Liquid Biopsy Overview, Challenges and New Solutions: Liquid Biopsy Series Part 1

Sample to Insight
Liquid biopsy overview, challenges and new solutions
Wei Cao, Ph.D.
Wei.Cao@qiagen.com
1

Sample to Insight
Legal disclaimer
Liquid biopsy overview 2
• 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.

Sample to Insight
Agenda
The main areas of liquid biopsy
• Circulating tumor cells
• Circulating tumor nucleic acid
• Exosomes, mRNA, miRNA and lncRNA
Solutions provided by QIAGEN
Questions
1
2
3

Sample to Insight
Agenda
Questions
1
2
3

Sample to Insight
Why liquid biopsy?
Biomarkers
Personalized
therapies
• Need to correlate mutation profiles with sensitivity or
resistance to specific therapies
• Molecular characterization of tumors allows progression from
“one-size-fits-all” strategy to “personalized medicine”
Monitoring of a disease is fundamental for successful treatment
Prognostic markersDiagnostic markers
Need novel non-invasive biomarkers – liquid biopsy as a game changer
Cancer
diagnosis
Cancer genome
analysis
Clinical
decision
Monitor
outcome
Dancey, J.E. (2012) The genetic basis for cancer treatment decisions. Cell 148, 409.
Personalized therapies for cancer patients

Sample to Insight
What is liquid biopsy?
A liquid biopsy is a liquid biomarker that can be isolated from body
fluids, such as blood, saliva, urine, ascites or pleural effusion. Like a
tissue biopsy, a liquid biopsy is representative of the tissue from
which it has spread.
Diaz, Jr., L.A. and Bardelli, A. (2014) Liquid biopsies: genotyping circulating tumor DNA.”Am. Soc. Clin. Oncol. 32, 579.
6
• Cancer is a heterogeneous disease
• Molecular properties differ within a
tumor
• Primary tumor biopsy may not reflect
current disease condition
• Therapy causes changes in tumor cells
• Biopsy is invasive
• May not be feasible based on patient
condition or tumor accessibility
• Impractical for periodic monitoring for
progression/ recurrence
• Biopsy tissue is limited
• Greater demand due to molecular
profiling
• Surgery is costly
Liquid biopsy overview
• Allows early disease detection
• Allows evaluation of metastasis in real-time and
monitoring of the actual treatment response
• Enables investigation of primary tumors and
metastases through simple, non-invasive blood
tests
• Enables assessment of tumor heterogeneity and
monitoring of tumor dynamics
• Enables study of the “tumor dormancy”
phenomenon
• Is much faster than classical biopsy testing
• Can be cheaper than classical biopsy testing
Liquid biopsy addresses all these limitations

Sample to Insight
Liquid biopsy: the 3 main areas
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Liquid biopsy
CTCs
(circulating
tumor cells)
ctNA
(circulating
tumor
nucleic
acids)
Exosomes
Tumors shed both intact cells (resulting in circulating tumor cells) as well as cellular components,
such as nucleic acids (resulting in cell-free DNA or RNA).
Diaz, Jr., L.A. and Bardelli, A. (2014) Liquid biopsies: genotyping circulating tumor DNA. Am. Soc. Clin. Oncol. 32, 579.
Cancer cells released
from primary tumor into
the bloodstream
ctDNA (circulating tumor
DNA), miRNAs, mRNA &
long non-coding RNA
Small membrane-derived
vesicles (40–100 nm) that
contain various molecules such
as signal protein, miRNA, mRNA,
lipid and exoDNA
CTCs ctNA, mainly ctDNA Exosomes, exoDNA, exoRNA
including miRNA and lncRNA
Samples: blood, serum/plasma, urine, CSF saliva
1 2 3

