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Sample to Insight
Sample Prep Solutions for Microbiome Research
Eddie Adams, Ph.D.
MO BIO Laboratories, a QIAGEN Company
Eddie.Adams@qiagen.com
Sample to Insight
Legal disclaimer
2
QIAGEN and MO BIO 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. MO BIO kit manuals are available
at www.mobio.com or can be requested from MO BIO Technical
Services.
Sample to Insight
Agenda
Introduction to the microbiome
Technologies for microbial community analysis
MO BIO’s sample prep solutions
Inhibitor Removal Technology® (IRT)
MO BIO’s microbiome product offerings
Questions
3
1
2
3
4
5
6
Sample to Insight
Agenda
Introduction to the microbiome
Technologies for microbial community analysis
MO BIO’s sample prep solutions
Inhibitor Removal Technology® (IRT)
MO BIO’s microbiome product offerings
Questions
4
1
2
3
4
5
6
Sample to Insight
Microbiome: Definition and background
5
What does “microbiome” mean?
“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 (Lederberg and
McCray 2001, The NIH HMP Working Group)
Kuczynski et al. Nature Reviews Genetics 13,47-58 (January 2012)
Sample to Insight
Human Microbiome Project
6
Microorganisms cluster by body site
Cataloguing efforts by the NIH
Human Microbiome Project
suggest:
• ~10,000 organisms live with us
• ~ 8 ×106 genes in this “second
genome”
Identifying microbiota in healthy
individuals revealed:
• Different body sites have
unique communities
• Race, age, gender, weight or
ethnicity have an effect
1 Hoffmann A.R., et al. “The Microbiome: The Trillionsof MicroorganismsThat Maintain Healthand Cause Disease in Humansand Companion Animals.” Vet Pathol.2015
2 http://commonfund.nih.gov/hmp/index
3 Structure, functionand diversity of the health human microbiome. The HumanMicrobiome Project Consortium. Nature,486, 207 -214 (14 June 2012). doi: 10.1038/nature11234
Sample to Insight
Earth Microbiome Project
7
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 diversity1:
• 160 distinct types of bacteria in 1 ml of ocean water
• 6,400 – 38,000 types of bacteria in 1 gram of soil
These are just estimates for bacteria alone; one still needs to consider viruses, archaea
and fungi
1Curtis, T. P.; Sloan, W. T.; Scannell, J. W. (2002). "Estimating prokaryotic diversity and its limits". Proceedings of the
National Academy of Sciences 99 (16): 10494–10499.
Sample to Insight
Agenda
Introduction to the microbiome
Technologies for microbial community analysis
MO BIO’s sample prep solutions
Inhibitor Removal Technology® (IRT)
MO BIO’s microbiome product offerings
Questions
8
1
2
3
4
5
6
Sample to Insight
Agenda
Introduction to the microbiome
Technologies for microbial community analysis
MO BIO’s sample prep solutions
Inhibitor Removal Technology® (IRT)
MO BIO’s microbiome product offerings
Questions
9
1
2
3
4
5
6
Sample to Insight
Molecular technologies for microbial communityanalysis
10
16S rRNA gene
sequencing
18S rRNA gene
sequencing
Total DNA sequencing
(shotgun)
Bacteria and
Archaea
Fungus
/ Yeast
Viruses Gene content
RNA expression profiling
(transcriptomics)
Gene expression
Metabolite, protein
characterization
Mass spectroscopy
(metabolomics &
proteomics)
Identify relative frequencies and pathways
Microbiome sample
Extract DNA Extract RNA Extract proteins & small
molecules
What organisms are present and
what is their relative abundance?
What are the functions of the community?
