Accelerate Your Discovery with QIAGEN Service Solutions for Biomarker Research: qPCR Technology Webinar Series Part 4

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QIAGEN Service Solutions for Biomarker Research


Samuel J. Rulli, Ph.D. Global Product [email protected]

mailto:[email protected]

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Legal disclaimer

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.


http://www.qiagen.com/


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Welcome to our four-part webinar series on qPCR

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qPCR technology overview, applications, data

analysis and service solutions

• Part 1: Introduction to Real-Time PCR (Q-PCR / qPCR/ qrt-PCR)

• Part 2: Advanced Real-Time PCR Array Technology – Coding and Noncoding RNA Expression Analysis

• Part 3: PCR Array Data Analysis Tutorial

• Part 4: Accelerate Your Discovery With QIAGEN Service Solutions for Biomarker Research


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Building a better biomarker4

Agenda

Introduction to QIAGEN Service Solutions1

Case study I: cancer biomarker 2

Case study II: miRNA as a biomarker3

More from QIAGEN5


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QIAGEN's service core teams


QIAGEN Corporate, Hilden, Germany

QIAGEN Sciences, Frederick, USA

Samples can be accessed globally using standardized protocols and products

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Why QIAGEN services?


Enable easy access to innovative QIAGEN technologies• Testing technologies before establishing in house• Alternative to investing in your own equipment • Outsourcing

QIAGEN service is flexible• Work on part of a project or deliver Sample to Insight solution

QIAGEN service cores• Located in Frederick, MD, U.S. and Hilden, Germany

• Provide flexibility for customers to accept samples globally and provide standardized results

Access to R&D facility and manufacturing in Frederick and Hilden • Custom kits, assays, products and protocols

• Quick design and delivery

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QIAGEN biomarker development


Exploratory: research use Registration: IVD for IUO and PMA

Phase I – discovery Phase II – market release

Biomarker profiling and

validation

QIAGEN service portfolio QIAGEN contractual, CDx CoE

QIAGEN specialty teams consult and deliver customized solutions

Final diagnostic – QIAGEN CDx

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Service core offerings


• Sample prep service (miRNA, RNA, DNA, exosomes)• Illumina microarray expression profiling• Targeted exon next-generation sequencing (QIAGEN DNAseq

Gene Panels)• Targeted RNA-seq (100 to 1000) RNA targets (~July 2015)• PCR arrays for profiling mRNA, lncRNA and miRNA• qPCR for somatic mutation, CNV and microbial analysis• Whole genome amplification (WGA, repliG)• DNA methylation detection by pyrosequencing• Sanger DNA sequencing

• Custom Bioinformatics solutionso Gene / pathway enrichment from discovery researcho Biomarker algorithm and validation

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Preclinical biomarker development: difficulties


Discovery Development Finalization

High-throughput

screening

Selection of targets

Assay selection &

optimization

2nd Sample collection

& assay

Data analysis

Preclinical biomarker generation

Topic selection &

sample collection

Sample collection

& assay

ModelModel--basedbaseddata data

analysisanalysis


Disease-specific PCR Array

Traditional

New

2-3 y

0.5-1 y

High-throughput

screening

Selection of targets

Assay selection &

optimization

2nd Sample collection

& assay

Data analysis


Topic selection &

sample collection

Sample collection

& assay

ModelModel--basedbaseddata data

analysisanalysis


Disease-specific PCR Array

Traditional

New

2-3 y

0.5-1 y

Development time

Gene expression biomarkers are increasingly being used in medical research. Unfortunately, several challenges have limited development of valid biomarkers into practical use

• Challenges in biomarker developmento Limited quality of biomarker candidate identification due to limited data analysis resourceso Limited or no quality-controlled assay developments for converting assays from the discovery

platform to more sophisticated assay types (e.g., real-time PCR) due to limited experienceo Available bioinformatics expertise is often limited in scope, restricting the identification process

and reducing the likelihood of producing an effective signature panel and classification algorithm

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Primary sample prep using QIAGEN reagents and instruments


Nucleic acid purification from biological samples using QIAGEN sample preparation kits and instruments

Purification of• Genomic DNA• RNA (mRNA and lncRNA) • miRNA (total RNA)

Purification from• Blood, serum, plasma and Paxgene-stabilized blood• Exosomes• Frozen tissue and FFPE tissue• Cultured cells• Buccal swabs and saliva• Feces, urine and other body fluids• Custom applications (e.g., dried blood)

