This presentation will focus on nucleic acid extraction tools developed by QIAGEN that facilitate accurate non-biased community analysis and eliminate common amplification problems via the depletion of endogenous polymerase inhibitors using our patented Inhibitor Removal Technology.

1. Sample to Insight
Overcome the challenges of isolating nucleic acids from inhibitor-
rich microbiome samples
Vishwadeepak Tripathi, Ph.D.
Global Market Manager
Vishwadeepak.Tripathi@qiagen.com
Overcome the challenges of isolating nucleic acids from inhibitor-rich microbiome samples 1

2. Sample to Insight
Legal disclaimer
QIAGEN products shown here are intended for molecular biology
applications. These products are not intended for the diagnosis,
prevention or treatment of a disease.
For up-to-date licensing information and product-specific
disclaimers, see the respective QIAGEN kit handbook or user
manual. QIAGEN kit handbooks and user manuals are available at
www.qiagen.com or can be requested from QIAGEN Technical
Services or your local distributor.
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3. Sample to Insight
Agenda
Introduction to microbiome1
Technologies for microbial community analysis2
QIAGEN's sample prep solutions3
Inhibitor removal technology (IRT)4
QIAGEN's microbiome product offerings5
Summary and questions6
Overcome the challenges of isolating nucleic acids from inhibitor-rich microbiome samples 3

4. Sample to Insight
Agenda
Introduction to microbiome1
Technologies for microbial community analysis2
QIAGEN's sample prep solutions3
Inhibitor removal technology (IRT)4
QIAGEN's microbiome product offerings5
Summary and questions6
Overcome the challenges of isolating nucleic acids from inhibitor-rich microbiome samples 4

5. Sample to Insight
What does microbiome mean?
Microbiome: Definition and background
• The microbiome is defined as the collective genomes of the microbes (composed of bacteria,
bacteriophage, fungi, protozoa and viruses) that live inside and on the human body – NIH, 2012
• Microbiota refers to the collection of microbial organisms that inhabits a certain environment
• Metagenomics is the study of the collective genomes of microorganisms from a sample without
cultivation – The NIH HMP Working Group
Sources:
• The NIH HMP Working Group et al. (2009) The NIH Human Microbiome Project. Genome Research 19, 2317–2323
• Kuczynski, J. et al. (2012) Experimental and analytical tools for studying the human microbiome. Nature Reviews Genetics 13, 47–58
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6. Sample to Insight
Human Microbiome Project
Microorganisms cluster by body site
• Cataloguing efforts by the NIH Human Microbiome Project suggest:
◦ Around 10,000 organisms live with us
◦ Second genome: outnumber our own genes by 150:1
• Identifying microbiota in healthy individuals revealed:
◦ Different body sites have unique communities
◦ Race, age, gender, weight or ethnicity have an effect
Sources:
• Hoffmann, A.R, Proctor, L.M, Surette and M.G, Suchodolski, J. (2015) The microbiome: The trillions of microorganisms that
maintain health and cause disease in humans and companion animals. Vet. Pathol. 53, 10–21
• Human Microbiome Project www.commonfund.nih.gov/hmp
• Huttenhower, C et al. (2012) Structure, function and diversity of the health human microbiome. The Human Microbiome Project
Consortium. Nature 486, 207–214
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7. Sample to Insight
Earth Microbiome Project
A multidisciplinary effort to survey the microbial composition of diverse environments across
the globe
• Aims to process 200,000 samples from these different biomes
• Generate a database of microbes and their gene products
◦ Will greatly enhance our understanding of the roles microbes play in ecology
◦ Will expand our understanding of microbial metabolism and gene products
• Estimates of bacterial diversity*:
◦ One-milliliter of ocean water contains 160 distinct types of bacteria
◦ One-gram of soil contains 6400–38,000 types of bacteria
These are just estimates for bacteria alone; one still needs to consider viruses, archaea and fungi
* Source: Curtis, T. P, Sloan, W. T and Scannell, J. W. (2002) Estimating prokaryotic diversity and its limit. Proceedings of
the National Academy of Sciences 99, 10494–10499
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8. Sample to Insight
Agenda
Introduction to microbiome1
Technologies for microbial community analysis2
QIAGEN's sample prep solutions3
Inhibitor removal technology (IRT)4
QIAGEN's microbiome product offerings5
Summary and questions6
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9. Sample to Insight
Molecular technologies for microbial community analysis
Microbiome sample
Extract DNA Extract RNA Extract proteins and small
molecules
16S rRNA gene
sequencing
Total DNA sequencing
(shotgun)
RNA expression profiling
(transcriptomics)
Mass spectroscopy
(metabolomics and proteomics)
Bacteria and
archaea
Fungus/yeast Viruses Gene
content
Gene expression Metabolite, protein characterization
Identify relative frequencies and pathways
What are the functions of the community?