Sample to Insight
Area 1: circulating tumor cells (CTCs)
8
Rolfo, C. et al. (2014) Liquid biopsies in lung cancer: the new ambrosia of researchers. Biochimica et Biophysica Acta 1846, 539.
• CTC enumeration: serves as a marker for tumor growth – higher CTC
counts mean negative cancer prognosis
• CTC characterization: protein and RNA expression, DNA aberrations
and propensity for metastatic colonization
Technologies to detect and capture CTCs:
• EpCAM-affinity based: CellSearch® system, AdnaTest BreastCancerDetect, CTC-Chip, Dynal®, MACS® (magnetic-
activated cell sorting system), MagSweeper, On-Q-Ity, CTC-ETI
• Physical properties-based: ISET (isolation by size of epithelial tumor cells), ScreenCell®, ApoStream™, density
gradient centrifugation
• Other methods: FAST (fiber-optic array scanning technology), EPISPOT (epithelial immunospot), FACS (flow
cytometry), PRO Onc Assay
Limitations:
• EpCAM-affinity based methods: low sensitivity, selection bias and poor specificity (false negatives and positives)
• Physical properties-based methods: elementary and imprecise, with low specificity
CTCs are extremely rare (~1 per 1 ml of blood), so optimization of CTC isolation and enrichment steps is essential.

Sample to Insight
CTCs: characterization and challenges
9
Molecular characterization of CTCs:
Paterlini-Bréchot, P. (2014) Circulating tumor cells: who is the killer?”Cancer Microenviron. 7, 161.
• Based on antibodies: EpCAM, cytokeratins (CK8, CK18, CK19), CD45-negative. Examples include CellSearch
• Based on transcripts: rely on transcripts, performed on the total RNA extracted from blood by RT-PCR. Examples
include AdnaTest BreastCancerDetect
• Based on functions: protein-based assays such as the EPISPOT (Epithelial ImmunoSPOT) assay
• Whole-genome amplifications: single-cell studies targeting CTC heterogeneity, or pooling CTCs to study the whole
tumor cell population
The clinical validity of CTCs as a modern, personalized, non-invasive and predictive test has been debated. Much
effort is still needed to determine whether they represent a potential surrogate for clinical endpoints.
Current clinical utility of CTCs
• CTCs are extremely rare
• CTCs are fragile
• CTCs are heterogeneous
Challenges
Sensitivity is directly linked to clinical benefit at early stages of the disease
Can easily vanish or morphology can be damaged during extraction from blood
Require the isolation of all types of CTC without any loss
Require broad-spectrum, specific cocktails of cell surface epithelial and
mesenchymal markers covering all potential CTC phenotypes

Sample to Insight
CTCs: detection/capture methods in clinical studies
10Liquid biopsy overview

Sample to Insight

Sample to Insight
12
Ignatiadis, M. et. al. (2015) “Circulating Tumor Cells and Circulating Tumor DNA: Challenges and Opportunities on the Path to Clinical Utility.”
Clinical Cancer Research, 21(21): 4786
Updated list of CTC assays that have been used to test patient samples within
the past 5 years

Sample to Insight
Area 2: circulating tumor nucleic acids (ctNAs)
13
• Biology of cell-free NA and clinical utilities
• Released from both healthy and tumor cells into circulation through various physiological events, such as
apoptosis, necrosis and secretion
• ctNAs reflect disease progression and treatment responses
• The clinical utility of ctDNA depends on reliable detection of ctDNA when it is present
• Cell-free vs. CTCs
• Shorter half-life (CTCs: 1–2.4 h vs. ctNAs: <1.5 h)
• Highly fragmented
Types of cell-free nucleic acids
 DNA (defined as cell-free DNA, cfDNA)
 miRNA (microRNA)
 mRNA (messenger RNA)
 Long non-coding RNAs (lncRNAs)
Schwarzenbach, H. et. al. (2011) Cell-free nucleic acids as biomarkers in cancer patients. Nat. Rev. Cancer 11, 426.
Diaz, Jr., L.A. and Bardelli, A. (2014) Liquid biopsies: genotyping circulating tumor DNA. Am. Soc. Clin. Oncol. 32, 579
Tumor cells
Apoptosis Necrosis/secretion
Exosome
free-circulating DNA
Normal circulating NA
Small RNA

Sample to Insight
ctDNA: detection and challenges
14
Methods for detecting circulating tumor DNA
based on the differences of ctDNA vs normal
cfDNA by cancer-associated genetic mutations:
• point mutations
• copy number variations
• chromosomal rearrangements
• methylation patterns
Challenges of current ctDNA assays
• Sensitivity: sometimes extremely low levels of ctDNA (1.0%) are present
• DNA extraction: the pre-analytical phases of cfDNA need to be better defined
• Assay standardization: procedures need to be standardized
• Quantification: accurate quantification is needed
• Variability of assay platform: different platform assays have different assay sensitivity and specificity
and analytical approach
Diaz, Jr., L.A. and Bardelli, A. (2014) Liquid biopsies: genotyping circulating tumor DNA. Am. Soc. Clin. Oncol. 32, 579.