Sample to Insight
Sample preparation requirements for successful microbiome studies
There are several areas where sample prep inefficiencies can bias a
microbiome/metagenomics study:
• Insufficient homogenization of sample matrix (to dislodge/disrupt cell:substrate
interactions)
• Insufficient cell lysis
◦ Can bias downstreamanalysis toward ‘the easily disrupted’population(s)
• Poor nucleic acid quality, extensive shearing
• Insufficient inactivation of nucleases/proteases
◦ Unintended destructionof 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
Sample to Insight
Agenda
Introduction to the microbiome
Technologies for microbial community analysis
MO BIO’s sample prep solutions
Inhibitor Removal Technology® (IRT)
MO BIO’s microbiome product offerings
Questions
12
1
2
3
4
5
6
Sample to Insight
Agenda
Introduction to the microbiome
Technologies for microbial community analysis
MO BIO’s sample prep solutions
Inhibitor Removal Technology® (IRT)
MO BIO’s microbiome product offerings
Questions
13
1
2
3
4
5
6
Sample to Insight
About MO BIO Laboratories:
• 22 year-old Carlsbad, CA molecular biology company
– Privately owned until acquisition by QIAGEN in November 2015
• Current catalog of products will be manufactured in Carlsbad until end of 2016;
QIAGEN (Hilden, Germany) will then manufacture all products from 2017 onward
• Customers will still be able to order MO BIO products through current website
(www.mobio.com) and products will be supported by current MO BIO Technical
Support staff
• ~50 employees
• Products sold in 90+ countries
MO BIO’s sample prep solutions
Sample to Insight
High Inhibitors
Difficult Lysis
MO BIO’s sample prep solutions
Easy Difficult
Lysis
Inhibitors
Low
High
Blood, animal
tissue & cells
Pure microbial
cultures
Soil microbesLeaf
tissue
Stool & gut
microbes
Biofilm
FFPE
Tissue
Food cultures
Sample to Insight
MO BIO’s sample prep solutions
Cell lysis with homogenous gene/community representation
• Mechanical homogenization with tailored lysis buffer formulations (DNA and RNA, DNA or RNA only,
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
Sample to Insight
Bead-basedhomogenization 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)
PowerLyzer24
High-speed homogenizer for
hard-to-lyse samples
(max 24 samples)
TissueLyser II
High-speed homogenizer for
hard-to-lyse samples
(up to 192 samples)
Sample to Insight
Sample-derived PCR/RT-PCR inhibitors
In the process of breaking open cells to
release nucleic acids, amplification
inhibitors are also released.
Inhibitors include humic/fulvic acids in
soil and bile, bilirubin and heme in stool.*
*Other examples for additional matrices given in Rådström, P. et al. (2004) Pre-PCR processing: Strategiesto
generate PCR-compatible samples. Mol. Biotechnol. 26, 133–46.
Sample to Insight
Sample-derived PCR/RT-PCR inhibitors
Sample-derived PCR inhibitors can act via several overlapping mechanisms
• Sequestration of reaction components/enzyme co-factors
◦ Mg2+ chelation by sample-derived carboxylates
• Competitive displacement of reaction co-factors
◦ Ca2+ displacement of Mg2+
• Direct interaction with template nucleic acids
◦ Competitive displacement of enzyme
◦ Steric occlusion of enzyme
• Direct interaction with polymerase
Sample to Insight
Agenda
Introduction to the microbiome
Technologies for microbial community analysis
MO BIO’s sample prep solutions
Inhibitor Removal Technology® (IRT)
MO BIO’s microbiome product offerings
Questions
20
1
2
3
4
5
6
Sample to Insight
Agenda
Introduction to the microbiome
Technologies for microbial community analysis
MO BIO’s sample prep solutions
Inhibitor Removal Technology® (IRT)
MO BIO’s microbiome product offerings
Questions
21
1
2
3
4
5
6
Sample to Insight
Inhibitor Removal Technology®
The inhibitor removal chemistry outlined in US 7,459,548 forms the basis for all
MO BIO “Power” kits (e.g., PowerSoil, PowerMicrobiome).
Sample to Insight
Inhibitor Removal Technology®
IRT removes the most challenging inhibitors
from lysates prior to nucleic acid purification.
Comparison of
samples with and
without IRT:
Inhibitors cause false negatives in qPCR,
RT-qPCR and sequencing analysis.
Sample to Insight
No IRT
IRT Method
IRT MethodNo IRT
No IRT
IRT
No IRT
No IRT
IRT
IRT
Why contaminationremoval is important
Sample to Insight
Low levels of contaminating inhibitors can lead to false negatives / aberrant
amplification
IRT + + - -
+ IRT
- IRT
Samples 260/280 260/230
IRT 1.91 2.03
IRT 1.92 1.99
No IRT 1.87 1.84
No IRT 1.85 1.53
Sample to Insight
Projects using MO BIO Kits with Inhibitor Removal Technology®
Sample to Insight
Agenda
Introduction to the microbiome
Technologies for microbial community analysis
MO BIO’s sample prep solutions
Inhibitor Removal Technology® (IRT)
MO BIO’s microbiome product offerings
Questions
27
1
2
3
4
5
6
Sample to Insight
Agenda
Introduction to the microbiome
Technologies for microbial community analysis
MO BIO’s sample prep solutions
Inhibitor Removal Technology® (IRT)
MO BIO’s microbiome product offerings
Questions
28
1
2
3
4
5
6
Sample to Insight
DNeasy PowerSoil Kit (formerly PowerSoil DNA Isolation Kit)*
29
MO BIO’s most important offeringto the microbiome community
*Though developed for soil, this kit is often used for stool, swabs,
and a host of other sample matrices. Often used interchangeably
with the QIAamp PowerFecal Kit (formerly PowerFecal DNA
Isolation Kit).
