacterium longum (gram+),
Faecalibacterium prausnitzii (gram+) or Bacteroides species(gram- genus) were measured in real-time PCR assays using specificprimer pairs and 10ng total DNA as template. Data presented hereis from a representative donor.

To evaluate the quality and integrity of the extracted
DNA, we checked samples for the presence of PCRinhibitors and measured DIN values (DNA Integrity

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APFPF-P

RBB+CZ

DIN
7.0

DIN
8.3

DIN
8.0

DIN
5.1

48500
15000
7000
4000
3000
2500
2000
1500
1200
900
600
400
250
100

BPF-P
1200
1000
800
PF
600

Z

M

RBB+C

400
200
0
900
1200
1500
2000
2500
3000
4000
7000
15000
48500

PowerFecal Pro600

PowerFecalFigure 1: Total DNA yields following extraction of a 250 µL
OMNIgene•GUT aliquot (representing ~50 mg of fecal material)from 8 different donors using either PowerFecal, PowerFecal Pro,
RBB+C or ZymoBIOMICS. DNA Extractions were performed as per
manufacturer’s instructions. The eluted DNA was quantified using
the PicoGreen® assay.

40002505100

10Molecular weight (bp)15Number). Fecal carry-over contaminants, such as
humic acids and polyphenols, can impact downstream
processing and analysis of the samples by interferingwith PCR and DNA library preparation for next
generation sequencing platforms. All DNA samples
extracted with commercially available kits were freeof inhibitors, but we were able to detect inhibitors in
10-20% of RBB+C extracted samples (data not shown).
Furthermore, our results demonstrate that PowerFecal,
PowerFecal Pro and RBB+C all produced high quality,
high molecular DNA with an average fragment sizeof >10 kbp and DIN values higher than 7. (Figure 3and Table 1). In comparison, ZymoBIOMICS
extractions yielded significantly sheared DNA withan average fragment size of 3–4 kbp and average
DIN values lower than 5.

Sample intensity (Normalized FU)

DNA yields (ug)20Size(bp)Figure 3: Integrity and average size of the DNA samples extracted fromOMNIgene•GUT samples using PowerFecal (PF), PowerFecal Pro (PF-P),
RBB+C or ZymoBIOMICS (Z) (M = Control Marker). Extracted DNA
samples were run on a genomic DNA ScreenTape (Agilent)to determine DNA integrity (A) and average fragment size (B).
Data presented here is from a representative donor.

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PowerFecal Pro

RBB+CZymoBIOMICSDNA yields

Low to medium

Medium to highLow to medium

Medium to highLysis efficacy

Limited on gram+ bacteria

GoodGoodGoodAverage fragment size
Average DIN

19.2 kbp ± 11.5
7.1 ± 1.0

19.3 kbp ± 2.3
7.8 ± 0.6

18.6 kbp ± 1.4
7.9 ± 0.3

3.2 kbp ± 0.6
4.8 ± 0.5

Presence of PCR/library
prep inhibitors

AbsentAbsentDetectable in
10-20% of samples

AbsentTable 1: Summary of the DNA yields, lysis efficacy, DNA quality and presence of PCR inhibitors obtained following DNA extraction fromOMNIgene•GUT stool samples (n=8) using various DNA extraction kits: QIAamp PowerFecal DNA Kit, the QIAamp PowerFecal Pro DNA Kit,the RBB+C protocol or the ZymoBIOMICS DNA Miniprep Kit.

It is worth noting that the performance of theextraction kits on fresh fecal samples was similar in
every aspect to OMNIgene•GUT collected samples(data not shown). This indicates that DNA Genotek’s
stabilizing chemistry has no impact on the overall
performance of the DNA extraction kits tested
in this application note.

Conclusions
Choosing a reliable stool DNA extraction protocol
is critical to any microbiome study. Efficient andconsistent lysis of the highly diverse microbial species
present in stool samples is a key consideration toensure that the extracted DNA captures an accurate
representation of the bacterial community present
in the sample with minimal bias. Suboptimal lysis,
such as seen with the PowerFecal kit, can significantly
impact the measured relative abundance of grampositive species such as Faecalibacterium prausnitzii
and Bifidobacterium longum. Interestingly, several
studies have reported a bias in F. prausnitzii4,5 in
OMNIgene•GUT collected samples. Multiple lines ofevidence indicate that these findings are derived from
suboptimal DNA extractions rather than actual changesin the sample taxonomic composition upon collection
and stabilization of the samples.
Isolation of high molecular weight DNA is arequirement to take advantage of the full suite ofavailable next generation sequencing technologies.
Long range sequencing applications such as fulllength 16S or Nanopore based sequencing,

MK-AN-00034 Issue 1/2019-01© 2019 DNA Genotek Inc., a subsidiary of OraSure Technologies, Inc., all rights reserved.

are dependent on the recovery of relatively intact andhigh quality DNA. Our data suggests that PowerFecal
Pro and RBB+C extractions provide a good balance
between lysis efficiency while preserving DNAintegrity, while the ZymoBIOMICS lysis is achievedat the expense of DNA integrity. This result has also
been observed in preliminary testing of Zymo
Research Quick-DNA™ Fecal/Soil Microbe Kit.
In summary, out of the extraction methods evaluated,
we have identified PowerFecal Pro as the best
performing stool DNA extraction kit, giving (i) highDNA yields from a wide range of donors, (ii) efficientlysis of gram+ bacteria, and (iii) recovery of highmolecular weight DNA free of inhibitors. Moreover,
our preliminary data also indicates that PowerFecal Pro
performs well with low biomass samples (such as infant
and donors undergoing dysbiosis), making thisextraction method a good choice for a wide variety ofgut microbiome studies. We highly recommend using
the OMNIgene•GUT collection and stabilization device
to effectively capture an accurate representation of thein vivo state of the donor microbial profile, without
introducing bias or loss of DNA integrity. For
extracting OMNIgene•GUT samples, we recommend
the PowerFecal Pro extraction kit in order to minimize
any potential extraction bias. This combination will
provide the most accurate and thorough representationof the microbial community present at the time ofsample collection.

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References:
1
2
3
4
5

Costea PI et al. Towards standards for human fecal sample processing in metagenomic studies. Nat Biotechnol. Nov;35(11):1069-1076 (2017).
OMNIgene•GUT enables reliable collection of high quality fecal samples for gut microbiome studies. DNA Genotek. PD-WP-00040. (2016).
Yu Z and Morrison M. Improved extraction of PCR-quality community DNA from digesta and fecal samples. Biotechniques
May;36(5):808-12. (2004).
Song EJ, Lee ES and Nam YD. Progress of analytical tools and techniques for human gut microbiome research. J Microbiol.
Oct;56(10):693-705. (2018).
Choo JM, Leong L and Rogers GB. Sample storage conditions significantly influence faecal microbiome profiles. Sci Rep.
2015 Nov 17;5:16350. (2015)

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