What organisms are present
and what are their relative
abundances?
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10. Sample to Insight
Sample preparation requirements for successful microbiome studies
Sample prep inefficiencies can influence a microbiome/metagenomics study
• Insufficient homogenization of sample matrix (to dislodge/disrupt cell substrate interactions)
• Insufficient cell lysis
◦ Can influence downstream analysis with a bias towards ‘the easily disrupted’ population(s)
• Poor nucleic acid quality, extensive shearing
• Insufficient inactivation of nucleases/proteases
◦ Unintended destruction of template molecules of interest
• Insufficient solubilization of analyte(s) of interest/separation from interacting cellular
components
• Unintended precipitation of nucleic acids via complexation with matrix-derived metals,
bioactive amines
• Low-yielding binding interactions with purification matrices
• Co-purification of small molecule inhibitors of amplification reactions (e.g., PCR)
◦ Decreases efficiency of amplification or can completely inhibit library prep reactions
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11. Sample to Insight
Agenda
Introduction to microbiome1
Technologies for microbial community analysis2
QIAGEN's sample prep solutions3
Inhibitor removal technology (IRT)4
QIAGEN's microbiome product offerings5
Summary and questions6
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12. Sample to Insight
QIAGEN’s sample prep solutions
High inhibitors;
difficult lysis
Easy Difficult
Lysis
Inhibitors
Low
High
Blood, animal
tissue and cells
Pure microbial
cultures
Soil microbesLeaf
tissue
Stool and gut
microbes
Biofilm
FFPE
Tissue
Food cultures
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QIAGEN’s sample prep solutions
13
Cell lysis with homogenous gene/community representation
• Mechanical homogenization with tailored lysis buffer formulations (DNA or RNA, DNA & RNA
and protein)
Increased purity
• Inhibitor removal technology – IRT (“Power” line of kits)
Customizable throughput
• From single silica spin filters to unique non-fouling ClearMag beads in high-throughput,
automated applications
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14. Sample to Insight
Bead-based homogenization for unbiased nucleic acid representation
Metal 2.38 mm
Ceramic 1.44 & 2.8 mm
Glass 0.1 & 0.5 mm
Carbide 0.25 mm
Garnet 0.15 & 0.70 mm
Vortex bead tube adapters
Inexpensive means for sample
homogenization
and lysis
(max. 24 samples)
PowerLyzer 24
High-speed homogenizer for
hard-to-lyse samples
(max. 24 samples)
TissueLyser II
High-speed homogenizer for
hard-to-lyse samples
(max. 192 samples)
PowerBead Tubes
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Vortex Genie 2

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Sample-derived PCR/RT-PCR inhibitors
*Examples for additional matrices: Rådström, P. et al. (2004) Pre-PCR processing: Strategies to
generate PCR-compatible samples. Mol. Biotechnol. 26, 133–46
In the process of breaking open cells to release nucleic acids, amplification inhibitors
are also released
Inhibitors include humic/fulvic acids in soil, polysaccharides/polyphenolics in plants
and bile, bilirubin and heme in stool*
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16. Sample to Insight
Sample-derived PCR/RT-PCR inhibitors
Sample-derived PCR inhibitors can act via several overlapping mechanisms
• Sequestration of reaction components/enzyme cofactors
◦ Mg2+ chelation by sample-derived carboxylates
• Competitive displacement of reaction cofactors
◦ Ca2+ displacement of Mg2+
• Direct interaction with template nucleic acids
◦ Competitive displacement of enzyme
◦ Steric occlusion of enzyme
• Direct interaction with polymerase
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17. Sample to Insight
Agenda
Introduction to microbiome1
Technologies for microbial community analysis2
QIAGEN's sample prep solutions3
Inhibitor removal technology (IRT)4
QIAGEN's microbiome product offerings5
Summary and questions6
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18. Sample to Insight
Inhibitor removal technology
The inhibitor removal chemistry outlined in US 7,459,548 forms the basis for all
“Power” kits (e.g., DNeasy PowerSoil, RNeasy PowerMicrobiome).
