automated nucleic acid purification from diverse sample types using dedicated microbiome kits on the...
TRANSCRIPT
Sample to Insight
Automated nucleic acid purification from diverse sample types using dedicated microbiome kits on the QIAcube.
Patrick Smith, PhD
Scientist R&D QIAGEN GmbH
Sample to Insight
Overview
Introduction
◦ Microbiome
◦ Inhibitor Removal Technology® (IRT)
QIAcube
◦ Introduction to nucleic acid purification with the QIAcube
Semi-automated nucleic acid isolation
◦ DNA isolation using dedicated microbiome kits
◦ RNA isolation using dedicated microbiome kits
Sample to Insight workflow
◦ DNA isolation with QIAamp PowerFecal kit on the QIAcube
◦ NGS library preparation (QIASeq)
◦ Bioinformatics with CLC Microbial Genomics Workbench
2
1
2
3
4
Sample to Insight
Overview
Introduction
◦ Microbiome
◦ Inhibitor Removal Technology® (IRT)
QIAcube
◦ Introduction to nucleic acid purification with the QIAcube
Semi-automated nucleic acid isolation
◦ DNA isolation using dedicated microbiome kits
◦ RNA isolation using dedicated microbiome kits
Sample to Insight workflow
◦ DNA isolation with QIAamp PowerFecal kit on the QIAcube
◦ NGS library preparation (QIASeq)
◦ Bioinformatics with CLC Microbial Genomics Workbench
3
1
2
3
4
Sample to Insight
Microbiome: Definition and background
4
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
5
Microorganisms cluster by body siteCataloguing efforts by the NIH Human Microbiome Project suggest:
• ~10,000 organisms live with us
• Second genome: outnumber
our own genes 150:1
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 Trillions of Microorganisms That Maintain Health and Cause Disease in Humans and Companion Animals.” Vet Pathol. 20152 http://commonfund.nih.gov/hmp/index 3 Structure, function and diversity of the health human microbiome. The Human Microbiome Project Consortium. Nature, 486, 207-214 (14 June 2012). doi: 10.1038/nature11234
Sample to Insight
Earth Microbiome Project
6
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
About MO BIO Laboratories:
• 22 year-old Carlsbad, CA molecular biology company
– Privately owned until acquisition by QIAGEN in November 2015
– World leader in DNA and RNA isolation tools for microbiome analysis
• Products now fully integrated with QIAGEN
– 9 kits available on QIAcube
– Custom protocols available
• Products sold in 90+ countries
MO BIO’s sample prep solutions
Sample to Insight
Projects using MO BIO Kits with Inhibitor Removal Technology®
Sample to Insight
Molecular technologies for microbial community analysis
9
16S rRNA gene
sequencingTotal DNA sequencing
(shotgun)
Bacteria and
Archaea
Fungus
/ Yeast
Viruses Gene content
RNA expression profiling
(transcriptomics)
Gene expressionMetabolite, 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:
• Co-purification of small molecule inhibitors of amplification reactions (e.g., PCR)
Decreases efficiency of amplification or can inhibit library prep reactions
• Insufficient cell lysis
Bias downstream analysis toward ‘the easily disrupted’ population(s)
• Reproducible isolation of high quality nucleic acids
• Poor nucleic acid quality, extensive shearing
• 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
• Insufficient homogenization of sample matrix (to dislodge/disrupt cell:substrate
interactions)
Sample to Insight
High Inhibitors
Difficult Lysis
MO BIO’s sample prep solutions
Easy DifficultLysis
Inh
ibit
ors
Low
High
Blood, animal
tissue & cells
Pure microbial
cultures
Soil microbesLeaf
tissue
Stool & gut
microbes
Biofilm
FFPE
Tissue
Food cultures
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.
*Other examples for additional matrices given in Rådström, P. et al. (2004) Pre-PCR processing: Strategies to
generate PCR-compatible samples. Mol. Biotechnol. 26, 133–46.
