Liquid biopsies enable us to monitor the evolution of genetic aberrations in primary tumors as they shed the tumor cells into the circulation. The limitation is the ability to detect these low frequency genetic aberrations in a consistent manner to understand short- and long-term implications and how this information will be used in the clinic. This slidedeck will cover the challenges and solutions associated with multiple steps as one starts with liquid biopsy and move towards finding a new biomarker.

1. Sample to Insight
- 1 -
Welcome!
Wei Cao, PhD
Wei.Cao@QIAGEN.com
Step by Step: From a Liquid Biopsy to a Genomic Biomarker
Contact Technical Support:
BRCsupport@QIAGEN.COM
1-800-362-7737
Webinar-related questions:
QIAwebinars@QIAGEN.com
Krishnan Allampallam, PhD
krishnan.allampallam@qiagen.com
1

2. Sample to Insight
- 2 -
Welcome to our four-part webinar series on liquid biopsies
Liquid biopsies: current progress, challenges and solutions
Step by step: from a liquid biopsy to a genomic biomarker
Circulating biomarkers: new solutions for DNA and RNA
Role of long non-Coding RNAs in cancer and microRNA regulation
Liquid biopsy technology:
overview, progress, challenges and new solutions
2
1
3
4
2

3. Sample to Insight
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.
Legal disclaimer
3

4. Sample to Insight
4
Liquid biopsy overview
Genotyping steps and challenges
Biomarkers and next-generation sequencing
Biomarkers and real-time PCR
1
2
3
4
Agenda

5. Sample to Insight
Liquid biopsy
Liquid biopsy:
• Is a minimally invasive technology to detect disease biomarkers particularly in
clinical cancer research and diagnostics
• Provides sensitive, specific and rapid sample purification, amplification and
detection technologies for:
• circulating tumor DNA (ctDNA)
• RNA from exosomes (exoRNA)
• NA from circulating tumor cells (CTCs)
5

6. Sample to Insight
Liquid biopsy: application areas
6
LS / translational research
• Currently, a strong focus on cancer related topics
• Neurodegenerativediseases
• Cardio vascular
• Transplant (early warning system)
Routine Dx
• Non-invasive pre-natal testing (NIPT)
• Cancer therapy monitoring

7. Sample to Insight
Tissue biopsy – traditional approach
Liquid biopsy: a game changer
Liquid Biopsy 2014 7
Liquid biopsies could save valuable time and costs in healthcare
Symptoms
First
analysis CT scan Biopsy
Histology
test
Molecular
test
Therapy
decision
Treatment
monitoring
Symptoms
Liquid
biopsy
Molecular
test
Therapy
decision
Treatment
monitoring
Liquid biopsy – new approach
• Answers multiple questions about a disease
• Much faster than traditional biopsy testing
• Can be more economical than traditional biopsy testing
• Is minimally invasive and less harmful
• Detects tumors from distant / multiple sites

8. Sample to Insight
Analytes in liquid biopsies
Analytes display different biological contents
CTC Circulating Cell-free NA
Content
HMW DNA
Total RNA
Protein
dsDNA (histones)
miRNA (RISC/Ago)
Exosomal content
Concentration
Very rare*
(<10 cp / Mio white blood cells
in metastatic patients,
1 CTC / 10 ml blood,
0.00001% CTC/WBC)
>50-fold increase over CTCǂ
(1.0-40.0 GE / 10 ml blood,
0.1 – 1 % ctDNA/ccfDNA)
Release process Unclear
Passive process
Apoptosis?
Focus for analysis
DNA and RNA
Tumor heterogeneity
Subclonal populations
Methods: broad spectrum
Detecting and counting genetic and
epigenetic aberrations
NIPT: trisomy
Methods: NGS, other
Clinical utility Monitoring therapies Therapy-monitoring diagnosis (NIPT)
*CTCs are undetectable in up to 35% of patients w ith metastatic disease
ǂRelative abundance of ctDNA/ccfDNA up to 87% in patients dying from cancer; for NIPT~
8

9. Sample to Insight
9
Liquid biopsy overview
Genotyping steps and challenges
Biomarkers and next-generation sequencing
Biomarkers and real-time PCR
2
3
4
Agenda
1

