This slidedeck presents an easy-to-use workflow that allows selective isolation of microbial DNA from samples that are intrinsically rich in host DNA. This protocol includes steps for efficient depletion of host DNA while providing optimized conditions specific for bacterial lysis. This workflow is also specific for the identification of live bacteria, avoiding false results due to nucleic acids from dead bacteria. Enriched microbial DNA can be directly used in other molecular methods such as whole genome sequencing, qPCR and microarray assays.
Microbiome Isolation and DNA Enrichment Protocol: Pathogen Detection Webinar Series Part 2
1. Sample to Insight
Microbiome: Isolate and enrich microbial DNA with our new protocol
isolate and enrich microbial DNA 1
Marco Polidori, Ph.D.
Global Product Manager
2. Sample to Insight
Welcome!
2
Pathogen detection: Microbial DNA isolation
and identification to characterization
• Part 1: Host-pathogen interactions: Molecular basis and host defense
mechanisms
• Part 2: Microbiome: Isolate and enrich microbial DNAwith our new
protocol
• Part 3: Microbiome: From identification to characterization
isolate and enrich microbial DNA
3. Sample to Insight
Legal disclaimer
3
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.
isolate and enrich microbial DNA
4. Sample to Insight
Agenda
4isolate and enrich microbial DNA
Introduction
• Host-microbe interactions
• Microbiome definition and background
• Technologies to analyze microbial communities
Data: QIAamp® DNAMicrobiome protocol
• Depletion of host DNA
• 16S rRNA gene sequencing data of the human oral
microbiome
• Maintenance of community composition
Summary
• Improved microbiome sequencing through host DNA
removal
Questions
1
2
3
4
5. Sample to Insight
Agenda
5isolate and enrich microbial DNA
Introduction
• Host-microbe interactions
• Microbiome definition and background
• Technologies to analyze microbial communities
Data: QIAamp® DNAMicrobiome protocol
• Depletion of host DNA
• 16S rRNA gene sequencing data of the human oral
microbiome
• Maintenance of community composition
Summary
• Improved microbiome sequencing through host DNA
removal
Questions
1
2
3
4
6. Sample to Insight
Microbiome: Definition and background
6isolate and enrich microbial DNA
“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)
What does “microbiome” mean?
Kuczynski et al. Nature Reviews Genetics 13,47-58 (January 2012)
7. Sample to Insight
Microbiome: Host-pathogen interaction
7isolate and enrich microbial DNA
Microbiota and the host peacefully coexist to achieve a mutually beneficial
relationship
When the mutualistic relationship is disrupted, the microbiota can cause or
contribute to diseases
Nature.com
Hybiske & Stephens Nature Reviews Microbiology 6, 99-110 (February 2008)
Host-pathogen interaction
Interaction of microbial
communities and impact on human
and animal health
8. Sample to Insight
Microbiome: Definition and background
8isolate and enrich microbial DNA
Consider this:
• We have about 10 times as many microbial cells as human cells
• 20% to 60% of all species may be uncultivable (The NIH HMP
Working Group)
• Each area of the body has its own microbiome (gut, mouth, skin etc.)
• Microbiomes can be linked to many diseases (cancer, infections,
metabolic disease)
Hallmark microbiome features
9. Sample to Insight
Microbiome: Definition and background
9isolate and enrich microbial DNA
• Gut
• Intestinal infections
• Obesity
• Inflammatory Bowel Disease
• Airway
• Pneumonia and other respiratory infections
• Chronic Obstructive Pulmonary Disease
• Cystic Fibrosis
• Urogenital
• Bacterial vaginosis
• Urinary Tract Infections
• Sexually Transmitted Disease
• Blood
• Sepsis/bloodstreaminfections
• Oral
• Periodontitis
• Gingivitis
• Neuronal
• Anxiety
• Depression
Association of the human microbiota and disease
http://commonfund.nih.gov/hmp/index
10. Sample to Insight
Technologies to analyze microbial communities
10isolate and enrich microbial DNA
• Gene cloning (Pan 16S rRNA) and sanger
sequencing
• Microarray
• MALDI
• Next generation sequencing
• 16S rRNA sequencing
• Whole genome sequencing
• qPCR - Target dependent
• 16S rRNA gene
• Other relevant gene (antibiotic
resistance gene, virulence factor gene)
11. Sample to Insight
Technologies to analyze microbial communities
11isolate and enrich microbial DNA
Next generation sequencing
16S rDNA sequencing
• What species are present?