Inhibitors include humic/fulvic acids in soil,
polysaccharides/polyphenolics in plants
and bile, bilirubin and heme in stool*
Sample to Insight
No IRT
IRT Method
IRT MethodNo IRT
No IRT
IRT
No IRT
No IRT
IRT
IRT
Why contamination removal 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
Low levels of inhibitors can also inhibit other enzymatic reactions commonly found in WGS library prep kits
Sample to Insight
Overview
Introduction
◦ Microbiome
◦ Inhibitor Removal Technology® (IRT)
QIAcube
◦ Introduction to nucleic acid purification with the QIAcube
Semi-automated nucleic acid isolation
◦ DNA isolation using dedicated microbiome kits
◦ RNA isolation using dedicated microbiome kits
Sample to Insight workflow
◦ DNA isolation with QIAamp PowerFecal kit on the QIAcube
◦ NGS library preparation (QIASeq)
◦ Bioinformatics with CLC Microbial Genomics Workbench
15
1
2
3
4
Sample to Insight
QIAcube: An in-depth look
QIAcube in-depth
16
Sample to Insight
QIAcube – Technical overview
17
Sample to Insight
QIAcube: Purification
QIAcube in NGS, Hilden, November 2015 18
Sample NGS run InsightSample prep
on QIAcube
▪ Low throughput (12 samples per run) automated nucleic acid purification
▪ DNA, RNA and protein purification
▪ No change from manual spin-column procedure
▪ Benchtop, plug and play
▪ Elimination of manual processing steps
▪ Standardized protocols ensure control
▪ 130 standardised protocols
▪ Customisable protocols also available
Sample to Insight
QIAcube: Purification setup
QIAcube in NGS, Hilden, November 2015 19
Load the sample Load the buffers Load the adaptors
Checking number and types of tips Checking after tip uptake
Verifying loading of centrifuge Checking for sufficient buffer volumesChecking samples in loaded shaker
Sample to Insight
QIAcube - Applications
20
Genomic DNA from human samples
QIAamp DNA Blood Mini Kit
QIAamp DNA Mini Kit
QIAamp DNA Stool Mini Kit
QIAamp DNA Micro Kit
QIAamp MinElute Media Kit
QIAamp DSP DNA Blood Mini kit [IVD]
QIAamp DSP DNA Mini kit [IVD]
QIAamp Circulating NA
PAXgene Tissue DNA Kit
QIAamp Fast DNA Stool Mini Kit
Forensic samples
QIAamp DNA Investigator Kit
Differential Wash protocol (custom.)
Viral DNA and RNA purification
QIAamp MinElute Virus Spin Kit
QIAamp Viral RNA Mini Kit
QIAamp DSP MinElute Virus Spin Kit
[IVD]
QIAamp DSP Virus Mini kit [IVD]
QIAamp DSP Viral RNA Mini kit [IVD]
QIAamp cador Pathogen Mini
FFPE samples
RNeasy Plus Universal Kit
RNeasy FFPE Kit
QIAamp DNA FFPE Kit
AllPrep DNA/RNA FFPE
miRNeasy FFPE
Plant or food samples
DNeasy Plant Mini Kit
RNeasy Plant Mini Kit
DNeasy mericon Food Kit
Bisulfite conversion and cleanup
EpiTect Bisulfite Kit
EpiTect Plus DNA Bisulfite Kit
EpiTect Fast DNA Bisulfite
Protein purification and serum depletion
Ni-NTA Spin Kit
Qproteome Albumin/IgG Depletion Kit
Qproteome Murine Albumin Depletion
Kit
Qproteome Total Glycoprotein Kit
Qproteome O-Glycan Glycoprotein Kit
Genomic DNA from animal samples
DNeasy Blood & Tissue Kit
Plasmid DNA purification
QIAprep Spin Miniprep Kits
DNA cleanup
QIAquick PCR Purification Kit
QIAquick Gel Extraction Kit
QIAquick Nucleotide Removal Kit
MinElute PCR Purification Kit
MinElute Reaction Cleanup Kit
MinElute Gel Extraction Kit
NGS specific Kits
GeneRead rRNA Depletion Kit
GeneRead Size Selection
GeneRead DNA Library I Core Kit
GeneRead DNA Library L Core Kit
Multiple analyte purification
AllPrep DNA/RNA Kits
AllPrep DNA/RNA FFPE Kit
AllPrep DNA/RNA Micro Kit
AllPrep DNA RNA miRNA Universal Kit
RNA purification from a range of
samples
RNeasy Mini Kit
RNeasy MinElute Cleanup Kit
RNeasy Plus Mini Kit
RNeasy Micro Kit
RNeasy Plus Micro Kit
RNeasy Lipid Tissue Mini Kit
RNeasy Fibrous Tissue Kit
RNeasy Protect Bacteria Mini Kit
RNeasy Protect Animal Blood Kit
miRNeasy Protect Animal Blood Kit
miRNeasy Serum/Plasma Kit
RNeasy Protect Animal Blood Kit
PAXgene Blood RNA Kit [IVD]
PAXgene Blood miRNA Kit
QIAamp RNA Blood Mini Kit
RNeasy Plus Universal Kit
RNeasy Microarray Tissue
Microbiome
MOBIO kits now available
on the QIAcube!