10. Sample to Insight
Liquid Biopsy to genomic biomarker ̶ steps
Steps in genotyping:
ccfNA
PAXgene,
ccfDNA tubes
.
QIAamp CAN,
miRNEasy,
QIAsymphony
GeneRead Gene
Panels,
qBiomarker SOM
Cancer workbench,
IVA
CTC REPLI-g
GeneRead Gene
Panels,
qBiomarker SOM
Cancer workbench,
IVA
QIAamp CNA Kit REPLI-g Kits
Pre-
amplification
Sample
collection
and
stabilization
Sample
enrichment
Sample
preparation
Assays
Data analysis
and
interpretation
10

11. Sample to Insight
Challenges in genotyping
Title, Location,Date 11
• Sample quantity and quality:
• Limited biopsy specimens
• Degraded nucleic acids – collection, storage, process
• Purity (genetic heterogeneity):
• Cancerous cells may be a minor fraction of total sample
• Multiple sub-clones of cancer cells may be present in a single
tumor sample
• Genomic alterations in cancer are found at low-frequency
• Data analysis:
• Biologically-interpretable data
• Fragmented workflow

12. Sample to Insight
• Sample collection and stabilization
• PAXgene Blood DNA Tubes
• Circulating nucleic acid from human plasma or serum
• QIAamp Circulating Nucleic Acid Kit
• Blood, tissues and cells
• DNeasy Blood & Tissue Kit
• Blood & Cell Culture DNA Kit (mini, midi, maxi)
• Quantification and qualification of amplifiable DNA prior to NGS
• GeneRead DNAQuantiMIZE Kits
• Whole genome amplification from single cell or limited sample
• REPLI-g WTASingle Cell Kit
- 12 -
Liquid biopsy to genomic biomarker ̶ steps
12
Pre-
amplification
Sample
collection
and
stabilization
Sample
enrichment
Sample
preparation
Assays
Data analysis
and
interpretation

13. Sample to Insight
REPLI-g technology
13
QIAGEN’s REPLI-g Single Cell technology:
For fast• and accurate genomic analysis of CTCs and single cells
DNA can be successfully amplified from just a single cell•
Problem
• Incomplete or biased genome amplification with missing or
underestimated sequence information
Solution
• REPLI-g Single Cell Kit
• Optimized multiple displacement amplification (MDA) process,
an innovative lysis process and an optimized form of Phi 29
DNA polymerase
Results
• High yield of high-molecular-weight DNA
• High fidelity amplification – minimal error rate
• Amplification of both DNA and RNA from a single sample for
direct analysis with high accuracy and minimal amplification bias

14. Sample to Insight
REPLI-g technology
14
Easy-to-use single-cell whole genome amplification (WGA) method:
• Offers complete genome coverage
• Minimizes sequence bias, allowing for discovery of cell heterogeneity
• Consistently yields of up to 40 μg of DNA from 1–1000 cells (average product length >10 kb)
Learn about the technology: http://www.qiagen.com/us/resources/technologies/wga
Read a white paper: “Genomic analysis of individual cells by NGS and real-time PCR”
15 min
Whole genome amplification workflow
The REPLI-g WGA Single Cell Kit:
For• whole genome amplification (WGA) from samples as small as a
single cell
The REPLI-g WTA Single Cell Kit:
For• transcriptome amplification (WTA) from samples as small as a
single cell
The REPLI-g Cell WGA & WTA Kit:
Enables• uniform WGA and WTA in parallel from a single sample
Starts• with the lysis of 25–1000 cells and yields up to 40 µg of gDNA
and cDNA, which can be used for comparative genome and
transcriptome analysis via NGS, qPCR or microarray analysis.
(Yield: 20-40 µg)

15. Sample to Insight
REPLI-g technology
15
Easy-to-use single-cell WGAor WTAmethod:
Offers• complete genome coverage
Minimizes• sequence bias, allowing for discovery of cell heterogeneity
Consistently• yields of up to 40 μg of DNA from 1–1000 cells (average product length >10 kb)
15 min
Whole genome amplification workflow
The REPLI-g WGA Single Cell Kit:
• For whole genome amplification (WGA) from samples as small as a
single cell
The REPLI-g WTA Single Cell Kit:
• For transcriptome amplification (WTA) from samples as small as a
single cell
The REPLI-g Cell WGA & WTA Kit:
• Enables uniform WGA and WTA in parallel from a single sample
• Starts with the lysis of 25–1000 cells and yields up to 40 µg of gDNA
and cDNA, which can be used for comparative genome and
transcriptome analysis via NGS, qPCR or microarray analysis.
(Yield: 20-40 µg)