Sample to Insight
In the literature:
Frossard, A. et al., (2015) Water regime history drives responses of soil Namib Desert microbial
communities to wetting events. Sci. Rep. 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 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
Sample to Insight
MagAttract PowerSoil DNA Kit (formerly PowerMag Soil DNA Isolation Kit)
• Uses the same chemistry as
PowerSoil/PowerFecal kits for lysis & IRT
• Magnetic bead purification of DNA enables
robotic automation of all steps post-IRT
Sample to Insight
In the literature:
Quinn, R.A. et al., (2016) From sample to multi-omics conclusions in under 48 hours.
mSystems, 1, 2.
Applied an integrated omics pipeline for
human and environmental samples & reported
full analysis of integrated data set w/in 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 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
Sample to Insight
RNeasy PowerMicrobiome Kit (formerly PowerMicrobiome RNA Isolation Kit )
Microbial RNA from (a) stabilized dog stool
and (b) dog stool transported on ice.
a b
Sample to Insight
In the literature:
Reck, M. et al., (2015) Stool metatranscriptomics: A technical guideline for mRNA stabilisation
and isolation. BMC Genomics 2015 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 unchanged for
up to 6 days at RT but a bias was clearly detected
(reduced abundance of Prevotellaceae transcripts &
depleted transcripts for COG category “Carbohydrate
transport & metabolism”
Conclusion: RNAProtect + PowerMicrobiome
yielded transcriptomes most similar to snap-frozen
controls
Sample to Insight
DNeasy PowerBiofilm Kit (formerly PowerBiofilm DNA Isolation Kit)
Sample to Insight
In the literature:
Abed, R.M. et al., (2014) Diversity, distribution and hydrocarbon biodegradation capabilities of
microbial communities in oil-contaminated cyanobacterial mats from a constructed wetland.
PLoS ONE 9(12)
Constructed wetland treatment systems are devised to exploit natural
processes for the cleanup of wastewaters.
“Producedwater” = water from underground formations brought to the
surface via wells during oil & gas production.
• Even after oil & gas separation, produced water is still contaminated w/residual
hydrocarbons.
• 50 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 are the major key players in
determining the spatial distribution of the wetland mats’ microbial
communities.
Mats contribute directly to the removal of hydrocarbons fromoil field
wastewaters..
Sample to Insight
Sample to Insight: MO BIO sample prep with QIAGEN’s microbial qPCR products
37
Microbiology: From identification to characterization
16S rRNA gene
- Conserved region - Variable region
Microbial qPCR assays and arrays for identification and profiling use probes and
primers against 16srRNA variable region.
Content: Largest microbiome portfolio; experimentally verified 580 assays
Custom: Select 8 to 384 microbial species for simultaneous detection / profiling
Control: Integrated controls ensure reliability of results
Sensitivity: Can detect as low as 10 copy numbers; data available
Sample to Insight
Overview of QIAGEN’s microbial qPCR products
38
Microbial DNA qPCR Array: Pre-printed assays profile up to 90 different species/genes
PCR plates (either 96-well or 384-well) are pre-printed with primers
and probes.
Each numbered well is a separate assay that tests the same sample.
Integrated control assays:
• Host assays detect genomic DNA to test sample collection
• Pan A/C is a pan- Aspergillus/Candida assay that detects
the presence of fungal rRNA
• PanB1 and PanB2 detect bacterial 16S rRNA to determine
bacterial load in the sample
• PPC is a positive PCR control reaction that tests if the PCR
reactions failed due to PCR inhibitors in the sample, etc.
Sample to Insight
Overview of QIAGEN’s microbial qPCR products
39
Layout of a Microbial DNA qPCR Array: Different arrays have different number of assays and
samples
Sample to Insight
Overview of QIAGEN’s microbial qPCR products
40
Microbial DNA qPCR Arrays and Assays
Profile or identify the presence of microbial DNA(from bacteria, fungi, virus, protist, antibiotic resistance
and virulence factors)
Identification experiment answers the following question:
Are any of these microbes or genes present in the sample?
• Must be compared against a known negative sample
• Run NTC as one sample
• Answers are Yes or No
Profiling experiment answers the following question:
Have the amounts of any of these microbes or genes changed?