Sample to Insight
Overview
Introduction
◦ Microbiome
◦ Inhibitor Removal Technology® (IRT)
QIAcube
◦ Introduction to nucleic acid purification with the QIAcube
Semi-automated nucleic acid isolation
◦ DNA isolation using dedicated microbiome kits
◦ RNA isolation using dedicated microbiome kits
Sample to Insight workflow
◦ DNA isolation with QIAamp PowerFecal kit on the QIAcube
◦ NGS library preparation (QIASeq)
◦ Bioinformatics with CLC Microbial Genomics Workbench
21
1
2
3
4
Sample to Insight
22
• All in one protocol for sample preparation and removal of inhibitors
• Sample material:
• Inherently rich in inhibitors: Mammalian stool (human, mouse, cow, horse), bird
stool, environmental samples (water, soil, biofilm, plant)
• Downstream application:
• Whole metagenome shotgun sequencing
• 16S rRNA gene sequencing
• qPCR, PCR genotyping
Microbial
Cell Lysis
Inhibitor
Removal
Microbial
DNA
Isolation
AnalysisSample
Collection
Nucleic Acid Isolation with Dedicated Microbiome Kits
Mechanical lysis Patented Inhibitor
Removal Technology
QIAamp PowerFecal Kit
Sample to Insight
Nucleic Acid Isolation with Dedicated Microbiome Kits
23
• Following mechanical lysis the supernatant is loaded directly into the QIAcube
• QIAcube performs all inhibitor removal, DNA binding, wash and elution steps
• Hands free high quality DNA is isolated in ~45-60 min, which can be used
immediately in downstream applications
Microbial
Cell Lysis
Inhibitor
Removal
Microbial
DNA
Isolation
AnalysisSample
Collection
Performed Manually Performed by QIAcube
Sample to Insight
Environmental Research
24
Soil
Distinct, diverse microbial species populate the soil of temperate, desert, tropical and arctic
regions
Soil biodiversity is influenced pH and the chemical composition of soil organic matter within the
soil environment
Isolation of nucleic acids from soil is complicated by the large amounts of humic acids
contained within
DNeasy PowerSoil Kit
Sample to Insight
DNeasy PowerSoil Kit
High quality DNA was isolated from a variety of samples including landfill, compost
and horse manure using the DNeasy PowerSoil Kit compared with two other
suppliers. All isolation were performed following the manufacturer’s protocols.
High yield, intact genomic DNA
Sample to Insight
DNeasy PowerSoil Kit
Complete removal of inhibitors allows for more accurate PCR and qPCR analyses
and reduces false negatives. PCR was performed using total bacterial primers on
1 ul of undiluted DNA.