16. Sample to Insight
REPLI-g technology
16
Unbiased amplification of DNA and RNA!
Complete genome coverage Maximized genome coverage
Analysis• of 267 loci spread out over different
chromosomes across the entire human
genome
Performed• using RT2
qPCR Primer Assays
(QIAGEN)
Low• and consistent CT values with no dropouts
from any marker, indicating successful DNA
amplification from all areas of the genome
Maximized transcript coverage
• Comparative genome and transcriptome analysis
after parallel WGA and WTA from the same
limited cell sample
• An RT2
Profiler PCR Array was used to analyze
over 60 genes and their corresponding transcripts
• Links genomes to corresponding gene expression
profiles and their phenotypes

17. Sample to Insight
17
Liquid biopsy overview
Genotyping steps and challenges
Biomarkers and next-generation sequencing
Biomarkers and real-time PCR
2
3
4
Agenda
1

18. Sample to Insight
Genotyping
Genotyping
aCGH
FISH
NGS, Sanger, Pyro
TAm-Seq
qPCR
SOM
CNV, ARMS, digital
Mass Spectrometry
18

19. Sample to Insight
Why NGS?
19
High throughput•
Sequence many genes simultaneously•
Assay several samples at the same time•
Cost effective•
Drastically decrease cost of sequencing•
Systematic and unbiased•
Detect several types of mutations•
Quantitative•
Easily quantify mutation frequency•

20. Sample to Insight
Which sequencing level do you need?
Choose the sequencing level that is appropriate for your application:
*30 genes
†
95 GB sequencing capacity
‡
7.5 GB sequencing capacity
VUSs – variants of uncertain significance
Attribute
Whole
genome
sequencing
Whole exome
sequencing
Targeted DNA
sequencing
Benefits of
targeted DNA
sequencing
Information
level
3 x 109 bps 5 x 107 bps 6 x 104 bps*
More relevant
data (VUSs)
Cost per
sample
$5000 $2000 $200
More cost-
effective
Coverage
achieved
30x 100x 1000x
Increased
confidence in
sequencing
results
DNA input 1 μg 0.5 – 1 μg 10 ng
Less DNA
required
No. of
samples
multiplexed
1†
2‡
96‡
Higher
multiplexing
capabilities
20

21. Sample to Insight
Steps in NGS from “Sample to Insight”
21
Sample
isolation
Targeted
enrichment
Library
construction
NGS run
Data
analysis
Biological
insightSample Insight
Sample QC
GeneRead DNA
QuantiMIZE System: rescue
low-quality DNA samples
Library QC
GeneRead DNAseq Library
Quant Kit: ensure equal library
loading for all samples
Variant confirmation
qBiomarker Somatic
Mutation Assays
Challenges
Isolating high
quality DNA
Amount of DNA,
Long turnaround time,
Panel contents,
Panel performance
Number of libraries
per sample
Platform
dependence
Massive amounts
of data require
expertise to yield
biological insight
Solutions
REPLI-g
Single Cell Kit
Repli-g Cell
WGA / WTA
Kit
GeneRead DNAseq
Targeted Panels V2
QIAamp DNA kits
GeneRead Size
Selection Kit
Compatible
with major
sequencing
platforms
GeneRead data
analysis portal
(free)
CLCbio Cancer
Genomics
Workbench
Ingenuity Variant
Analysis

22. Sample to Insight
Hybridization Hybridization, Ligation + PCR MultiplexPCR
Which is most suitable target enrichment technology?
22

