overcome the challenges of nucleic acid isolation from pcr inhibitor-rich microbiome samples -...

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Overcome the challenges of isolating nucleic acids from inhibitor-rich microbiome samples

Vishwadeepak Tripathi, Ph.D.

Global Market Manager

[email protected]

Overcome the challenges of isolating nucleic acids from inhibitor-rich microbiome samples 1

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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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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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Agenda








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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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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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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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Agenda








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Molecular technologies for microbial community analysis

Microbiome sample

Extract DNA Extract RNA Extract proteins and small

molecules

16S rRNA gene

sequencingTotal 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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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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Agenda








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QIAGEN’s sample prep solutions

High inhibitors;

difficult lysis

Easy DifficultLysis

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

Overcome the challenges of isolating nucleic acids from inhibitor-rich microbiome samples

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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 adaptersInexpensive means for sample

homogenization

and lysis

(max. 24 samples)

PowerLyzer 24 High-speed homogenizer for

hard-to-lyse samples

(max. 24 samples)

TissueLyser IIHigh-speed homogenizer for

hard-to-lyse samples

(max. 192 samples)

PowerBead Tubes


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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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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Agenda








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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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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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Projects using QIAGEN microbiome kits


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Agenda








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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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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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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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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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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.


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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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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Agenda








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Microbiome workflow

Sample

collection/

stabilization

Sample

preparation

Library

constructionNGS run

Data analysis

and

interpretation

Instruments

KitsLifeGuard 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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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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November 2017 – a 3-part microbiome webinar series


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


Part III: Microbiome profiling with the Microbial Genomics Pro Suite


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Thank you for your attention!

Questions? [email protected] or [email protected]

Telephone: +1 888-503-3187

Contact QIAGEN: www.qiagen.com/about-us/contact/global-contacts/


overcome the challenges of nucleic acid isolation from pcr inhibitor-rich microbiome samples -...

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