More accurate PCR analysis
Sample to Insight
DNeasy PowerSoil Kit – DNA Yield
manual extraction
QIAcube extraction
A B
C D
Estuary Compost
manual extraction
QIAcube extraction
manual extraction
QIAcube extraction
A B
C D
Estuary Compost
manual extraction
QIAcube extraction
• 250mg of either Estuary or compost soil
was used as input material
• There were no differences in yield or
quality between manual and QIAcube
protocol
• Purified DNA had high 260/280 and
260/230 ratios
• Average 260/280 = 1.8
• Average 260/230 = 1.7
Sample to Insight
DNeasy PowerSoil Kit – PCR Inhibition
• PCR inhibition was measured using
QIAGEN QuantiFast Pathogen PCR + IC
Kit
• 5-10ul of DNA eluate was added to each
rxn
• If inhibitors are present change in Ct value
is typically between 3 and 15
• DNA is ready for immediate use in
downstream applications
0,00
5,00
10,00
15,00
20,00
25,00
30,00
35,00
40,00
Ct
valu
e
Estuary
manual extraction QIAcube estraction
05
10152025303540
Ct
va
lue
Compost
Sample to Insight
Environmental Research
29
Water
Distinct, diverse microbial species populate the water of Oceans, Lakes, Rivers, Marshes and
arctic regions
Water contains large amounts of humic acids, heavy metals, chemical and biological waste
QIAcube automation of the DNeasy PowerWater Kit
Carlsbad Lagoon, Carlsbad, Ca
Pacific Ocean, Encinitas, Ca
Sample to Insight
DNeasy PowerWater Kit – DNA Yield
• 75ml of lagoon water or 150ml Ocean
water as input material
• There were no differences in yield or
quality between manual and QIAcube
protocol
• Purified DNA had high 260/280 and
260/230 ratios
• Average 260/280 = 1.8
• Average 260/230 = 1.5
0
0,5
1
1,5
2
2,5
3
3,5D
NA
yie
ld (
ug)
Lagoon water
manualprotocol
QIAcubeprotocol
0
0,02
0,04
0,06
0,08
0,1
0,12
0,14
0,16
0,18
Ocean water
DN
A y
ield
(u
g)
Sample to Insight
DNeasy PowerWater Kit – PCR Inhibition
• PCR inhibition was measured using
QIAGEN QuantiFast Pathogen PCR + IC
Kit
• 5-10ul of DNA eluate was added to each
rxn
• If inhibitors are present change in Ct value
is typically between 3 and 15
• DNA is ready for immediate use in
downstream applications
Manual extraction QIAcube extraction
0
5
10
15
20
25
30
35
40
1 2 3 4 5 6 control
Ct
va
lue
Lagoon Water
Manual
QIAcube
Sample to Insight
Environmental Research
32
Biofilm
Biofilms are diverse mixtures of microbes that can be very difficult to lyse
Microbial Mats, Stream/Lagoon Rocks, Gypsum Crust, Sink Pipe
Lysis difficulty is due to the formation of extracellular polymeric substances
DNA, Proteins and polysaccharides
QIAcube automation of the DNeasy PowerBiofilm Kit
Sample to Insight
DNeasy PowerBiofilm Kit – DNA Yield
• 0.25mg of concrete Biofilm
• There were no differences in yield or
quality between manual and QIAcube
protocol
• Purified DNA had high 260/280 and
260/230 ratios
• Average 260/280 = 1.9
• Average 260/230 = 1.9
0
1
2
3
4
5
6
Concrete Biofilm
DN
A Y
ield
(u
g)
ManualProtocol
QIAcubeProtocol
Manual Extraction QIAcube Extraction
Sample to Insight
DNeasy PowerBiofilm Kit – PCR Inhibition
• No Inhibition in either the manual or QIAcube protocols
Manual extraction QIAcube extraction
0
5
10
15
20
25
30
35
40
1 2 3 4 5 Control
Ct
Val
ue
Concrete Biofilm
Manual Protocol
QIAcube Protocol
Sample to Insight
Biological Research
35
RNA from human and animal stool
Can be difficult to lyse without introducing microbial biases
Can contain high amount of inhibitors, heme, bile acids, polysaccharides
Intact high quality RNA can be difficult to obtain due to the amount of RNases and other
degrading enzymes present in stool
QIAcube automation of the RNeasy PowerMicrobiome Kit
Sample to Insight
RNeasy PowerMicrobiome Kit – DNA Yield
• 200mg of human stool as input
• Isolated a high yield of high quality RNA
• There were no differences in yield or quality between manual and QIAcube