QIAGEN has over 20 years experience isolating nucleic acid from difficult samples for the most demanding downstream applications

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QIAGEN services for biomarker discovery


QIAGEN is more than “sample isolation”

microRNA • miScript miRNA PCR Array / Assay

DNA variants• GeneRead DNAseq Gene Panel• qBiomarker Somatic Mutation

Array / Assay• qBiomarker Copy Number

Array / Assay

mRNA/lncRNA detection• Illuminia microArray

• RT2 PCR Arrays (lncRNA / mRNA)

• qBiomarker hydrolysis probe qPCR

assays

• dRNA-seq

mRNA /lncRNA

DNA variants

miRNA

Computational analysis

Computational analysis / biomarker development• Custom pathway analysis from genome-wide data• Literature- and data-mining for focused biomarker

discovery• Biomarker algorithm and refinement• Ingenuity IPA consulting and professional services

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Agenda




More from QIAGEN5


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Case study I: cancer biomarker


• Estimated 60220 new cases of cancer (45310 women and 14910 men) and 1850 deaths in 2013

• The mortality from cancer has essentially remained unchanged over the last decade

• About one million people each year will have a nodule requiring further investigation

• Challenges and needs:o Separate tumor and non-tumor sample well from surgical samples.o Separate board line tumor from benign module with samples o Prognostic potentialo A qPCR based gene expression signature is good for fast diagnosis

help.

Carcinoma

Normal tissue

Clinical needs

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Total RNA discovery using the QIAGEN service core


From sample to biomarker Sample receiving

RNA isolation

Quality control of extracted RNA

Custom RT2 Biomarker PCR Array

Researcher shippedRNA sample

Illuminia microarray /RNA-seq

Custom pathway enrichment / feature ranking

Targets (47000)

Targets (96–384)

Biomarker signature (2–12) and algorithm

Microarray / RNA-seq validation and algorithm training

Microrray or RNA-seq

Laboratory- validated SYBR

Hydrolysis probe

Custom RT2 Biomarker PCR Array

Biomarker shortlist for independent validation and algorithm refinement

qPCR instrument (384-well)and / or Fluidigm 96 x 96

Optimization for specific platform (POC testing)

Researcher microarray or RNA-seq data

Assay transfer for LDT/ MDx

Ingenuity Professional Service

Ingenuity Professional Service

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Whole transcriptome profiling by microarray


Final data delivers:• All quality control and quantity of total RNA and amplified RNA

samples

• A Sample Probe Profile (generated using GenomeStudio) containing raw data for each gene that can be directly uploaded into GeneSpring for further analysis

• A Microsoft Excel workbook (generated from GeneSpring) containing the normalized expression data, fold change for each gene and list of two-fold and significant up- or downregulated genes

• A pathway-focused gene expression profile that can be provided to the customer upon request.

• Scatter plots, bar plots, line plots, box plots, heat maps and cluster analysis dendrograms are available as custom data analysis options

• All original image data files (.idat) can be provided to the customer upon request

Illumina CSPro certified in 2011 to provide Infinium Genotyping and Direct Hybridization gene expression

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Cancer biomarker


Researcher microarray or RNA-seq dataWhole-genome screening data selection and quality control


• Microarray dataset o Provided by researcher

and/or GEO database

• Selection and QCo Identify “outliers” in the

dataset

o Does not include the two outliers for downstream analysis


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Cancer biomarker


Whole-genome screening data selection and quality control Data mining, gene selection, assay development

Gene selection:• Multiple algorithms for the selection

o Random foresto Nearest shrunken centroid o Support vector machine

• Uses all plus genes from literature-mining

• Use custom RT2 Profiler PCR Array

• Assay verified by specificity and sensitivity

50

RF

SVMNSC51 865

6413

Microarray Data

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QIAGEN RT2 and miScript PCR Arrays


QIAGEN PCR arrays offer a standardized workflow on any qPCR instrument• Results obtained in QIAGEN’s service core can be repeated

• Patented controls ensure reliability every time samples are run

PCR arrays – panels of pre-validated qPCR assays. One manufacturing lot can be sent to different sites to ensure the same qPCR performance for multi-site studies

In-process control assays: for data normalization, sample quality and process robustness

Any qPCR instrument

Pathway / disease content: Laboratory-validated qPCR primer assays for most relevant genes involved in pathway / disease or custom content

QIAGEN received U.S. Patent (8,597,938) for the “system for providing control reactions for real time RT-PCR”

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RT2 Profiler / lncRNA Catalog PCR Arrays – over 170 pathways