• Must be compared against a reference sample
• Answers are fold change
Sample to Insight
Integration of MO BIO products into the Sample to Insight workflow
41
Sample to Insight : Inhibitor-depleted nucleic acids  Microbial qPCR Assays and Arrays
QIAsymphony/QIAcube/QIAcube HT QIAgility Rotor-Gene Q
DNA
Isolation
Assays
and Arrays
Data
Analysis
• DNeasy PowerSoil
• DNeasy PowerFecal
• RNeasy
PowerMicrobiome
• DNeasy PowerBiofilm
DNA
• RNeasy PowerBiofilm
• MagAttract PowerSoil
DNA
• DNeasy Microbial DNA
• Microbial DNA
qPCR Arrays
• Microbial DNA
qPCR Assay Kits
• Microbial DNA
qPCR Assays
• Microbial qPCR
Multi-Assay Kits
• Custom Microbial
DNA qPCR Arrays
• GeneGlobe Data
Analysis Center
Sample Insight
Sample to Insight
Overview of QIAGEN’s Microbial qPCR products
Microbial NGS (microbiome / pathogen): MO BIO + QIAGEN Tools
Sample
Disruption
Sample
Preparation
Library
construction
NGS run Data analysis
Validation by
PCR
• TissueLyserII;
TissueLyser LT;
TissueRuptor;
PowerLyzer 24;
Vortex adapters
• Pathogen Lysis
Tubes
• Power Bead
Tubes
Data Analysis Software
• CLC Bio
Genomics
workbench
• Microbial Genome
Finishing module
Predesigned & custom
arrays / assays
for verification and
focused microbiome
analyses
• Microbial DNA
qPCR Arrays
• Microbial DNA
qPCR Multi-Assay
Kits
• Microbial DNA
qPCR Assay Kits
• Microbial DNA
qPCR Assays
• QIAamp
PowerFecal
• DNeasy
o PowerSoil
o PowerWater
o PowerBiofilm
• RNeasy
PowerMicrobiome
• AllPrep
PowerFecal
• Repli-g Single Cell
Kit (Limited
primary sample
material)
• QIASeq FXDNA Library Kit
for high-complexity metagenome
libraries
• REPLI-g Single Cell Kit
for whole genome amplification
from unculturable organisms or
limited samples
• QIASeq 1-Step Amplicon Kit
the fastest solution for targeted
metagenomics libraries
• QIAGEN Multiplex PCR kits
for effective 16s, 18s or ITS
amplification with minimal PCR-
bias.
Sample to Insight
Want to stay up-to-date?
Visit www.mobio.com to see our complete kit
catalog, find Technical Support FAQs, and read
our microbiome blog with great guest writer
entries.
Stay up to date with developments in microbiome
news at the Microbiome Project
(http://news.microbiomeproject.com/).
All products are now on www.qiagen.com
@mobio
Follow us:
MO BIO Laboratories
Sample to Insight
Agenda
Introduction to the microbiome
Technologies for microbial community analysis
MO BIO’s sample prep solutions
Inhibitor Removal Technology® (IRT)
MO BIO’s microbiome product offerings
Questions
44
1
2
3
4
5
6
Sample to Insight
Agenda
Introduction to the microbiome
Technologies for microbial community analysis
MO BIO’s sample prep solutions
Inhibitor Removal Technology® (IRT)
MO BIO’s microbiome product offerings
Questions
45
1
2
3
4
5
6
Sample to Insight
Thank you for coming
Any questions?
Contact us
QIAGEN
Telephone: 888-503-3187
Webinar questions:
qiawebinars@QIAGEN.com
Technical support:
techservice-na@qiagen.com
Contact us
MO BIO Laboratories, Inc.
Toll-free in USA: 800-606-6246
Outside USA: 760-929-9911
46

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Sample Prep Solutions for Microbiome Research

  • 1. Sample to Insight Sample Prep Solutions for Microbiome Research Eddie Adams, Ph.D. MO BIO Laboratories, a QIAGEN Company Eddie.Adams@qiagen.com
  • 2. Sample to Insight Legal disclaimer 2 QIAGEN and MO BIO 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. MO BIO kit manuals are available at www.mobio.com or can be requested from MO BIO Technical Services.