• At what ratio?
• How do they react to changes?
• Correlation with health/disease status?
Whole metagenome shotgun sequencing
• Detailed analysis of unculturable microbes
• Novel species/strains
• Identify virulence factors (disease-related genes)
• Functional analysis of bacterial community/metabolic networks
qPCR - Target dependent
• 16S rRNA gene
• Other relevant gene (antibiotic resistance gene, virulence factor gene)
12. Sample to Insight
Technologies to analyze microbial communities
12isolate and enrich microbial DNA
• Methods for microbiome analyses:
• 16s rRNA gene sequencing for community composition
• Workflow:
Mixed sample DNA isolation —
host and microbes
16S rDNA amplification
Amplicon sequencing
Community composition
Diversity within and
in between samples
Ashelford et al., Appl. Environ. Microbiol.71 7724-7736 (December 2005)
13. Sample to Insight
Technologies to analyze microbial communities
13
• Methods for microbiome analyses:
• Whole metagenome shotgun sequencing for additional information regarding function
• Workflow:
Comparison to reference
databases and/or genomes
Mixed sample DNA isolation —
host and microbes
Library preparation
from fragmented
DNA
Whole genome
sequencing
Community composition
Genome assemblyPresence of metabolic
pathways and
functions
De novo
assembly
isolate and enrich microbial DNA
14. Sample to Insight
Technologies to analyze microbial communities
14
Learnings from the HMP
• Human Microbiome Project:
• Up to 99 % human reads depending on
sample material
• Cost intensive waste of sequencing
capacity
• Additional filtering to obtain microbial
data sets
• Lower sequencing depth for microbial
DNA of interest
Human
QC filtered
Usable
Percentage%
A framework for human microbiome research,
Nature. 2012 June 13; 486(7402): 215–221.(7402): 215–221.
isolate and enrich microbial DNA
15. Sample to Insight
Technologies to analyze microbial communities
15
DNA extraction can introduce community composition bias
Willner et al. PLOS One, 2012; DOI: 10.1371/journal.pone.0034605
A bias in community composition may be introduced
by sample preparation depending on:
• Cell wall morphology (e.g., Gram,
Actinomycetes, EPS)
• Lysis methods used (e.g., mechanical vs.
enzymatic)
• Sample type (solid, viscous, host cell rich)
isolate and enrich microbial DNA
16. Sample to Insight
Agenda
16isolate and enrich microbial DNA
Introduction
• Host-microbe interactions
• Microbiome definition and background
• Technologies to analyze microbial communities
Data: QIAamp® DNAMicrobiome protocol
• Depletion of host DNA
• 16S rRNA gene sequencing data of the human oral
microbiome
• Maintenance of community composition
Summary
• Improved microbiome sequencing through host DNA
removal
Questions
1
2
3
4
17. Sample to Insight
Human DNA depletion: QIAamp DNA Microbiome
17
Overview sample preparation for whole metagenome shotgun sequencing:
• Mechanical dispruption
• Chemical lysis
• Mild lysis conditions to
keep bacteria intact
• QIAamp protocol
• All in one protocol for sample preparation and depletion of host DNA in about 180
minutes
• Sample material:
• Inherently rich in host DNA: buccal swabs, saliva, BAL, tissue, sputum, vaginal
and nasopharyngeal swabs etc.