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19. Sample to Insight
Inhibitor removal technology
Inhibitors result in false negatives in qPCR, RT-qPCR and sequencing analysis
A comparison of samples
with and without IRT
IRT removes the most challenging inhibitors from lysates prior to nucleic acid
purification.
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Why contamination removal is important
Sample
ID
ng/µl A260 A280 260/280 260/230 Constant Cursor
pos.
Cursor
abs.
340 raw
No IRT 59.38 1.188 0.807 1.47 0.78 50.00 230 1.527 0.696
No IRT 60.71 1.214 0.818 1.48 0.78 50.00 230 1.565 0.852
No IRT 60.30 1.206 0.828 1.46 0.78 50.00 230 1.539 0.768
IRT 25.58 0.512 0.268 1.91 1.63 50.00 230 0.314 0.056
IRT 24.60 0.492 0.262 1.88 1.60 50.00 230 0.308 0.043
IRT 26.97 0.539 0.287 1.88 1.55 50.00 230 0.349 0.052
No IRT
IRT method
IRT methodNo IRT
NanoDrop data
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Why contamination removal is important
Low levels of contaminating inhibitors can lead to false negatives/aberrant amplification
IRT M + + – –
Samples 260/280 260/230
IRT
IRT
1.91
1.92
2.03
1.99
No IRT
No IRT
1.87
1.85
1.84
1.53
+ IRT
- IRT
Amplification plot
ΔRn
Cycle
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22. Sample to Insight
Projects using QIAGEN microbiome kits
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23. Sample to Insight
Agenda
Introduction to microbiome1
Technologies for microbial community analysis2
QIAGEN's sample prep solutions3
Inhibitor removal technology (IRT)4
QIAGEN's microbiome product offerings5
Summary and questions6
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24. Sample to Insight
DNeasy PowerSoil Kit*
QIAGEN’s most important offering to the microbiome community
Optimized
lysis for tough
samples
Inhibitor
removal
technology
Rapid protocol
Isolates pure DNA from soil, stool and
other environmental samples
Eliminates humic substances and other
PCR inhibitors for DNA that is ready to
use in PCR, qPCR, Sanger sequencing
and NGS
Enables isolation of high-quality, pure
DNA from 250 mg samples in just 30
minutes
*Though developed for soil, this kit is often used for stool, swabs and a host of other
sample matrices; often used interchangeably with the PowerFecal DNA isolation kit.
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25. Sample to Insight
DNeasy PowerSoil Kit: An example from literature
Frossard, A., Ramond, J.-B., Seely, M and Cowan, D. A. (2015). Water regime history drives responses of soil
Namib Desert microbial communities to wetting events. Scientific Reports 5, 12263.
• The effects of wetting event frequency and
intensity on Namib Desert microbial
communities from two soils with different water-
regime histories were tested over 36 days
• The intensity of the water pulses (i.e., the
amount of water added) rather than the
frequency of wetting events had the greatest
effect in shaping bacterial and fungal community
structures
• In contrast to microbial diversity, microbial
activities (enzyme activities) showed very little
response to the wetting events and were mainly
driven by soil origin
Conclusion: while microbial community structures might be irreversibly altered by the
successive dry and wet cycles, microbial activities are expected to be more resilient,
suggesting functional redundancy of the microbial communities.