  • 3. Sample to Insight Agenda Introduction to the microbiome Technologies for microbial community analysis MO BIO’s sample prep solutions Inhibitor Removal Technology® (IRT) MO BIO’s microbiome product offerings Questions 3 1 2 3 4 5 6
  • 4. Sample to Insight Agenda Introduction to the microbiome Technologies for microbial community analysis MO BIO’s sample prep solutions Inhibitor Removal Technology® (IRT) MO BIO’s microbiome product offerings Questions 4 1 2 3 4 5 6
  • 5. Sample to Insight Microbiome: Definition and background 5 What does “microbiome” mean? “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 (Lederberg and McCray 2001, The NIH HMP Working Group) Kuczynski et al. Nature Reviews Genetics 13,47-58 (January 2012)
  • 6. Sample to Insight Human Microbiome Project 6 Microorganisms cluster by body site Cataloguing efforts by the NIH Human Microbiome Project suggest: • ~10,000 organisms live with us • ~ 8 ×106 genes in this “second genome” Identifying microbiota in healthy individuals revealed: • Different body sites have unique communities • Race, age, gender, weight or ethnicity have an effect 1 Hoffmann A.R., et al. “The Microbiome: The Trillionsof MicroorganismsThat Maintain Healthand Cause Disease in Humansand Companion Animals.” Vet Pathol.2015 2 http://commonfund.nih.gov/hmp/index 3 Structure, functionand diversity of the health human microbiome. The HumanMicrobiome Project Consortium. Nature,486, 207 -214 (14 June 2012). doi: 10.1038/nature11234
  • 7. Sample to Insight Earth Microbiome Project 7 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 diversity1: • 160 distinct types of bacteria in 1 ml of ocean water • 6,400 – 38,000 types of bacteria in 1 gram of soil These are just estimates for bacteria alone; one still needs to consider viruses, archaea and fungi 1Curtis, T. P.; Sloan, W. T.; Scannell, J. W. (2002). "Estimating prokaryotic diversity and its limits". Proceedings of the National Academy of Sciences 99 (16): 10494–10499.
  • 8. Sample to Insight Agenda Introduction to the microbiome Technologies for microbial community analysis MO BIO’s sample prep solutions Inhibitor Removal Technology® (IRT) MO BIO’s microbiome product offerings Questions 8 1 2 3 4 5 6
  • 9. Sample to Insight Agenda Introduction to the microbiome Technologies for microbial community analysis MO BIO’s sample prep solutions Inhibitor Removal Technology® (IRT) MO BIO’s microbiome product offerings Questions 9 1 2 3 4 5 6
  • 10. Sample to Insight Molecular technologies for microbial communityanalysis 10 16S rRNA gene sequencing 18S rRNA gene sequencing Total DNA sequencing (shotgun) Bacteria and Archaea Fungus / Yeast Viruses Gene content RNA expression profiling (transcriptomics) Gene expression Metabolite, protein characterization Mass spectroscopy (metabolomics & proteomics) Identify relative frequencies and pathways Microbiome sample Extract DNA Extract RNA Extract proteins & small molecules What organisms are present and what is their relative abundance? What are the functions of the community?
  • 11. Sample to Insight Sample preparation requirements for successful microbiome studies There are several areas where sample prep inefficiencies can bias a microbiome/metagenomics study: • Insufficient homogenization of sample matrix (to dislodge/disrupt cell:substrate interactions) • Insufficient cell lysis ◦ Can bias downstreamanalysis toward ‘the easily disrupted’population(s) • Poor nucleic acid quality, extensive shearing • Insufficient inactivation of nucleases/proteases ◦ Unintended destructionof 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
  • 12. Sample to Insight Agenda Introduction to the microbiome Technologies for microbial community analysis MO BIO’s sample prep solutions Inhibitor Removal Technology® (IRT) MO BIO’s microbiome product offerings Questions 12 1 2 3 4 5 6
  • 13. Sample to Insight Agenda Introduction to the microbiome Technologies for microbial community analysis MO BIO’s sample prep solutions Inhibitor Removal Technology® (IRT) MO BIO’s microbiome product offerings Questions 13 1 2 3 4 5 6
  • 14. Sample to Insight About MO BIO Laboratories: • 22 year-old Carlsbad, CA molecular biology company – Privately owned until acquisition by QIAGEN in November 2015 • Current catalog of products will be manufactured in Carlsbad until end of 2016; QIAGEN (Hilden, Germany) will then manufacture all products from 2017 onward • Customers will still be able to order MO BIO products through current website (www.mobio.com) and products will be supported by current MO BIO Technical Support staff • ~50 employees • Products sold in 90+ countries MO BIO’s sample prep solutions
  • 15. Sample to Insight High Inhibitors Difficult Lysis MO BIO’s sample prep solutions Easy Difficult Lysis Inhibitors Low High Blood, animal tissue & cells Pure microbial cultures Soil microbesLeaf tissue Stool & gut microbes Biofilm FFPE Tissue Food cultures
  • 16. Sample to Insight MO BIO’s sample prep solutions Cell lysis with homogenous gene/community representation • Mechanical homogenization with tailored lysis buffer formulations (DNA and RNA, DNA or RNA only, 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
  • 17. Sample to Insight Bead-basedhomogenization 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) PowerLyzer24 High-speed homogenizer for hard-to-lyse samples (max 24 samples) TissueLyser II High-speed homogenizer for hard-to-lyse samples (up to 192 samples)
  • 18. Sample to Insight Sample-derived PCR/RT-PCR inhibitors In the process of breaking open cells to release nucleic acids, amplification inhibitors are also released. Inhibitors include humic/fulvic acids in soil and bile, bilirubin and heme in stool.* *Other examples for additional matrices given in Rådström, P. et al. (2004) Pre-PCR processing: Strategiesto generate PCR-compatible samples. Mol. Biotechnol. 26, 133–46.
