Semi Automated Low-throughput Workflow for Microbial Analyses of Human Stool

Sample to Insight
Semi-automated low-throughput workflow for microbial analyses of
human stool
Patrick Smith PhD, R&D Scientist, QIAGEN GmbH, Patrick.Smith@qiagen.com

Sample to Insight
Overview
Introduction
◦ Microbiome
◦ Inhibitor Removal Technology (IRT)
Semi-automated DNA purification
◦ QIAamp® PowerFecal® Kit
◦ QIAcube® automation
Library preparation
◦ QIAseq™ 1-Step Amplicon Library Kit (16S)
◦ QIAseq FX DNA Library Kit (WGS)
Bioinformatics – CLC Genomics Workbench
◦ CLC Microbial Genomics Module
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1
2
3
4

Sample to Insight
Overview
Introduction
◦ Microbiome
QIAamp PowerFecal Kit
QIAcube automation
Library preparation
QIAseq 1-Step Amplicon Library Kit (16S)
QIAseq FX DNA Library Kit (WGS)
CLC Microbial Genomics Module
3
1
2
3
4

Sample to Insight
The human super-organism
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Cataloging efforts by the NIH Human Microbiome Project (1) suggest:
• ~10,000 organisms live with us
• Bacterial genes outnumber our own 150:1
Identifying microbiota in healthy individuals revealed:
• Different body sites have unique communities
• Race, age, gender, weight or ethnicity have an effect
1. The Human Microbiome Project Consortium. (2012) Nature. 486, 207.

Sample to Insight
Association of human microbiota and disease
If the host/microbiota relationship is disrupted, gut microbiota can contribute to disease
• 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 diseases
• Blood
◦ Sepsis/bloodstream infections
• Oral
◦ Periodontitis
◦ Gingivitis
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Sample to Insight
Molecular technologies for microbial community analysis
6
16S 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 and
proteomics)
Identify relative frequencies and pathways
Microbiome sample
Extract DNA Extract RNA Extract proteins and
small molecules
What organisms are present and
what is their relative abundance?
What are the functions of the community?

Sample to Insight
Sample preparation for successful microbiome studies
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
◦ Biases downstream analysis toward ‘the easily disrupted’ population(s)
• Reproducible purification 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)
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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 complex polysaccharides, bile,
bilirubin and heme in stool*
Other examples for additional matrices given in Rådström, P. et al. (2004) Mol. Biotechnol. 26, 133.
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Sample to Insight
Effect of contaminating inhibitors
Low levels of contaminating inhibitors can lead to false negatives/aberrant amplification
IRT + + − −
+ IRT
−IRT
Low levels of inhibitors can also inhibit other enzymatic reactions commonly found in WGS
library prep kits
Samples A260/A280 A260/A230
IRT 1.91 2.03
IRT 1.92 1.99
No IRT 1.87 1.84
No IRT 1.85 1.53
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Sample to Insight
Overview
Introduction
◦ Microbiome
◦ QIAamp PowerFecal Kit
◦ QIAcube automation
Library preparation
◦ QIAseq 1-Step Amplicon Library Kit (16S)
10
2
1
3
4

Sample to Insight
Microbial
DNA
isolation
Sample
collection
DNA purification using the QIAamp PowerFecal Kit
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Microbial
cell lysis
Inhibitor
removal
Library
prep
• Mechanical lysis • Patented Inhibitor
Removal
Technology
• QIAamp
PowerFecal Kit
• All-in-one protocol for sample preparation and removal of inhibitors
• Sample material inherently rich in inhibitors: human stool, mammalian stool (mouse,
cow, horse), bird stool, environmental samples (DNeasy® PowerSoil® Kit)
• Downstream applications:
◦ Whole metagenome shotgun sequencing
◦ 16S rRNA gene sequencing
◦ qPCR

Sample to Insight
QIAcube: Purification
QIAcube in NGS, Hilden, November 2015 12
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 standardized protocols
• Customizable protocols also available