protocol
0
5
10
15
20
25
30
35
40
45
50
Human Stool
RN
A Y
ield
(u
g)
0
0,5
1
1,5
2
2,5
3
Human Stool
26
0/2
30
nm
Ra
tio
0
0,5
1
1,5
2
2,5
Human Stool
26
0/2
80
nm
Ra
tio
Manual Protocol QIAcube Protocol
Sample to Insight
RNeasy PowerMicrobiome Kit– PCR Inhibition
• Comparable Inhibition in either the manual or QIAcube protocols
Manual extraction QIAcube extraction
0
5
10
15
20
25
30
35
40
45
1 2 3 4 5 6 No_IRT Control
Ct
Val
ue
Manual Protocol
QIAcubeProtocol
Sample to Insight
Overview
Introduction
◦ Microbiome
◦ Inhibitor Removal Technology® (IRT)
QIAcube
◦ Introduction to nucleic acid purification with the QIAcube
Semi-automated nucleic acid isolation
◦ DNA isolation using dedicated microbiome kits
◦ RNA isolation using dedicated microbiome kits
Sample to Insight workflow
◦ DNA isolation with QIAamp PowerFecal kit on the QIAcube
◦ NGS library preparation (QIASeq)
◦ Bioinformatics with CLC Microbial Genomics Workbench
38
1
2
3
4
Sample to Insight
Semi-automated DNA Isolation With QIAamp PowerFecal Kit
39
Performed Manually Performed by QIAcube
Microbial
Cell Lysis
Inhibitor
Removal
Microbial
DNA
Isolation
Library
Prep
Sample
Collection
• Human Stool, N = 10
• Aged samples:
female (77), female
(78), female (74),
male (77), male (65)
• Young samples:
female (28), female
(49), male (28), male
(28), male (37)
• Additional Metadata
– race, diet
• Use samples directly
for 16s and whole
genome sequencing
applications
• Analyze data with
CLC Microbial
Genomics Pro Suite
Sample to Insight
Semi-automated DNA Isolation With QIAamp PowerFecal Kit
40
0
5
10
15
Old Young
DN
A (
ug
)
Isolation of high quality DNA
• 200mg human stool lysed in PowerBead tubes for
10 minutes using a vortex adapter
• Supernatant was then placed in QIAcube for DNA
isolation
• Agarose gel electrophoresis shows isolation of
high molecular weight genomic DNA with minimal
degradation
• For each sample, several μg of DNA was isolated
as measured by a flourescence based assay
Sample to Insight
16S rRNA gene library prep
41
Methods for microbiome analyses:
16s rRNA gene sequencing for community composition
Workflow:
Mixed sample DNA isolation —
host and microbes16S rDNA amplification
Ashelford et al., Appl. Environ. Microbiol. 71 7724-7736 (December 2005)
Community composition
Diversity within and
between samples
Amplicon
SequencingData
Analysis
Sample to Insight
16S rRNA gene library prep
42
• 16S PCR amplicons – 22 cycles
Young SamplesOld Samples Blanks
-500
-300
-1000
bp
Inhibitor removal from DNA allows efficient amplification/yield of V4 16S region
Sample to Insight
16S rRNA gene library prep
43
• 50 ng inhibitor free DNA in 16s rDNA PCR
• Modified 515f PCR primer and 806r PCR primer
• Addition of adapters with indices using QIAseq 1-step Amplicon kit
• 2x250 bp on Illumina MiSeq
• 500 ng amplicon as input, all libraries > 10 nM final concentration
Sample to Insight
Whole Genome Sequencing library prep
44
Purified
gDNA(1ng
-1μg)
Single
Tube FX
Rxn
(60min)
Purification
sequencin
g
Adapter
ligation
(45 min)
• Accepts wide
range of input
DNA
• Easily
customizable
fragment sizes
• Adapter ligation in
a single step, with
dual indices
• Fully compatible
with Illumina
sequencing
Sample to Insight
16S and WGS Sequencing Statistics
45
• 16s - 2 x 250 bp with Illumina MiSeq
◦ Generated > 22 million paired end reads
◦ ~ 90% reads passed quality filter, %>Q30 85
◦ ~ 88% of reads were assigned to an index
◦ Of reads identified, 9 of 10 samples were between 9-11% of total reads
• WGS – 2 x 250bp with illumina MiSeq
◦ Generated > 27 million paired end reads
◦ 97% reads passed quality filter, %>Q30 88
◦ 88% of reads were assigned to a dual index
◦ Of reads identified, 9 of 10 samples were between 8-10% of total reads
• DNA isolation through library prep and sequencing worked great!