Adherens junctionsAdipogenesisAllergies and asthmaAlzheimer‘s diseaseAmino acid metabolism (I / II)AngiogenesisAngiogenic growth factorsAntibacterial responseAntifungal responseAntiviral responseApoptosis AtherosclerosisAutophagyBreast cancercAMP calcium signaling pathwayCancer drug resistanceCancer drug targetsCancer PathwayFinderCardiotoxicityCell cycleCell Death PathwayFinderCell Junction PathwayFinderCell lineage identificationCell motilityCell surface markersCellular senescenceCellular stress responsesChemokines and receptorsCrohn‘s diseaseCircadian rhythmsComon cytokinesCystic fibrosisCytokines and chemokinesCytoskeleton regulatorsDendritic and antigen-presenting cellsDiabetesDNA damage signaling pathway

DANN repairDopamine and serotonin pathwaysDrug metabolismDrug metabolism phase I / II enzymesDrug transportersEGF PDGF signaling pathwayEmbryonic stem cellsEndothelial cell biologyEpigenetic chromatin modification enzymesEpigenetic chromatin remodeling factorsEpithelial to mesenchymal transitionEstrogen receptor signalingExtracellular matrix and adhesion moleculesFatty acid metabolismFatty liverFemale infertilityFibrosisFocal adhesionsGABA and glutamateGap junctionsGlucocorticoid signalingGlucose metabolismGlycosylationG protein-coupled receptorsGPCR PathwayFinderGrowth factorsHeat shock proteins and chaperonesHedgehog signaling pathwayHematopoiesisHepatoxicityHIV host responseHomeobox HOX genesHousekeeping genesHuntington‘s diseaseHypertensionHypoxia signaling pathwayIL6 / STAT3 signaling pathway

InflammasomesInflammatory cytokines and receptorsInflammatory response and autoimmunity Innate and adaptive immune responsesInsulin resistanceInsulin signaling pathwayInterferons and receptorsJAK / STAT signaling pathwayLet-7 targetsLeukemiaLipoprotein signaling and cholesterolLiver cancerLung cancerLymphomaMacular degenerationMale infertilityMAP kinase signaling pathwayMesenchymal stem cellsmiR-1 and miR-206 targetsmiR-146a targetsmiR-153 targetsmiR-155 targetsmiR-17 and miR-20a targetsmiR-181 targetsmiR-21 targetsmiR-210 targetsmiR-221 and miR-222 targetsmiR-29 targetsmiR-34a targetsmiR-4514 and miR-4692 targetsMitochondriaMitochondrial energy metabolismMolecular Toxicology PathwayFindermTOR signalingMultiple sclerosisNecrosisNephrotoxicity

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20QIAGEN Service Solutions for Biomarker Research

NeurogenesisNeuronal ion channelsNeurotoxicityNeutrotransmitter receptorsNeurotrophins and receptorsNF-kB signaling pathwayNF-kB signaling targetsNitric oxide signaling pathwayNotch signaling pathway / targetsNuclear receptors and coregulatorsObesityOncogenes and tumor suppressor genesOsmotic stressOsteogenesisOsteoporosisOxidative stressP53 signaling pathwayPain neuropathy and inflammatoryParkinson‘s diseasePI3K / AKT signaling pathwayPolycomb and trithorax complexesPolycystic kidney diseasePPAR targetsPre-eclampsiaPrimary ciliaProstate cancerProtease activated receptor signalingProtein phosphatasesRNA QCSignal Transduction PathwayFinderSkeletal muscle myogensis and myopathyStem cell / signalingStem cell transcription factors

Stress and toxicity pathway finderSynaptic plasticityT cell anergy and immune toleranceT cell and B cell activationTelomeres and telomeraseTerminal differentiation markersTGFb / BMP signaling pathwayTGFb signaling targetsT helper cell differentiationTh1 and Th2 responsesTh17 responseTight junctionsTNF signaling pathwayToll-like receptor signaling pathwayTranscription factorsTransplant rejectionTumor metastasisType I interferon responseTyrosine kinasesUbiquitin ligasesUbiquitinationUnfolded protein responseVEGF signalingWNT signaling pathway / targetsWound healing

lncRNA FinderlncRNA CancerFinderlncRNA development / differentiationlncRNA autoimmunity / inflammation

RT2 Profiler/lncRNA Catalog PCR Arrays – over 170 pathways

• Expertly curated by PhD scientists

• Perfect for discovery or validation studies

• Economical for large studies

• Quick turnaround time

• Run multiple panels on the same sample for deeper understanding

• Results can quickly be turned into custom biomarker panels or transferred to hydrolysis assays