Sample to Insight
Microbial
DNA
isolation
Sample
collection
DNA purification using the QIAamp PowerFecal Kit
13
Microbial
cell lysis
Inhibitor
removal
Library
prep
Performed
manually
Performed by QIAcube
• Following mechanical lysis the supernatant is loaded directly into the QIAcube
• QIAcube performs all inhibitor removal, DNA binding, wash and elution steps
• High-quality DNA is purified (hands-free) in ~45–60 min and can be used
immediately in downstream applications

Sample to Insight
Semi-automated DNA purification: QIAamp PowerFecal Kit (1)
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• 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 collected: Ethnicity, diet
Microbial
DNA
isolation
Sample
collection
Microbial
cell lysis
Inhibitor
removal
Library
prep
Performed
manually
Performed by QIAcube

Sample to Insight
15
Purification of high-quality DNA
• 200 mg human stool lysed in PowerBead tubes
for 10 minutes using a vortex adapter
• Supernatant was then placed in QIAcube for DNA
purification
• Agarose gel electrophoresis shows purification of
high-molecular-weight genomic DNA with minimal
degradation
• For each sample, several micrograms of DNA
was purified, as measured by a fluorescence-
based assay
0
5
10
15
Old Young
DNA(µg)

Sample to Insight
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Purification of high-quality DNA
• Purified DNA had high A260/A280 and A260/A230
ratios:
◦ Average A260/A280 = 1.8
◦ Average A260/A230 = 1.7
• PCR inhibition was measured using QIAGEN
QuantiFast® Pathogen PCR + IC Kit
• 5 µl DNA eluate was added to each reaction
• If inhibitors are present, change in CT value is
typically between 3 and 15 CTs
• DNA is ready for immediate use in downstream
applications

Sample to Insight
Overview
Introduction
◦ Microbiome
Library preparation
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1
4
3
2

Sample to Insight
Whole genome sequencing library prep
Methods for microbiome analyses
16s rRNA gene sequencing for community composition
Amplicon
sequencing
Data
analysis
Mixed sample DNA purification −
host and microbes
16S rDNA
amplification
Community composition
Diversity within and
between samples
Ashelford, K.E., et al. (2005). Appl. Environ. Microbiol. 71, 7724.
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Sample to Insight
16S rRNA gene library prep (1)
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• 50 ng inhibitor-free DNA in 16s rDNA PCR
• Modified 515f PCR primer and 806r PCR primer
• Addition of adapters with indices using the QIAseq 1-Step Amplicon Kit
• 2 x 250 bp on Illumina® MiSeq®
Forward primer
5'
Reverse primer
Adapter Adapter
V4
BC 3'
BC 5'3'
About 350 bp
Caporaso, J. G., et al. (2012). ISME J. 6, 1621.

Sample to Insight
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• 16S PCR amplicons – 22 cycles
• Inhibitor removal from DNA allows efficient amplification/yield of V4 16S region
Young samplesOld samples Blanks
-500
-300
-1000
bp

Sample to Insight
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• Addition of adaptors with indices using QIAseq 1-Step Amplicon Kit
• 500 ng amplicon as input, all libraries >10 nM final concentration

Sample to Insight
Overview
Introduction
◦ Microbiome
Library preparation
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1
4
3
2

Sample to Insight
Whole genome sequencing library prep (1)
Pure
gDNA
(1 ng−
1 μg)
Single-
tube FX
reaction
(60 min)
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
Fragment peak size 250 bp 350 bp 450 bp 550 bp
Fragmentation time (min) at 32°C
10 ng input DNA 24 16 14 10
100 ng input DNA 16 10 8 6
1000 ng input DNA 14 8 6 4
Guideline for choosing the initial fragmentation time
Purification
sequencing
23

Sample to Insight
Whole genome sequencing library prep (2)
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Purified librariesStandards
500-
300-
1000-
bp
• 500 ng input DNA – target fragment size = 350 bp
• Agarose gel shows amplicons from qPCR assay
using the QIAseq Library Quant Assay Kit
• qPCR primers bind to adapters
Pure
gDNA
(1 ng−
1 μg)
Single-
tube FX
reaction
(60 min)
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
Purification
sequencing

Sample to Insight
16S and WGS sequencing statistics
25
• 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 and 11% of total reads
• WGS: 2 x 250b p 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 and 10% of total reads
• DNA preparation through library prep and sequencing worked well!