23. Sample to Insight
23
Multiplex PCR technology
Hybridization
Hybridization,
ligation + PCR
Multiplex PCR
Workflow
1. Library construction
(4 hours)
2. Hybridization
with probes
(24-48 Hours)
3. PCR and indexing
(2 hours)
TOTAL = 30-54 hours
1. Hybridization
with probes
(16 hours)
2. Ligation
(1 hour)
3. PCR and indexing
(2 hours)
TOTAL = 19 hours
1. PCR amplification
(3 hours)
2. Library
construction
(4 Hours)
(Simple workflow)
TOTAL = 7 hours
DNA input 1-3 μg 200-400 ng <100 ng (Low)
Time from
DNA sample
to NGS library
2-3 days 2 days 1 day (Rapid TAT)
Optimal
target size
2 MB-exome 2 MB-exome < 2 MB
Types of variants
1. Point mutations
2. Large indels
3. Structural variations
1. Point mutations
2. Large indels
3. Structural variations
1. Somatic mutations
2. CNV
Which is most suitable target enrichment technology?

24. Sample to Insight
Gene 1 Gene 2
Primer design algorithm
GeneRead DNAseq Panels V2
Multiplex PCR-basedenrichment of gene(s) or genomic region(s)
PCR Chemistry
Primer separation algorithm
GeneRead DNAseq Panel V2
(sets of primer pools / tubes)
GeneRead DNAseq
PCR Kit V2
Number of pools depends on how far apart the targeted regions are (up to 2500 pairs/pool)
Division of gene primer sets into 4 pools increases amplification specificity
24

25. Sample to Insight
What do the panels do?
25
ACCAGTGAC
TATAGCTAG
GTCCTATTG
CCGGTGTAC
Variants Report
Ref: AAGTCT
Case: AACTCT
GeneRead DNAseq Panel V2 GeneRead DNAseq PCR Kit V2
DNA
Amplicons
Library
Reads
GeneReadDNAseqpanels enrich genomic targets by
generating amplicons (small dsDNA fragments) using PCR
150 bp amplicons for fragmented DNA (FFPE samples)
225 bp amplicons for intact DNA (blood samples)
Sample
isolation
Targeted
enrichment
Library
preparation
NGS run
Data
analysis
InterpretationSample Insight

26. Sample to Insight
What are the panels?
26
Breast cancer
Colorectal cancer
Myeloid neoplasms
Liver cancer
Lung cancer
Ovarian cancer
Prostate cancer
Gastric cancer
Cardiomyopathy
Only 40 ng of DNA needed per panel
(except the Actionable Mutations panel which needs a total of 20 ng)
Largest collection of pre-designed panels suitable for a wide range of applications
(Custom and Mix-n-Match)
GeneRead
DNAseq Panels V2
Wet-bench-validated
panels requiring
minimal DNA input
Comprehensive
Cancer Panel &
several others
Actionable
Mutations
Panel
Clinically
Relevant
Tumor Panel
Cancer-specific
panels

27. Sample to Insight
GeneRead DNAseq Targeted Panels V2
27
Application Panel name
#
genes
Target region
(bases)
Coverage
(%)
Specificity
(%)
Uniformity
(%)
Solid tumors
Tumor Actionable Mutations 8 7,104 100.0 98.2 91
Clinically Relevant Tumor 24 39,603 98.1 95.3 90
Hematologic
malignancies
Myeloid Neoplasms 50 236,319 98.1 97.4 94
Disease-specific
Breast Cancer 44 268,621 98.2 96.8 91
Colorectal Cancer 38 182,851 98.7 98.3 95
Liver Cancer 33 191,170 99.0 96.4 96
Lung Cancer 45 332,999 97.5 98.1 90
Ovarian Cancer 32 189,058 98.9 96.6 96
Prostate Cancer 32 167,195 98.4 97.3 94
Gastric Cancer 29 222,333 98.1 98.5 93
Cardiomyopathy 58 249,727 96.3 96.7 87
Comprehensive
Carrier Testing 157 664,735 97.5 97.9 91
Cancer Predisposition 143 620,318 98.3 96.8 93
Comprehensive Cancer 160 744,835 98.0 97.7 92
Panel optimization results in outstanding experimental performance metrics

28. Sample to Insight
Build your unique panel
28
GeneReadDNAseq Mix-n-Match Panel V2
Pool of 570
wet-bench-validated
gene designs
Gene(s) of interest (GOI)
Wet-bench-validated panel
(BRC.Custom@qiagen.com)
Quick manufacturing turnaround time: three days
Maximize
sequencing capacity:
Build your gene panel
from a pool of
wet-bench-validated
designs and focus on GOI
Flexibility:
Choose any genes from
this wet-bench validated
pool of genes