• Downstream application:
• Whole metagenome shotgun sequencing
• 16S rRNA gene sequencing
• qPCR
isolate and enrich microbial DNA
Host cell
lysis
Degradation
of host cell
nucleic
acids
Microbial
cell lysis
Isolation of
microbial
DNA
Library
preparation
18. Sample to Insight
Human DNA depletion: Data
18
Quantification of human DNAdepletion
• Reduction of human reads from over 90% to under 5%
• At least 20x enrichment of sequences derived from the oral flora
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Lib 1 AHL Lib 2 QIAamp Lib 3 Molzym
Percentreads
Successful depletion of host DNA with the QIAamp DNA
Microbiome protocol
Human Oral
Microbiome
human
QIAamp DNA
Microbiome
No host DNA
depletion Kit 1
Host DNA
depletion Kit 2
• Pooled buccal swab samples
• GeneRead Library Preparation
• 2x151 bp Illumina®
MiSeq ®
• FastQC for quality control
• Mapping to hg19 with Bowtie 2
isolate and enrich microbial DNA
19. Sample to Insight
Human DNA depletion: Data
19
Retainment of community composition
Species Gram %GC Size [Mb] CFU persample STDEV
Bacillus subtilis + 43.5 4.22 1.08E+05 2.16E+03
Corynebacterium glutamicum + 53.8 3.31 1.32E+05 5,72E+03
Escherichia coli - 50.8 4.56 1.24E+05 5,79E+03
Micrococcus luteus + 73.0 2.5 7.11E+04 9,05E+03
Rhodococcus erythropolis + 62.3-62.5 6.4-6.9 1.86E+05 1,65E+04
Xanthomonas campestris - 65.1 5.08 9.64E+04 4,44E+03
Whole Metagenome Shotgun Sequencing:
• Quantify depletion of host DNA
• Determine maintenance of bacterial community composition
Sample Preparation:
• Samples: buccal swabs with 7.2 x 105 colony forming units of mock community per sample
• Comparison to alternative protocols
isolate and enrich microbial DNA
Buccal
swab
swirled in
PBS
Addition of
mixed
community
Isolation of
genomic
DNA
QIAGEN
GeneRead
Library Prep
2x151 bp
Illumina
MiSeq
Count
CFUs for
each
species
20. Sample to Insight
20
Sequencing results
QIAamp Microbiome Kit 1 Kit 2 Kit 3
B. subtilis 4.66E+04 1.38E+04 3.61E+04 1.15E+04
C. glutamicum 2.28E+05 8.01E+03 2.34E+03 3.94E+03
E. coli 2.05E+05 1.50E+04 8.10E+03 1.38E+04
M.luteus 6.02E+04 3.45E+04 1.37E+05 3.88E+04
R.erythropolis 3.76E+05 1.61E+04 3,63E+03 8.66E+03
X.campestris 1.14E+05 1.10E+04 8,56E+03 1.22E+04
unmapped 8.34E+06 4.98E+05 6,12E+05 2.97E+05
Human hg19 4.18E+05 1.07E+07 6,44E+06 6.77E+06
total reads 9.79E+06 1.13E+07 7.24E+06 7.15E+06
total reads on bacterial
references
1.03E+06 9.84E+04 1.95E+05 8.89E+04
Data Analysis (about 35.5 Mio reads):
• Quality control (FastQC)
• Mapping against reference genomes (Bowtie 2)
• Normalization to genome size for community composition data
• Ongoing Blast search for reads not mapping to reference genomes (oral microbiome)
Human DNA depletion: Data
isolate and enrich microbial DNA
21. Sample to Insight
Human DNA depletion: Data
21
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Lib4-QIAamp DNA
Microbiome
Lib8-MoBio
percentreads
Host DNA depletion allows deeper analysis of microbial
community
X. campestris
R.erythropolis
M. luteus
E. coli
C. glutamicum
B. subilits
Human Oral Microbiome
Human
QIAamp DNA
Microbiome
No host DNA
depletion
Kit 3
Quantification of human DNAdepletion
Depletion of host DNA provides valuable data for in depth analysis of the oral
microbiome.