Sample to Insight
QIAGEN Microbial Genomics Pro Suite
46
From Data to Discovery
• Integrated analytics deliver research continuity
◦ All analytics for microbial genomics and metagenomics comes fully integrated
◦ Fully Scalable with data and sample-metadata management included
• Focus on what matters
◦ Streamlined workflow allows users to focus on interpretation of results
• High performance algorithms
◦ Designed to save time and compute resources
◦ Accessible to bioinformatics experts and non-bioinformaticians alike
Sample to Insight
QIAGEN Microbial Genomics Pro Suite
47
Powered by Quality Components
• Microbial genmoics Pro Suite Expands upon CLC
genomic Workbench
• Plugins and modules add a layer of specialization
◦ Download CLC microbial genomics module,
CLC genom finishing module,
MetaGeneMarkPlugin
• Upload data and with customizable workflows data
is just a few clicks away
• Easy to use Graphical User Interface
• Quick and helpful customer service
Sample to Insight
QIAGEN Microbial Genomics Pro Suite
48
16S and WGS workflows
Sample to Insight
QIAGEN Microbial Genomics Pro Suite
49
16S OTU and taxonomic assignment workflow
• Includes quality filtering
• For OTU picking, sequences were mapped
against the greengenes data base and clustered
at 97% identity
• Unmapped sequences were then clustered de
novo
• Results shown are summarized at the genus
level
• Highly customizable outputs
• Differential Abundance analysis (not shown)
Sample to Insight
QIAGEN Microbial Genomics Pro Suite
50
Alpha and Beta Diversity Workflow
• Young individuals have a higher alpha diversity
compared with old as measured by observed
OTUs
• Other metrics include, Shannon and Simpson
Diversity, phylogenetic diversity
• Highly customizable output
• Beta diversity: unweighted UniFrac Analysis
• Young and old gut communities are significantly
different. P = 0.047 Permanova analysis,
corrected for multiple comparisons
• Other metrics include weighted UniFrac,
gneralized UniFrac, Bray-Curtis, Euclidean
• Highly customizable outputs
Sample to Insight
QIAGEN Microbial Genomics Pro Suite
51
WGS read mapping and Taxonomic assignment workflow
• Includes quality filtering and read merging
• The taxonomic profiler maps each read
against the chosen database
• Output is an abundance table including
graphical representation
• Highly customizable output
• Graph shown at left is summarized at the
genus level
• CLC Microbial Genomics Pro Suite can also
alpha and beta diversity analyses (not shown)
Sample to Insight
QIAGEN Microbial Genomics Pro Suite
52
WGS De novo assembly and Functional assignment workflow
• Includes quality filtering
• De novo assembly of reads, identification and
annotation of genes and coding sequences
• Perform functional and statistical analyses
• Highly customizable output
• Samples are clustered by functional similary
using Euclidean distances
• CLC Microbial Genomics Pro Suite can also
perform taxonomic assignment (not shown)
Sample to Insight
QIAGEN Microbial Genomics Pro Suite
53
WGS De novo assembly and Functional assignment workflow
Name
Max group
mean
Log₂ fold
change Fold change P-value FDR p-value