29. Sample to Insight
The power of Mix-n-Match pool of validated genes
29
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Experimental Coverage Uniformity
PDGFRA TP53RB1PIK3CA PTENNRASMET RET
Superior assay design and wet-bench validation deliver
consistent gene performance across different panels

30. Sample to Insight
Build your unique panel
30
GeneReadDNAseqCustom Panel V2
Customization tools:
Increase breadth of coverage
Adjust amplicon length
Allow denser tiling
Add QC primers
Adjust exon padding
Add UTRs to CDS
Gene(s) or Genomic region(s) of interest (GROI)
Fully customized panel
(BRC.Custom@qiagen.com)
Choose any
gene from the
human genome
OR almost any
genomic region
Build a custom
panel with a
turnaround time of
two weeks to
maximize
sequencing
capacity by
focusing on GROI
Flexibility: customization tools to match unique customer requirements

31. Sample to Insight
GeneRead DNAseq Targeted Panels V2
Simple protocol:
GeneRead DNAseq Targeted Panel V2
PCR primer mix
Add genomic DNA (10 ng/reaction); Use
GeneRead DNAseq Panel PCR Kit V2
Pool reactions for each sample
and purify (AMPure® bead purification)
PCR amplification
3 hours
NGS library preparation (One library preparation per sample)
31

32. Sample to Insight
GeneRead DNAseq Targeted Panel V2: workflow
32
AMPure bead purification
GeneRead Size Selection Kit
DNA quantification & QC
Data analysis
Day 1
Day 2
Day 3
Turnaround time for 12 samples:
Four days
Day 4
GeneRead QuantiMIZE Kit
• DNA quantification
• Provides QC score
• Optimized target enrichment
Step Time (hh:mm)
DNA from liquid biopsy 3:45
DNA quantification and QC 2:00
GeneRead DNAseq Panel V2
+ mastermix
3:00
AMPure®
bead purification 1:00
GeneRead Library Core Kit
+ Adapter Set
2:00
GeneRead Size Selection Kit 1:15
AMPure® bead purification 1:00
GeneRead amplification 0:45
QIAquick PCR Purification Kit 0:30
GeneRead Library Quant Kit 3:00
Sequencing 24:00
Data analysis 5:00

33. Sample to Insight
Compatibilitywith major sequencing platforms
Same panel for different platforms
IlluminaQIAGEN
Ion
GeneRead® DNAseq Panels V2
HiSeq 2500
MiSeq
PGM
Proton
Panels are platform-independent

34. Sample to Insight
Why choose GeneRead DNAseq Panels?
34
Features Benefits
Platform-independent
No need to switch panels
if you use different NGS platforms
Conveniently cataloged off-the-shelf panels
as well as custom panels with quick turn-
around time
Save time and
start your NGS analysis quickly
Panels are cost-effective and
free data analysis is provided Reduce costs
Simple workflow
Easy to implement in
any lab and core facility
GeneRead® DNAseq Panels V2

35. Sample to Insight
35
Why choose GeneRead DNAseq Panels V2?
Input
• FASTQ files
• Panel used
• Job type
• Single
• Matched tumor / normal
• Analysis mode
• Somatic
• Germline
Added value: powerful and easy-to-use data analysis at no cost
Output
• BAM and VCF files
• Link to IVA (automatic upload of files)
• Sequencing metrics
• Variants detected
• Copy number alterations

36. Sample to Insight
What can they be used for?
Title, Location,Date 36
Detect known and discover novel variants
Actionable
mutations
Clinically
relevant
Disease-
specific
Comprehensive
Detection
Discovery
Multiplexing
Target size
Content that fits a wide range of biological and clinical applications
Custom and Mix-n-Match
PanelsApplicationsSpecifications
Clinical research Translational & Discovery research

37. Sample to Insight
GeneRead DNAseq Panels V2: Application data
37
Matched normal / tumor lung FFPE samples
Normal
Isolate gDNA
Targeted enrichment
Library construction
Barcode
Tumor
Isolate gDNA
Targeted enrichment
Library construction
Barcode
MiSeq sequencing
Comprehensive Cancer Panel
Lung Cancer Panel
Variant report