Sample to Insight
Area 3: exosomes in cancer progression
15
Exosomes: small membrane vesicles (30–100 nm), secreted by most cell types into the bloodstream.
• Exosomes play a central role in cell-to-cell communication.
• The majority of DNA associated with tumor exosomes is double-stranded, representing whole
genomic DNA.
• Biological molecules (protein, RNA and miRNA) contained in exosomes are well protected by a lipid
bilayer membrane that confers a high degree of stability.
• Exosomal RNAs contain fingerprints for various diseases, so have potential as liquid biopsy
Functional biomolecules:
• DNA fragments (exosomal DNA,
exoDNA)
• Proteins and/or peptides
• Lipids
• mRNA (exoRNA)
• microRNA (miRNA)
• Long non-coding RNA (lncRNA)
Klevebring, D. et. al. (2014) Evaluation of exome sequencing to estimate tumor burden in plasma. PLOS One 9, e104417.

Sample to Insight
Exosomes: isolation methods and challenges
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Challenges: improve and standardize methods for exosomes isolation
• Many current protocols to isolate exosomes use ultracentrifugation based on size
• Long processing time
• Irreproducible and not selective for tumor exosomes
• Different RNA isolation methods give extensive variation in exosomal RNA yield and patterns
• Often fail to distinguish between differently-sized exosomes and membrane-free macromolecular aggregates, so
should be taken cautiously
Current methods
• Differential
centrifugation
• Size exclusion
• Immunoaffinity isolation
• Microfluidic devices
• Polymeric precipitation
(ExoQuick)
Issues
• Long processing time
• Cannot achieve absolute
separation
• Low reproducibility
• High contamination
• Low yields
• Low purity
Needs
• Rapid, exosome-
specific extraction
• Simple, effective
release from
exosomes
• Highly sensitive,
specific detection
Exosomal RNAs characterization methods: RNA-seq, microarray and real-time PCR based approaches.

Sample to Insight
Exosomal RNAs
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mRNAs:
• mRNAs released into the circulation are stable because they are protected from
degradation by being packaged into exosomes
• The level of exosomal RNA implies genetic information
miRNAs:
• miRNAs are ~21 nt and have the potential to serve as diagnostic markers
• miRNA expression is frequently deregulated in cancer
• In blood, miRNAs are highly stable because most of them are included in
apoptotic bodies, microvesicles or exosomes and can withstand known mRNA
degradation factors
Long non-coding RNAs (lncRNAs):
• Play regulatory roles in cancer progression and metastasis
• The long non-coding PCA3 RNA-based urine test is the first FDA-approved test
for the diagnosis of prostate cancer patients
Schwarzenbach, H. et. al. (2013) Cell-free nucleic acids as biomarkers in cancer patients. Nat. Rev. Cancer 11, 426.
Rönnau, C.G.H. (2014) Noncoding RNAs as novel biomarkers in prostate cancer. Biomed. Res. Int. 2014; 591703: 17

Sample to Insight
Agenda
The main areas of Liquid Biopsy
 Circulating tumor cells
 Circulating tumor nucleic acid
 Exosomes, miRNA, and lncRNA
Questions
1
2
3

Sample to Insight
Liquid biopsy solutions at QIAGEN – Sample to Insight
For non-invasive biomarker discovery: Sample to Insight
Free-circulating nucleic acids
• QIAamp Circulating NA Kit
• QIAsymphony Circulating NA Kit
• miRNeasy Serum/Plasma Kit
Exosomes
• exoEasy Maxi Kit for intact exosome isolation
• exoRNeasy Serum/Plasma Kits for exoRNA
isolation
.
Circulating tumor cells
• Single cell analysis for amplification
• The AdnaTest for CTC detection
Sample
Separate plasma
Isolate analytes
Real-time PCR
NGS technology
Analytical
Blood draw
(venipuncture)
Pre-analytical Insight

Sample to Insight
Solutions for circulating tumor cells
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REPLI-g® Single Cell technology to analyze circulating tumor cells