0008452 // RNA ligase activity 151.2 -1.64 -3.12 0.000216912 0.27
0071804 // cellular potassium ion transport 47 5.04 32.84 0.000765205 0.27
0071805 // potassium ion transmembrane transport 47 5.04 32.84 0.000799437 0.27
0016407 // acetyltransferase activity 1229.8 -0.38 -1.30 0.001037548 0.27
0016410 // N-acyltransferase activity 1039 -0.43 -1.35 0.001044564 0.27
0046353 // aminoglycoside 3-N-acetyltransferase activity 22.6 -7.56 -188.37 0.001054322 0.27
0034069 // aminoglycoside N-acetyltransferase activity 22.6 -7.56 -188.37 0.001060815 0.27
0008080 // N-acetyltransferase activity 1039 -0.43 -1.35 0.001068129 0.27
0008234 // cysteine-type peptidase activity 615.2 0.57 1.48 0.00138698 0.28
0006276 // plasmid maintenance 145.2 3.68 12.85 0.001412554 0.28
0010393 // galacturonan metabolic process 16.8 -7.10 -137.19 0.002238434 0.33
0016837 // carbon-oxygen lyase activity, acting on polysaccharides 16.8 -7.10 -137.19 0.002247484 0.33
0045490 // pectin catabolic process 16.8 -7.10 -137.19 0.002280259 0.33
0045488 // pectin metabolic process 16.8 -7.10 -137.19 0.002313312 0.33
0047487 // oligogalacturonide lyase activity 15.8 -7.02 -129.63 0.002642413 0.35
0006787 // porphyrin-containing compound catabolic process 15.2 -6.93 -121.66 0.003588363 0.38
0046149 // pigment catabolic process 15.2 -6.93 -121.66 0.003699807 0.38
0033015 // tetrapyrrole catabolic process 15.2 -6.93 -121.66 0.00381645 0.38
0015994 // chlorophyll metabolic process 15.2 -6.93 -121.66 0.003925426 0.38
0047746 // chlorophyllase activity 15.2 -6.93 -121.66 0.003935538 0.38
0015996 // chlorophyll catabolic process 15.2 -6.93 -121.66 0.003991415 0.38
0033926 // glycopeptide alpha-N-acetylgalactosaminidase activity 10.2 -7.30 -157.15 0.00434989 0.39
0036211 // protein modification process 1168.6 -0.43 -1.34 0.004493368 0.39
0006464 // cellular protein modification process 1168.6 -0.43 -1.34 0.004604395 0.39
0019439 // aromatic compound catabolic process 891.6 -0.51 -1.42 0.006839066 0.53
1901361 // organic cyclic compound catabolic process 890.6 -0.50 -1.42 0.006882415 0.53
0046700 // heterocycle catabolic process 890.6 -0.50 -1.42 0.007291632 0.54
0044270 // cellular nitrogen compound catabolic process 898.2 -0.50 -1.42 0.007732012 0.54
0009236 // cobalamin biosynthetic process 1097.4 -0.35 -1.28 0.007825894 0.54
Differential Abundance Analysis
Sample to Insight
Semi-automated low-throughput workflow for microbial analyses
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• QIAcube isolated DNA hands-free in ~1hr (cat no. 9001882)
• Sample Prep kits available on the QIAcube
◦ DNeasy PowerSoil
◦ DNeasy PowerLyzer PowerSoil
◦ QIAamp PowerFecal kit (cat no. 12830-50)
◦ DNeasy PowerWater
◦ DNeasy PowerPlant Pro
◦ DNeasy PowerClean Pro
◦ DNeasy PowerBiofilm
◦ DNeasy Ultraclean Microbial
◦ RNeasy PowerMicrobiome
• Easy, fast high quality DNA library prep
◦ QIAseq 1-step amplicon kit (16S) (cat no. 180412/180415)
◦ QIAseq FX DNA library kit (WGS) (cat no. 180473)
• QIAGEN Microbial Genomics Pro Suite provides user-friendly, highly accurate microbial
analysis
◦ 16S
◦ WGS
Sample to Insight
Questions
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