  • 19. Sample to Insight Sample-derived PCR/RT-PCR inhibitors Sample-derived PCR inhibitors can act via several overlapping mechanisms • Sequestration of reaction components/enzyme co-factors ◦ Mg2+ chelation by sample-derived carboxylates • Competitive displacement of reaction co-factors ◦ Ca2+ displacement of Mg2+ • Direct interaction with template nucleic acids ◦ Competitive displacement of enzyme ◦ Steric occlusion of enzyme • Direct interaction with polymerase
  • 20. Sample to Insight Agenda Introduction to the microbiome Technologies for microbial community analysis MO BIO’s sample prep solutions Inhibitor Removal Technology® (IRT) MO BIO’s microbiome product offerings Questions 20 1 2 3 4 5 6
  • 21. Sample to Insight Agenda Introduction to the microbiome Technologies for microbial community analysis MO BIO’s sample prep solutions Inhibitor Removal Technology® (IRT) MO BIO’s microbiome product offerings Questions 21 1 2 3 4 5 6
  • 22. Sample to Insight Inhibitor Removal Technology® The inhibitor removal chemistry outlined in US 7,459,548 forms the basis for all MO BIO “Power” kits (e.g., PowerSoil, PowerMicrobiome).
  • 23. Sample to Insight Inhibitor Removal Technology® IRT removes the most challenging inhibitors from lysates prior to nucleic acid purification. Comparison of samples with and without IRT: Inhibitors cause false negatives in qPCR, RT-qPCR and sequencing analysis.
  • 24. Sample to Insight No IRT IRT Method IRT MethodNo IRT No IRT IRT No IRT No IRT IRT IRT Why contaminationremoval is important
  • 25. Sample to Insight Low levels of contaminating inhibitors can lead to false negatives / aberrant amplification IRT + + - - + IRT - IRT Samples 260/280 260/230 IRT 1.91 2.03 IRT 1.92 1.99 No IRT 1.87 1.84 No IRT 1.85 1.53
  • 26. Sample to Insight Projects using MO BIO Kits with Inhibitor Removal Technology®
  • 27. Sample to Insight Agenda Introduction to the microbiome Technologies for microbial community analysis MO BIO’s sample prep solutions Inhibitor Removal Technology® (IRT) MO BIO’s microbiome product offerings Questions 27 1 2 3 4 5 6
  • 28. Sample to Insight Agenda Introduction to the microbiome Technologies for microbial community analysis MO BIO’s sample prep solutions Inhibitor Removal Technology® (IRT) MO BIO’s microbiome product offerings Questions 28 1 2 3 4 5 6
  • 29. Sample to Insight DNeasy PowerSoil Kit (formerly PowerSoil DNA Isolation Kit)* 29 MO BIO’s most important offeringto the microbiome community *Though developed for soil, this kit is often used for stool, swabs, and a host of other sample matrices. Often used interchangeably with the QIAamp PowerFecal Kit (formerly PowerFecal DNA Isolation Kit).
  • 30. Sample to Insight In the literature: Frossard, A. et al., (2015) Water regime history drives responses of soil Namib Desert microbial communities to wetting events. Sci. Rep. 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 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
  • 31. Sample to Insight MagAttract PowerSoil DNA Kit (formerly PowerMag Soil DNA Isolation Kit) • Uses the same chemistry as PowerSoil/PowerFecal kits for lysis & IRT • Magnetic bead purification of DNA enables robotic automation of all steps post-IRT
  • 32. Sample to Insight In the literature: Quinn, R.A. et al., (2016) From sample to multi-omics conclusions in under 48 hours. mSystems, 1, 2. Applied an integrated omics pipeline for human and environmental samples & reported full analysis of integrated data set w/in 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 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
  • 33. Sample to Insight RNeasy PowerMicrobiome Kit (formerly PowerMicrobiome RNA Isolation Kit ) Microbial RNA from (a) stabilized dog stool and (b) dog stool transported on ice. a b
  • 34. Sample to Insight In the literature: Reck, M. et al., (2015) Stool metatranscriptomics: A technical guideline for mRNA stabilisation and isolation. BMC Genomics 2015 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 unchanged for up to 6 days at RT but a bias was clearly detected (reduced abundance of Prevotellaceae transcripts & depleted transcripts for COG category “Carbohydrate transport & metabolism” Conclusion: RNAProtect + PowerMicrobiome yielded transcriptomes most similar to snap-frozen controls
  • 35. Sample to Insight DNeasy PowerBiofilm Kit (formerly PowerBiofilm DNA Isolation Kit)
  • 36. Sample to Insight In the literature: Abed, R.M. et al., (2014) Diversity, distribution and hydrocarbon biodegradation capabilities of microbial communities in oil-contaminated cyanobacterial mats from a constructed wetland. PLoS ONE 9(12) Constructed wetland treatment systems are devised to exploit natural processes for the cleanup of wastewaters. “Producedwater” = water from underground formations brought to the surface via wells during oil & gas production. • Even after oil & gas separation, produced water is still contaminated w/residual hydrocarbons. • 50 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 are the major key players in determining the spatial distribution of the wetland mats’ microbial communities. Mats contribute directly to the removal of hydrocarbons fromoil field wastewaters..