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Solutions for CTCs
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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”
Problem
• Incomplete or biased genome amplification with
missing or underestimated sequence information
Solutions
• REPLI-g Single Cell Kit
• Optimized Multiple Displacement Amplification
(MDA) process with 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.
QIAGEN’s REPLI-g Single Cell technology:
• For fast and accurate genomic analysis of CTCs and single cells
• DNA or RNA can be successfully amplified from just a single cell

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

Sample to Insight
REPLI-g Single Cell technology
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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 dropout
from any marker, indicating a 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
• Link the genome to the corresponding gene
expression profile and to its phenotype

Sample to Insight
Applications of REPLI-g Single Cell Kits
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WGA
or
WTA
Whole Genome Sequencing
• Detect variability in genome sequence (SNV, microsatellites, etc.)
• Variability in genome structure (CNV, structural rearrangements, aneuploidy)
• De novo sequencing of new, unidentified, and unculturable organisms
Targeted DNA Sequencing
• Detect variability in a target set of genes or region of the genome
Microarrays
• Use SNP-chips to genotype thousands of loci
RNA-seq
• Detect variability in transcript abundance for all expressed genes
• Detect variability in isoform structure and abundance
qRT-PCR profiling
• Profile gene expression for a targeted set of transcripts
• Accurately quantify specific splice-junctions, isoforms or other structural
features

Sample to Insight
AdnaTest: the CTC revolution
Blood sampling Determination of
prognostic or
predictive
biomarkers (liquid
biopsy)
Turn around time: 5 h
CTC enrichment
using multi AB
labelled
magnetic beads
Lysis of the
enriched cells
RT and multiplex
PCR
Two-step procedure for the enrichment of CTCs from blood samples
1. Immunomagnetic capturing using a multi-antibody mix
2. Multiplex RT-PCR to determine a number of tumor associated transcripts
• Every AdnaTest uses different antibody mixtures optimized for each tumor entity
• This method is an easy and straightforward manual assay
Type AdnaTest into the questions box to request further information

Sample to Insight
Solutions for circulating nucleic acid (ctNA)
26
Isolation of free-circulating DNA and RNA

Sample to Insight
Circulating nucleic acids: analysis workflow
Sample
Separate plasma
Extract circulating nucleic acids:
• Real-time PCR
• NGS
• Digital PCR
therascreen assays
Analytical
Blood draw
(venipuncture)
QIAamp Circulating Nucleic Acid Kit:
For isolating free-circulating DNA and RNA from human plasma or serum and other
cell-free body fluids

Sample to Insight
QIAamp Circulating Nucleic Acid Kit
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Simple procedure and high performance:
• 4 steps: lyse, bind, wash and elute
• Allows simultaneous processing of multiple samples
• Provides nucleic acids in less than 2 hours per 24 samples
• No organic extraction or ethanol precipitation
• Removal of contaminants and inhibitors
• Suitable for human plasma, serum and urine
Applications:
Purified and concentrated circulating DNA and RNA is free of proteins, nucleases and
other impurities, and is ready to use in wide range of downstream applications, including:
• Biomarker research and verification for blood-based cancer detection
• PCR and quantitative real-time PCR and RT-PCR
• Viral nucleic acid detection
http://www.qiagen.com/search/qiaamp-circulating-nucleic-acid-kit/#orderinginformation
Purify & concentrate:
Concentration of nucleic acids, with high input (5 ml) and low elution volumes (20–150 µl).

Sample to Insight
QIAamp Circulating Nucleic Acid Kit
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http://www.qiagen.com/search/qiaamp-circulating-nucleic-acid-kit/#orderinginformation
Improved recovery of fragmented DNA
Reproducible recovery of circulating DNA
Efficient purification with reproducible yields
provides a representative population of the
circulating nucleic acids in blood.
Advanced technology involving selective
binding to a silica-based membrane enables
improved recovery of fragmented nucleic acids.
Simplify your analysis of tumor-specific extracellular DNA fragments and mRNAs in the blood!