38. Sample to Insight
38
Sequencing metrics (Comprehensive Cancer Panel)
Metric Control sample (normal lung) Case samples (lung cancer)
Total reads 16,887,660 10,952,708
Specificity 94.3% 94.2%
Median read depth 1,986 1,199
Uniformity (coverage at 0.2x median) 91% 90%
% bases covered at >=100x 96 95
# bases in target region: 745 kb
# amplicons = ~8000
Sequencing metrics that enable you to detect variants with confidence
GeneRead DNAseq Panels V2: Application data

39. Sample to Insight
Variant analysis
39
Identification of known and novel variants
Sample
Chromosome
Position
Gene
Reference
Variant
dbSNPID
COSMICID
Filtered
coverage
Variant
Frequency
AAChange
snpEffEffect
snpEffImpact
Ingenuity
CancerDriver
Protein
domain
1 17 7577581 TP53 A G COSM238606 1497 0.314 p.Y234H
NON
SYNONYMOUS
CODING
MODERATE
p.Y234H;
p.Y195H;
p.Y102H;
p.Y75H
zinc
binding
site
2 17 37880995 ERBB2 C CTGT novel novel 2477 0.612 p.A775AV
CODON
INSERTION
MODERATE
p.A745_G
746insV;
p.A775_G
776insV
active site,
ATP
binding
site
Extensive variant annotation
Primary analysis
Secondary analysis

40. Sample to Insight
Confirmation using Sanger sequencing
40
Known TP53 mutation in lung cancer cells
Sequence in non-cancerous cells:
GTAGTGG
Sequence in tumor cells:
GTGGTGG

41. Sample to Insight
41
Sequence in non-cancerous cells:
ACCAGCCATCA
Sequence in tumor cells:
ACCAACAGCCATCA
Novel ErbB2 insertion in lung cancer cells
Confirmation using Sanger sequencing

42. Sample to Insight
Biological significance
42
Insertion creates a putative Erk1 phosphorylation site
Kinase Site Score Percentile Sequence SA
Cdk5 Kinase S17 0.6116 3.26% YVMAGVGSPYVSRLL 0.406
Cdc2 Kinase S17 0.6353 3.08% YVMAGVGSPYVSRLL 0.406
Erk D-domain I26 0.8066 4.87% YVSRLLGICLTSTVQ 0.084
Kinase Site Score Percentile Sequence SA
Cdk5 Kinase S18 0.6246 3.68% VMAVGVGSPYVSRLL 0.406
Cdc2 Kinase S18 0.6471 3.45% VMAVGVGSPYVSRLL 0.406
Erk1 Kinase S18 0.6713 4.76% VMAVGVGSPYVSRLL 0.406
Erk D-domain I27 0.8066 4.87% YVSRLLGICLTSTVQ 0.084
Normal cells
Tumor cells
Determined by Scansite

43. Sample to Insight
Discover novel mutations
43
Gene panels can facilitate discovery of novel mutations
Notably, this study facilitated the identification of BRCA2 Thr9976, which is
the strongest genetic association with lung cancer reported so far.
For a smoker carrying this variant (2% of the population), the risk of developing
lung cancer is approximately doubled, which may have implications for identifying
high-risk ever-smoking subjects for lung cancer screening.
Additionally, future study of the effects of PARP inhibition in smokers with
lung cancer carrying BRCA2 Thr9976 may be warranted

44. Sample to Insight
Why choose GeneRead DNAseq Panels V2?
44
An intersectionof solutions to address NGS challenges
GeneRead
DNAseq
Panels V2
Broadest
collection of
wet-bench-
verified
panels
Free data
analysis
& biological
interpretation
Short time to
library
generation
& one library
per sample
Works with
multiple NGS
platforms
Fully
customized
& low DNA
requirements
Outstanding
experimental
performance