  • 37. Sample to Insight Sample to Insight: MO BIO sample prep with QIAGEN’s microbial qPCR products 37 Microbiology: From identification to characterization 16S rRNA gene - Conserved region - Variable region Microbial qPCR assays and arrays for identification and profiling use probes and primers against 16srRNA variable region. Content: Largest microbiome portfolio; experimentally verified 580 assays Custom: Select 8 to 384 microbial species for simultaneous detection / profiling Control: Integrated controls ensure reliability of results Sensitivity: Can detect as low as 10 copy numbers; data available
  • 38. Sample to Insight Overview of QIAGEN’s microbial qPCR products 38 Microbial DNA qPCR Array: Pre-printed assays profile up to 90 different species/genes PCR plates (either 96-well or 384-well) are pre-printed with primers and probes. Each numbered well is a separate assay that tests the same sample. Integrated control assays: • Host assays detect genomic DNA to test sample collection • Pan A/C is a pan- Aspergillus/Candida assay that detects the presence of fungal rRNA • PanB1 and PanB2 detect bacterial 16S rRNA to determine bacterial load in the sample • PPC is a positive PCR control reaction that tests if the PCR reactions failed due to PCR inhibitors in the sample, etc.
  • 39. Sample to Insight Overview of QIAGEN’s microbial qPCR products 39 Layout of a Microbial DNA qPCR Array: Different arrays have different number of assays and samples
  • 40. Sample to Insight Overview of QIAGEN’s microbial qPCR products 40 Microbial DNA qPCR Arrays and Assays Profile or identify the presence of microbial DNA(from bacteria, fungi, virus, protist, antibiotic resistance and virulence factors) Identification experiment answers the following question: Are any of these microbes or genes present in the sample? • Must be compared against a known negative sample • Run NTC as one sample • Answers are Yes or No Profiling experiment answers the following question: Have the amounts of any of these microbes or genes changed? • Must be compared against a reference sample • Answers are fold change
  • 41. Sample to Insight Integration of MO BIO products into the Sample to Insight workflow 41 Sample to Insight : Inhibitor-depleted nucleic acids  Microbial qPCR Assays and Arrays QIAsymphony/QIAcube/QIAcube HT QIAgility Rotor-Gene Q DNA Isolation Assays and Arrays Data Analysis • DNeasy PowerSoil • DNeasy PowerFecal • RNeasy PowerMicrobiome • DNeasy PowerBiofilm DNA • RNeasy PowerBiofilm • MagAttract PowerSoil DNA • DNeasy Microbial DNA • Microbial DNA qPCR Arrays • Microbial DNA qPCR Assay Kits • Microbial DNA qPCR Assays • Microbial qPCR Multi-Assay Kits • Custom Microbial DNA qPCR Arrays • GeneGlobe Data Analysis Center Sample Insight
  • 42. Sample to Insight Overview of QIAGEN’s Microbial qPCR products Microbial NGS (microbiome / pathogen): MO BIO + QIAGEN Tools Sample Disruption Sample Preparation Library construction NGS run Data analysis Validation by PCR • TissueLyserII; TissueLyser LT; TissueRuptor; PowerLyzer 24; Vortex adapters • Pathogen Lysis Tubes • Power Bead Tubes Data Analysis Software • CLC Bio Genomics workbench • Microbial Genome Finishing module Predesigned & custom arrays / assays for verification and focused microbiome analyses • Microbial DNA qPCR Arrays • Microbial DNA qPCR Multi-Assay Kits • Microbial DNA qPCR Assay Kits • Microbial DNA qPCR Assays • QIAamp PowerFecal • DNeasy o PowerSoil o PowerWater o PowerBiofilm • RNeasy PowerMicrobiome • AllPrep PowerFecal • Repli-g Single Cell Kit (Limited primary sample material) • QIASeq FXDNA Library Kit for high-complexity metagenome libraries • REPLI-g Single Cell Kit for whole genome amplification from unculturable organisms or limited samples • QIASeq 1-Step Amplicon Kit the fastest solution for targeted metagenomics libraries • QIAGEN Multiplex PCR kits for effective 16s, 18s or ITS amplification with minimal PCR- bias.