Sample to Insight
miRNeasy Serum/Plasma Kit:
For purification of circulating RNA – primarily miRNAs and other small RNAs –
from plasma, serum, CSF, urine, saliva, etc.
Sample
Separate plasma
• Real-time PCR
• NGS
• Digital PCR
therascreen assays
Analytical
Blood draw
(venipuncture)

Sample to Insight
miRNA & mRNA isolation from blood
31
• Purifying RNA and miRNA from 200 µl serum or plasma
• Minimal elution volume (14 µl)
• Easy procedures and automatable protocol
• High-purity RNA suitable for a variety of downstream applications:
• Biomarker discovery
• Northern blot analysis
• Quantitative, real-time RT-PCR using the miScript PCR system
• Microarray analysis
http://www.qiagen.com/search/mirneasy-serumplasma-kit/#orderinginformation

Sample to Insight
Solutions for exosomes
32
Isolation of exosomes and exoRNAs

Sample to Insight
Isolation of exosomes and exoRNAs
33
exoRNeasy Serum/Plasma Kits
• Isolate RNA from exosomes and other extracellular vesicles from serum or plasma
samples
• Novel and efficient workflow: from sample to extracellular vesicle RNA in just 1 h
https://www.qiagen.com/us/shop/sample-technologies/rna/rna-preparation/exorneasy-serum-plasma-kits/
exoEasy Maxi Kit
Isolate intact exosomes and other extracellular vesicles from:
• Plasma
• Serum
• Cell culture supernatant
https://www.qiagen.com/shop-old/sample-technologies/exoeasy-maxi-kit/

Sample to Insight
exoEasy Maxi Kit: principle and protocol
34
Starting material: serum, plasma or cell culture supernatant
• Isolation of bioactive exosomes and
extracellular vesicles
• Can be used for cell culture work
• Speed: 25 minutes – no time-consuming
procedure
• Allows much shorter time-to-analysis
• Ease of use allows processing of multiple
samples in parallel
• Standardization
• Highest specificity for vesicle isolation
through affinity membrane-binding
technology
• Excludes non-vesicular protein complexes
and results in cleaner preparations

Sample to Insight
exoEasy Maxi Kit: elute intact vesicles
35
Highest specificity through affinity membrane-binding technology to ensure delivery of
intact vesicles of the expected size
• Scanning electron microscopy images of isolated vesicles from ultracentrifugation and eluate
from exoEasy column
• exoRNeasy Serum/Plasma Kits yield a cleaner preparation

Sample to Insight
exoRNeasy Serum/Plasma Kits: principle and protocol
36
Pre-filter
plasma
to remove
particles
larger than
0.8 μm
Microvesicle isolation: 20 minutes exoRNA isolation: 35 minutes

Sample to Insight
*Arroyo, J.D. et al. (2011) Argonaute2 complexes carry a population of circulating microRNAs independent of vesicles in human
plasma. Proc. Natl. Acad. Sci. USA 108, 5003.
mRNAs exclusively within vesicles – near 100% bound
miRNA in vesicular and non-vesicular fractions (e.g., free Ago2 complexes)
exoRNeasy: captures all mRNAs and vesicle-specific miRNAs

Sample to Insight
Sample
Separate plasma
• Real-time PCR
• NGS
• Digital PCR
therascreen assays
Analytical
Blood draw
(venipuncture)

Sample to Insight
Solutions for circulating miRNA biomarkers
39
miScript miRNA PCR Arrays

Sample to Insight
miScript miRNA PCR system
40
• miRNome
• Human: miRBase v21, covers 2402 primer assays
• Mouse: miRBase v21, covers 1765 primer assays
• Rat: 653 primer assays
• Dog: 277 primer assays
• Rhesus macaque: 469 primer assays
• Cow: 744 primer assays
• Pathway-focused arrays (>20 arrays)
• Serum and plasma
• miFinder
• Cancer PathwayFinder
• Liver miFinder
• Brain cancer
• Breast cancer
• Ovarian cancer
• Prostate cancer
• Cancer stem cells
• Apoptosis
miScript PreAMP Kit
• Optional step for small or precious samples
• Full miRNome profiling from as little as 1 ng RNA
Pre-formatted, single-use PCR arrays with wet-lab verified assays

Sample to Insight
miScript miRNA PCR Array workflow
41
Workflow
1. Isolate total RNA
2. Perform reverse transcription
3. Prepare PCR pre-mix
4. Load PCR arrays and perform
real-time PCR
5. Analyze data
 1 hour
 2 minutes
 2 hours
 15 minutes
1.
2.
3.
4.
5.
Request webinar slides for miRNA webinar series to learn more  type “miRNA webinar” in questions box.