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26. Sample to Insight
MagAttract PowerSoil DNA Kit
Optimized for
tough samples
ClearMag
technology
Hands-free
purification
Inhibitor
removal
technology
Automated isolation of high-quality DNA
from soil, stool and environmental
samples
Novel magnetic particle technology
captures DNA without binding organic
inhibitors, facilitating isolation of pure
DNA
Optimized for use with automated
processing and liquid handling systems
Removes PCR-inhibiting compounds,
including humic substances, phenolics
and polysaccharides
• Uses the same chemistry as PowerSoil/PowerFecal Kits for lysis and IRT.
• Magnetic bead purification of DNA enables robotic automation of all steps post-IRT.
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MagAttract PowerSoil DNA Kit: An example from literature
Quinn, R. A et al. (2016) From sample to multi-omics conclusions in under 48 hours. mSystems 1, e00038–16.
• Applied an integrated omics pipeline for human
and environmental samples and reported full
analysis of integrated data set within 48 hours
◦ 16S rRNA gene sequencing
◦ Inferred gene function profiles
◦ LC-MS/MS metabolomics
• Swab samples from skin, feces, oral cavity,
fermented foods and household surfaces
• Conclusions:
◦ Human samples clustered with corresponding
types in American Gut Project data set
◦ Fermented foods produced a distinct cluster
◦ Microbial communities of household surfaces
derived primarily from fermented foods
◦ Modified and unmodified metabolites derived from
fermented foods detected in stool
◦ Multi-omics analysis achieved on time scales
similar to classical microbiological culturing
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28. Sample to Insight
RNeasy PowerMicrobiome Kit
Optimized for
samples high
in PCR
inhibitors
Inhibitor
removal
technology
Rapid protocol
Isolates total RNA from stool, gut
material, dried feces, contaminated
buccal swabs and secretions
Eliminates inhibitory substances,
including heme, lipids and
polysaccharides, for RNA that is
ready to use in RT-qPCR and RNA-
seq
Enables isolation of high quality total
RNA in less than 45 minutes
Microbial RNA from (a) stabilized dog stool
and (b) dog stool transported on ice
a b
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29. Sample to Insight
RNeasy PowerMicrobiome Kit: An example from literature
Reck, M. et al. (2015) Stool metatranscriptomics: A technical guideline for mRNA stabilization and isolation.
BMC Genomics 16, 494.
• Authors present a workflow for the stabilization
of stool microbial cells/nucleic acids and their
extraction
• Compared 3 commercial kits and one literature
methodology
◦ Determined that the PowerMicrobiome kit
performed the best with respect to RNA yield and
purity
• Paired the PowerMicrobiome kit with several
commercial stabilization reagents and analyzed
extracted mRNA via Illumina HiSeq compared to
snap-frozen controls
◦ mRNA transcripts preserved in RNAlater remained
unchanged for up to 6 days at RT but a bias was
clearly detected (reduced abundance of
Prevotellaceae transcripts and depleted transcripts
for COG category “carbohydrate transport and
metabolism”
Conclusion: RNA Protect + PowerMicrobiome yielded transcriptomes most similar to
snap-frozen controls.
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Absorbance
ratio
260/280 260/230

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DNeasy PowerBiofilm Kit
Novel lysis
method
Inhibitor
removal
technology
Optimized for
challenging
biofilms
A combination of pre-treatment,
mechanical and chemical lysis
ensures high DNA yields from the
toughest biofilm samples
Eliminates PCR inhibitors including
humic acids, metals, salts and
pesticides, for DNA that is ready to
use in PCR, qPCR, Sanger
sequencing and NGS
Isolates pure DNA from all biofilms,
including dental plaques and
microbial mats
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31. Sample to Insight
DNeasy PowerBiofilm Kit: An example from literature
Abed, R. M. M, Al-Kharusi, S, Prigent, S and Headley, T (2014) Diversity, distribution and hydrocarbon
biodegradation capabilities of microbial communities in oil-contaminated cyanobacterial mats from a constructed
wetland. PLoS ONE 9, e114570.