45. Sample to Insight
Complete NGS solutions from “Sample to Insight”
45
Sample
isolation
Targeted
enrichment
Library
construction
NGS run
Data
analysis
Biological
insightSample Insight
Sample QC
GeneRead DNA
QuantiMIZE System: rescue
low-quality DNA samples
Library QC
GeneRead DNAseq Library
Quant Kit: ensure equal library
loading for all samples
Variant confirmation
qBiomarker Somatic
Mutation Assays
Challenges
Isolating high
quality DNA
Amount of DNA,
Long turnaround time,
Panel contents,
Panel performance
Number of libraries
per sample
Platform
dependence
Massive amounts
of data require
expertise to yield
biological insight
Solutions
REPLI-g
Single Cell Kit
Repli-g Cell
WGA / WTA
Kit
GeneRead DNAseq
Targeted Panels V2
QIAamp DNA kits
GeneRead Size
Selection Kit
Compatible
with major
sequencing
platforms
GeneRead data
analysis portal
(free)
CLCbio Cancer
Genomics
Workbench
Ingenuity Variant
Analysis

46. Sample to Insight
46
Liquid biopsy overview
Genotyping steps and challenges
Biomarkers and next-generation sequencing
Biomarkers and real-time PCR
2
3
4
Agenda
1

47. Sample to Insight
Genotyping
47
Analyzing somatic mutations
Discovery
• SNP chips
• NGS
Validation
• qPCR
• Sequencing
• Mass spectroscopy
Pre-screen
Diagnostic

48. Sample to Insight
Methods to analyze DNA mutations
Method Benefits Drawbacks
Point mutation analysis Exact Time consuming
Mass spectrometry Exact
Time consuming
Need instrument / training
High resolution melt
Inexpensive
Fast
Simple
Base change identity not
obvious
Microarray Large coverage
Sensitivity
Protocol complex
Need instrument / training
Next-generation sequencing Exact
Time consuming
Need instrument / training
Quantify DNATargets
 Real-time PCR
Fast
Reliable
High-throughput
48

49. Sample to Insight
Comparing different technologies
qPCR
Sequenome
OncoCarta*
Sanger sequencing NGS
Technology
Real-time PCR based:
• Single-step handling
• Close tube
Multiplex PCR,
extension, mass spec:
• Multi-step handling
• Risk of contamination
Individual PCR, clean-
up, extension, CE:
• Multi-step handling
• Risk of contamination
Sequence enrichment,
library construction, NGS:
• Lengthy procedure
• High cost for high
coverage
Coverage
Focused content:
• Expanding coverage
• Relevant mutations
• Can be customized
• Profile
~300 mutations across
19 genes:
• Include some very
rare mutations
• Not so easy to
customize
Homebrew assay, low
gene coverage, variable
levels of mutation
discovery
Genome wide, but
depends on sequencing
depth
(Detecting 1% mutation =
1000X sequencing depth)
Samples
5-10 ng fresh / frozen
200-500 ng FFPE
500 ng fresh or FFPE Fresh / frozen, FFPE
Several micrograms of
DNA
Sensitivity <1% 10% >20%
Depends on sequencing
depth
Throughput
High-throughput based
on content
>96/d
High sample / low content
throughput
Variable, lengthy wait
Instrument
Real-time PCR cycler:
• Widely accessible
Sequenome mass
spectrometer:
• Limited access
CE sequencer
NGS sequencer:
• Limited access
Data analysis Simple Complicated Simple Complicated
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50. Sample to Insight
qBiomarker Somatic Mutation Arrays
Disease or pathway-focused mutation profiling
Isolate genomic DNA from fresh, frozen
or FFPE samples using QIAamp or
DNeasy Kits recommended in the handbook.
Upload CT values to Data Analysis Webportal
Standard 40 cycle PCR on most real-time
thermocyclers
One sample per plate
Add qBiomarker Probe mastermix to genomic DNA
• 200–500 ng fresh / frozen DNA
• Less DNA requires the WGAKit
• 500–3000 ng FFPE DNA
50
1
2
3
4
5

51. Sample to Insight
qBiomarker Somatic Mutation PCR Array layout
Greek symbols = gene
# = mutation
• For normalization
• Assays detect non-variable
region (not ARMS-based
design)
• Designed to control for
differences in sample input
and quality
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52. Sample to Insight
qBiomarker Somatic Mutation Arrays
Focusedarrays for somatic mutation screening
Pathway Cancer
EGFR Breast cancer
FGFR Colon cancer
RTK - Panel I Hematopoietic neoplasms
RTK - Panel II Lung cancer
Ras ̶ Raf Skin cancer
ErbB2 Brain cancer
KIT Lymphoid neoplasms
APC / CTTNNB1 Thyroid cancer
FLT3 Melanoma
c-MET Liver cancer
p53 / Rb Ovarian cancer
PDGFR Gastric cancer
PI3K-AKT signaling pathway Esophageal cancer
DNA - QC Head and neck cancer
52