Sample to Insight
miScript miRNA PCR Array applications
42
miScript Serum & Plasma 384HC PCR Array profiled 381 miRNAs isolated using
exoRNeasy Kits
miRNA distribution
Abundance of miRNAs inside and
outside extracellular vesicles

Sample to Insight
Solutions for lncRNAs: novel biomarkers in cancer
43
RT2 lncRNA PCR Arrays and Assays

Sample to Insight
RT2 lncRNA qPCR system
44
• lncRNA databases: in-house database at QIAGEN GeneGlobe provides
cover > 40,000 human and 27,000 mouse lncRNA targets.
• RT2 lncRNA assays: laboratory-verified for optimal qPCR performance –
high specificity, amplification efficiency and sensitivity.
• RT2 lncRNA qPCR Arrays: pathway or disease relevant lncRNA assays
• RT2 lncRNA Cancer PathwayFinder Array (human and mouse)
• RT2 lncFinder PCR Array (human and mouse)
• Custom option: flexible custom design from the lncRNA and qPCR
databases to profile mRNA and lncRNA simultanously.
• lncRNA isolation: exoRNeasy or miRNeasy Kits
• Data analysis: free online data analysis tool
http://www.qiagen.com/us/landing-pages/lncrna/

Sample to Insight
RT2 lncRNA qPCR Array: applications
45
Flexible layout and patented controls
Each 96-well plate has 84 lncRNA-specific assays, 5 reference genes and genomic DNA,
reverse transcription and PCR controls. Arrays are also available in 384-well plates and
100-well ring discs for the Rotor-Gene Q.
http://www.qiagen.com/us/landing-pages/lncrna/
Volcano plot of lncRNA gene expression changes in stage II
prostate cancer tissue compared with normal tissue.

Sample to Insight
Solutions for next-generation sequencing
46
NGS portfolio: complete solutions for NGS

Sample to Insight
Complete NGS solutions from “Sample to Insight”
47
• Streamlined, standardized and automated sample-to-insight workflow
• Overcome challenges at every step in your DNA-NGS workflow
Isolate 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 get biological insight
REPLI-g Single Cell Kit
REPLI-g Cell WGA/WTA Kit
GeneRead DNAseq
Targeted Panel V2
DNA Amp Kit GeneRead
Size Selection Kit
Compatible with major
sequencing platforms
GeneRead data analysis portal (free)
CLCbio Cancer Genomics Workbench
Ingenuity® Variant
Analysis™
Challenges
Solutions
NGS webinar series: learn more  type “NGS webinar” in the questions box to request webinar slides.
Sample
isolation
Targeted
enrichment
Library
construction
NGS run
Data
analysis
InterpretationSample Insight
Sample
QC Library QC
Variant
confirmation
qBiomarker Somatic Mutation AssaysGeneRead DNAseq Library Quant KitGeneRead DNA QuantiMIZE System

Sample to Insight
We provide service – send samples to us & receive results
48
. Whole genome
• Illumina gene expression profiling
• Illumina genotyping
. Pathway/focused panels
• Mutation profiling
• Methylation
• PCR arrays
• miRNA PCR arrays
• NGS
. Individual gene/locus
• Mutation detection
• Methylation
• qPCR
• NGS
. Sample preparation – DNA, RNA extraction and purification
• Cells, tissues or biofluids
• Fixed tissue
• Small samples
. http://www.qiagen.com/products/catalog/services/

Sample to Insight
Summary of products offered at QIAGEN
49
1 Single-cell technology – REPLI-g WGA Single Cell Kit
2 CTC enrichment and detection – AdnaTest and AdnaPanel
3 Sample isolation technology
1. QIAamp Circulating NA Kit
2. miRNeasy Serum/Plasma Kit
3. exoRNeasy Serum/Plasma Kit
4 Assay technology
1. miScript miRNA PCR Arrays for circulating miRNA biomarker discovery
2. RT2 Profiler lncRNA PCR Arrays for lncRNA biomarker discovery
3. Complete NGS workflow to allow Sample to Insight
5 Service – We provide expert service

Sample to Insight
Agenda
Questions
1
2
3

Sample to Insight
51
Thank you for attending today’s webinar!
Contact QIAGEN
Call: 1-800-426-8157
Email: BRCsupport@QIAGEN.com
Wei Cao, PhD
Wei.Cao@QIAGEN.com
QIAwebinars@QIAGEN.com
Questions?
Thank you for attending

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