• Constructed wetland treatment systems are devised to exploit
natural processes for the cleanup of wastewaters
• “Produced water” = water from underground formations
brought to the surface via wells during oil and gas production
◦ Even after oil and gas separation, produced water is still
contaminated with residual hydrocarbons
◦ Fifty million m3/day of produced water = environmental challenge
for energy industry
• Authors studied cyanobacterial mats in BAUER-
commissioned wetland system that treats 95,000 m3 of oil-
field production water/day
• Conclusions:
◦ The wetland mats were able to degrade 53–100% of C12–C30
alkanes after 6 weeks of incubation under aerobic conditions
◦ Oil and ammonia concentrations determine the spatial distribution
of the wetland mats’ microbial communities
◦ Mats contribute directly to the removal of hydrocarbons from oil
field wastewaters
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32. Sample to Insight
Agenda
Introduction to microbiome1
Technologies for microbial community analysis2
QIAGEN's sample prep solutions3
Inhibitor removal technology (IRT)4
QIAGEN's microbiome product offerings5
Summary and questions6
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33. Sample to Insight
Microbiome workflow
Sample
collection/
stabilization
Sample
preparation
Library
construction
NGS run
Data analysis
and
interpretation
Instruments
Kits
LifeGuard Soil
Preservation Solution
Disruption
• PowerBead Tubes
• Pathogen Lysis
Tubes L/S
Purification
AllPrep
• PowerViral DNA/RNA
• PowerFecal DNA/RNA
DNeasy
• PowerSoil
• PowerMax Soil
• PowerLyzer PowerSoil
• PowerSoil HTP 96
• PowerWater
• PowerWater Sterivex
• PowerBiofilm
QIAamp
• PowerFecal DNA
• 96 PowerFecal QIAcube HT
• BiOstic Bacteremia
RNeasy
• PowerMicrobiome
• PowerSoil Total RNA
• PowerWater
• PowerBiofilm
MagAttract
• PowerSoil DNA
• PowerWater DNA
• PowerMicrobiome DNA/RNA
QIAseq
• FX DNA Library Kit
• 1-Step Amplicon Kit:
• Ultralow Input Library Kit
• FX single cell DNA
Library Kit
Microbial Genomics Pro Suite
Disruption
• TissueLyser II
• PowerLyzer 24
• TissueLyser LT
• Vortex adapters
Purification
• QIAcube
• QIAcube HT
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34. Sample to Insight
Want to stay up to date?
Visit www.mobio.com to see our complete kit
catalog, find technical support FAQs at
mobio.com/faq, learn about our Microbiome
Awards, and read our microbiome blog
mobio.com/blog/cat/blog with great guest
writer entries
Stay up to date with developments in
microbiome news at the Microbiome Project
(news.microbiomeproject.com/)
@QIAGENsciences
QIAGEN Life Sciences
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35. Sample to Insight
November 2017 – a 3-part microbiome webinar series
Overcome the challenges of isolating nucleic acids from inhibitor-rich microbiome samples 35
Analyzing the microbiome – a 3-part
microbiome webinar series
Part I: Overcome the challenges of nucleic acid isolation from inhibitor-rich microbiome samples
Date and time: Nov 2, 2017 at 2:30 p.m. CET
Part II: QIAseq technologies for metagenomics and microbiome NGS library prep
Date and time: Nov 9, 2017 at 3:30 p.m. CET
Part III: Microbiome profiling with the Microbial Genomics Pro Suite
Date and time: Nov 16, 2017 at 3:30 p.m. CET

36. Sample to Insight
Thank you for your attention!
Questions? qiawebinars@qiagen.com or brcsupport@QIAGEN.com
Telephone: +1 888-503-3187
Contact QIAGEN: www.qiagen.com/about-us/contact/global-contacts/
Overcome the challenges of isolating nucleic acids from inhibitor-rich microbiome samples 36