Further
analyzed
by 16S
isolate and enrich microbial DNA
22. Sample to Insight
16S rRNA gene sequencing — data
22
16s rRNA gene sequencing
Caporaso et al., The ISME Journal (2012) 6, 1621–1624
• Buccal swab samples
• 10 ng DNA in 16S rDNA PCR
• Modified 515f PCR Primer and 806r PCR primer
• 2x151 bp on Illumina MiSeq
• Mapping with Bowtie2 to reference data from RDP database
Fwd Primer
5‘
Rev Primer
Adapter Adapter
V4
BC
Read 1
Index Read
Read 2
3‘
BC 5‘3‘
About 350 bp
isolate and enrich microbial DNA
23. Sample to Insight
16S rRNA gene sequencing: Data
23
16s rRNA gene sequencing
Caporaso et al., The ISME Journal (2012) 6, 1621–1624
Depletion of human DNA improves amplification/yield of V4 16S region
• PCR amplicons with modified 515f and 806r PCR primer
• 25 cycles
-750
-500
-250
-1000
bp
QIAamp DNA
Microbiome
QIAamp DNA
Microbiome
Kit 3 Kit 1 Kit 2
Sample 2Sample 1
isolate and enrich microbial DNA
24. Sample to Insight
16S rRNA gene sequencing: Data
24
0% 20% 40% 60% 80% 100%
Sample 1
Sample 2
Detection of bacterial families corresponding to the human oral microbiome
Cardiobacteriaceae
Coriobacteriaceae
Bacteroidaceae
Clostridiales Family XIII. Incertae Sedis
Bacillaceae
Erysipelotrichaceae
Peptostreptococcaceae
Flavobacteriaceae
Campylobacteraceae
Eubacteriaceae
Lachnospiraceae
Enterococcaceae
Prevotellaceae
Fusobacteriaceae
Porphyromonadaceae
Leptotrichiaceae
Actinomycetaceae
Carnobacteriaceae
Micrococcaceae
Neisseriaceae
Streptococcaceae
Veillonellaceae
Pasteurellaceae
Corynebacteriaceae
DNA from buccal swabs isolated with the QIAamp DNA Microbiome protocol
V4 Sequencing confirms representatives of the oral microbiome
isolate and enrich microbial DNA
25. Sample to Insight
Maintenance of community composition — data
25
Comparisonmapped reads to colony forming units in input material
0%
20%
40%
60%
80%
100%
120%
Cfus in input
sample
QIAamp
Microbiome
Q M PS
readsmappedtoreferencegenome
Community composition with QIAamp DNAMicrobiome protocol
X. campestris
R. erythropolis
M. luteus
E. coli
C. glutamicum
B. subtilis
Best correlation for community composition with QIAamp DNA Microbiome protocol
No host DNA
depletion
Kit 3
Host DNA
depletion
Kit 2
No host DNA
depletion
Kit 1
QIAamp DNA
Microbiome
CFUs per
sample
Improved microbiome sequencing through host DNA removalisolate and enrich microbial DNA
26. Sample to Insight
Summary: QIAamp DNA Microbiome main points
26
Improved microbiome sequencing through hostDNAremoval
• Perfect for samples that contain human or animal cells (like
swabs, body fluids)
• Depletes >95% host DNA
• Enables whole genome sequencing
• High sensitivity in 16S analysis
• Thorough lysis protocols minimize bias
• Contains QIAamp UCP columns
isolate and enrich microbial DNA
27. Sample to Insight
QIAamp DNA Microbiome Kit specifics
27
Features
Catalogue number: 51704
Time to extract: ca. 3h total
Number of reactions: 50
Material included:
QIAamp UCP columns, Pathogen Lysis Tubes, buffers, enzymes,
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.comor can be
requested from QIAGEN TechnicalServices or your local distributor.
Trademarks: QIAamp®
, GeneRead™ (QIAGEN Group); Illumina®
, MiSeq®
(Illumina, Inc.).
isolate and enrich microbial DNA
28. Sample to Insight
Thank you for coming
isolate and enrich microbial DNA
Contact QIAGEN Technical Service
Call: 1-800-426-8157 for US
Call: +49 2103-29-12400 for EU
Email:
techservice-na@qiagen.com
techservice-eu@qiagen.com
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