Sample to Insight
Overview
Introduction
◦ Microbiome
Library preparation
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1
2
3
4

Sample to Insight
QIAGEN Microbial Genomics Pro Suite (1)
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From data to discovery
• Integrated analytics deliver research continuity
◦ All analytics for microbial genomics and
metagenomics 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
28
Powered by quality components
• Microbial Genomics Pro Suite expands
upon the CLC Genomics Workbench
• Plug-ins and modules add a layer of
specialization:
◦ CLC Genome Finishing Module
◦ MetaGeneMark plug-in
• Upload data and using customizable
workflows, data is just a few clicks away
• Easy-to-use graphic user interface
• Quick and helpful customer service

Sample to Insight
• 16S: Greengenes/SILVA
• WGS: GO Pfam
Add
database
16S and WGS workflows
Data
import
and QC
Meta-
genome
assembly
Meta-
genome
analysis
OTU
clustering
Alpha,
beta
diversity
• Trim and quality filter raw reads
• Merge paired-end reads
• De novo assembly
• Identify gene and CDS
• Annotate CDS
• Build functional profile (GO)
• Statistical analysis
• Visualize data
• Open and closed OTU picking
• OTU table filtering
• Tree building
• Phylogenic diversity
• UniFrac distances
• Statistical analyses
29

Sample to Insight
30
16S OTU and taxonomic assignment workflow
• Includes quality filtering
• For OTU picking, sequences were
mapped against the greengenes
database and clustered at 97%
identity
• Unmapped sequences were then
clustered de novo
• Results shown are summarized at
the genus level
• Highly customizable outputs

Sample to Insight
31
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,
generalized UniFrac, Bray-Curtis, Euclidean
• Highly customizable outputs

Sample to Insight
32
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
• The graph shows a summary at the
genus level
• CLC Microbial Genomics Pro Suite
can also display alpha and beta
diversity analyses (not shown)

Sample to Insight
33
WGS de novo assembly and functional assignment workflow
• Includes quality filtering
• De novo assembly of reads,
identification and annotation of genes
and coding sequences
• Performs functional and statistical
analyses
• Samples are clustered by functional
similarity using Euclidean distances
• CLC Microbial Genomics Pro Suite can
also perform taxonomic assignment
(not shown)

Sample to Insight
34
WGS de novo assembly and functional assignment workflow – differential abundance analysis
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

Sample to Insight
Semi-automated low-throughput workflow for microbial analyses
35
Summary
• Semi-automated purification of high-quality, inhibitor-free DNA
◦ QIAamp PowerFecal kit (cat no. 12830-50)
◦ DNA purified, hands-free, on QIAcube in ~1 hour (cat. no. 9001882)
◦ DNA can be used immediately in downstream applications
• 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
36
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.
Trademarks: QIAGEN®, Sample to Insight®, QIAamp®, QIAcube®, QIAseq™, DNeasy®, Inhibitor Removal Technology®,
PowerFecal®, PowerSoil®, QuantiFast® (QIAGEN Group); Illumina®, HiSeq®, MiSeq® (Illumina, Inc). Registered names,
trademarks, etc. used in this document, even when not specifically marked as such, are not to be considered
unprotected by law. 05/2017 PROM-10910-002

Semi Automated Low-throughput Workflow for Microbial Analyses of Human Stool

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