53. Sample to Insight
Copy number analysis
53
We can use different methods to answer different experimental questions
Discovery
• Array CGH
• SNP chips
• NGS
Validation
• FISH
• qPCR
Pre-screen
Diagnostic Test

54. Sample to Insight
Method of choice: qPCR
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• Determine the gene and reference genome
• Design primers, amplify genes using PCR, determine Ct value
• As copy number increases Ct value will decrease
1x
2x
3x
Sample 1
Sample 2
Sample 3
A B C D
A B C DB
A B C DB B
Gene ‘B’
copy number
(Highest Ct value)
(Lowest Ct value)

55. Sample to Insight
Multicopy reference assay (MRef)
55
Benefits of a well-designed reference assay:
• Superior assay for input normalization
• Lower error rate in variant calling
The ideal reference assay should fulfill the following criteria:
• Not be affected by local changes in the genome
• Copy number or SNP
• Copy number
• >20 copies in a diploid genome
• Location distribution
• Located on different chromosomes
• ≤ 10% copies concentratedon a single chromosome
• For copies on the same chromosome, preferably on different arms
• Sequence
• Sequence-stable in human genome
Key challenge: error in variant calling
Need: better reference assays

56. Sample to Insight
Multicopy reference assays yield increased accuracy
CNA Experimental Setup
56
• Genomic DNA samples
• Liposarcoma samples
• Gene Of Interest (GOI)
• Reference genome
• Multicopy reference assay

57. Sample to Insight
CNV in liposarcoma — aCGH to qBiomarkerArrays
Thirty liposarcoma samples were tested by
aCGH for copy number events
Results from one of those samples (T50) is
shown
• Analysis with Partek software
• Deletions on Chromosome 11
• Amplifications on Chromosome 12
Initial screen yielded a list of 23 genes with
copy number changes
All samples were re-tested using Custom
Copy Number PCR Array
Data courtesy of Kara Pascarelli and Dominique Broccoli, Memorial University Medical Center, Savannah, GA, USA; and Lesley Ann Hawthorne,
Medical College of Georgia, Georgia HealthSciences University, Augusta, GA, USA)
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58. Sample to Insight
0
5
10
15
20
25
30
35
CopyNumber
**
*
*
*
* *
*
*
* *
***
qBiomarker
aCGH
Copy Number PCR Array Data for Sample T50
Data courtesy of Kara Pascarelli and Dominique Broccoli, Memorial University Medical Center, Savannah, GA, USA; and Lesley Ann Hawthorne,
Medical College of Georgia, Georgia HealthSciences University, Augusta, GA, USA)
CNV in liposarcoma — aCGH to qBiomarker arrays
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59. Sample to Insight
We provide service: send us samples and receive results
. Whole genome
• Illumina gene expression profiling
• Illumina genotyping
. Pathway / focused panels
• Mutation profiling
• Methylation profiling
• PCR arrays
• miRNA PCR arrays
• NGS
. Individual gene / locus
• Mutation detection
• Methylation
• qPCR
• NGS
. Sample prep. – DNA and RNA extraction and purification
• Cells, tissues or biofluids
• Fixed tissue
• Small samples
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60. Sample to Insight
Liquid biopsy to genomic biomarker
Single-cell assays
• Amplify the whole genome and from a
single cell from liquid biopsy
Sample stabilization & isolation
• Achieve high-quality and high-yield
sample for downstreamapplications
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NGS solutions
• Speed up and simplify your NGS
workflow with our innovative technology
CNV/CAN analyses
• Profiling copy number variations and
alterations
Somatic mutation analyses
• Rapid and accurate profiling of somatic
DNA mutations with focused panels

61. Sample to Insight
Thank you for attending today’s webinar!
Contact QIAGEN
Call: 1-800-426-8157
Email: BRCSupport@qiagen.com
Questions?
Krishnan Allampallam, PhD
krishnan.allampallam@qiagen.com
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