  • 43. Sample to Insight Want to stay up-to-date? Visit www.mobio.com to see our complete kit catalog, find Technical Support FAQs, and read our microbiome blog with great guest writer entries. Stay up to date with developments in microbiome news at the Microbiome Project (http://news.microbiomeproject.com/). All products are now on www.qiagen.com @mobio Follow us: MO BIO Laboratories
  • 44. Sample to Insight Agenda Introduction to the microbiome Technologies for microbial community analysis MO BIO’s sample prep solutions Inhibitor Removal Technology® (IRT) MO BIO’s microbiome product offerings Questions 44 1 2 3 4 5 6
  • 45. Sample to Insight Agenda Introduction to the microbiome Technologies for microbial community analysis MO BIO’s sample prep solutions Inhibitor Removal Technology® (IRT) MO BIO’s microbiome product offerings Questions 45 1 2 3 4 5 6
  • 46. Sample to Insight Thank you for coming Any questions? Contact us QIAGEN Telephone: 888-503-3187 Webinar questions: qiawebinars@QIAGEN.com Technical support: techservice-na@qiagen.com Contact us MO BIO Laboratories, Inc. Toll-free in USA: 800-606-6246 Outside USA: 760-929-9911 46

Editor's Notes

  1. During cell lysis, which is the first step in DNA isolation, we use bead beating or mechanical lysis to break open microbes and release the DNA and this is the best way to lyse the most cells with the least amount of bias. In this process of bead beating, the inhibitors from the sample are released. In stool samples, this includes substances such as bile, bilirubin, and dead red blood cells as well as food as diet plays a major role in the content of the gut sample.
  2. And this is where the Inhibitor Removal Technology comes in and is used to remove the inhibitory compounds before isolation of the nucleic acids so that the purification results in high quality DNA and RNA. This is a patented process that is utilized in all of the “Power” kits by MO BIO. To give you an idea of how important it is to remove these impurities, here is an example of how inhibitors will impact the perceived DNA yields and cause problems.
  3. Here we are comparing the isolation of DNA from the same samples – prepared with and without IRT. The nanodrop data is what most people look at first and looking at this data, if we focus on yields only, when inhibitors are present it appears as if the samples prepared without inhbititor removal have higher DNA yields. If you look across at the purity readings, we see some problems- the combination of a low 260/230 ratio with a high 340 ratio tells us this co-contaminant is not just salt. It is compounds from the sample. If it were just salt, we’d see only a low 260/230 ratio. If we look at the plot data from the Nanodrop, we clearly see that the inhibitors are causing amplified absorbance across the entire range of wavelengths including the 260 reading. To confirm the nanodrop data we always run an agarose gel to check fro integrity and to make sure the yield data correlates with the gel picture. And clearly it does not. In fact the samples prepped with IRT have higher DNA yields than the samples without. We see some degraded RNA in these samples which will cause more 260 wavelength signal and a false DNA yield reading. Going into your next experiment, your input DNA is going to be inaccurate if you have DNA containing inhibitors. This will lead to inconsistences in the data going forward and problems with accuracy later when it comes time to analyze the results of qPCR or sequencing. So the take home message is that contamination removal is very important! This next slide demonstrates how inhibitors can impact qPCR.
  4. https://www.whitehouse.gov/the-press-office/2015/03/27/fact-sheet-obama-administration-releases-national-action-plan-combat-ant https://www.beefusa.org/newsreleases1.aspx?NewsID=4966 Antibiotic resistance is a huge problem globally. This just gives you a background about some issues that we are experiencing just in the US with antibiotics resistance. Just in 2015, it was estimated by the CDC (Center for Disease Control in the US) that antibiotic-resistance would cause 2 million people to be sick, and about 23,000 deaths per year. In March of 2015, The Obama Administration released an action plan that spans over 5 years to help combat the continued emergence of antibiotic resistant bacteria both domestically and internationally. They set a lot of different goals with this plan, such as reducing unnecessary antibiotic use in patients and eliminating the use of certain antibiotics in meat animals that are just for growth promotion, and developing a more robust monitoring system for antibiotic usage. There are also more goals they plan to develop internationally as well and you can look through this entire plan online. On June 2, 2015, The National Cattlemen’s Beef Association participated in the White House Forum on Antibiotic Stewardship, and they were able to highlight how this industry is contributing to the fight against antibiotic resistance, and their plan to eliminate the use of medically-important antibiotics to increase feed efficiency or growth promotion**, which they plan to implement by December 2016. NOTES: **” intent of enhancing growth (to make animals grow faster) or to improve feed efficiency (the animals need less food to gain weight).”