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Cancer biomarker

Whole-genome screening data selection and quality controlLiterature-mining – gene-pathway-disease network

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Custom RT2 Biomarker PCR Array – next-generation PCR arrays

• Proprietary qPCR algorithm designed to create a hydrolysis probe assay that works under both SYBR® and hydrolysis probe cycling conditions for all genes in species

• For initial studies, while in SYBR® Green mode, the probe is not used. qPCR analysis is done using SYBR® Green Master Mix

• For biomarker validation, the hydrolysis probe is added back to the qPCR assay and hydrolysis master-mix is used and optimized for performance in specific instrument platforms

• This product and qPCR design algorithm are currently used exclusively for service and biomarker development projects

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Cancer biomarker


qPCR biomarker validation

Importance

NPC2 CHI3L1

S100A11 TBPSDC4 RPL13ACD53 RPS13MET HSP90AB1

GCSH YWHAZ

Prediction

Accuracy 91.7%

Sensitivity 87.5%

Specificity 100.0%

PPV 100.0%

NPV 80.0%

Sample collection Assay setup Feature ranking

Algorithm trainingBiomarker shortlistIndependent validation

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Agenda




More from QIAGEN5


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Case study II: miRNA as a non-invasive biomarker for acetaminophen poisoning


In order to use a biomarker for diagnostics, the sample material must be as easy to obtain as possible

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miRNAs are being pursued as biomarkers in many diseases


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Stable extracellular miRNAs in blood


Exosomes & microvesicles1,2,3

miRNA

Ago

Ago-2-miRNA complexes4

HDL mediatedmiRNA transport5

Other ‘protective’protein6

High levels of nucleases present in plasma • Freely circulating RNA should be rapidly degraded• Surprisingly, stabile miRNA can be detected in serum and plasma• QIAGEN has expertise in plasma, serum & exosomal RNA isolation

and detection

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miRNA detection and quantification by qPCR


QIAGEN specializes in using miRBase 21

Find new miRNAs or establish superior intellectual property around your discovery!

Other species available – dog, monkey, cow, corn, etc.

Species Mature miRNAs

Human 2402

Mouse 1146

Rat 653

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Does a miRNA signature for acetaminophen poisoning exist?


Tylenol® overdose is one of the most common causes of poisoning worldwide. In the US and UK it is the most common cause of acute liver failure.

With Dr. James Dear, Edinburgh University

Hours 0–24• Mild symptoms

Hours 24–48• Symptoms of acute liver poisoning

Beyond 48 hours• Complete hepatic failure, kidney failure, cerebral edema,

sepsis, multiple organ failure and death

http://en.wikipedia.org/wiki/Acute_liver_failure

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microRNA biomarker strategy


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Phase one: determine expressed miRNAs


Survey the miRNA landscape for your system

Which miRNAs were selected?• miRNAs expressed in all four pools• miRNAs expressed in three pools• miRNAs expressed in two pools• miRNAs expressed in one pool

~377 assays

• Plasma samples (54):o Control samples (27)o Liver injury samples (27)

• Total RNA Isolation: miRNeasy Serum / Plasma Kito Note: perform QC at this step to ensure samples are devoid of inhibitors

• Prepare random pools with 10 samples per pool:o Two control poolso Two patient pools

• miRNA Expression Profiling: miScript PCR System and Human miRNome (1850 assays)

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Phase two: determine differentially expressed miRNAs


Secondary screen of individual samples from phase one• Plasma samples (54):

o Control samples (27)o Liver injury samples (27)

• miRNA Expression Profiling: miScript PCR System and expressed miRNAs (377 assays)

Which miRNAs were differentially expressed?

Log10 (Control Group 2-ΔCT)

Log 1

0 (Liv

er In

jury

Gro

up 2

-ΔCT

)

miR-122-5p

miR-885-5p

Log2 (FC of Liver Injury Group/Control Group)

-Log

10 (P

-val

ue

miR-122-5p

miR-885-5p

Scatter Plot Volcano Plot

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Phase three: statistical power


• Samples: Screen larger cohort of affected individuals• Assays: Re-array differentially expressed miRNAs• Continually refine classifier model

o Original 54 samples, top 16 miRNAs

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Validate miRNA signature on naïve samples

Things to consider Strong changes might be general indicators or even

non-specific (e.g., other liver disease or stress markers)

Relatively weak changes may add specificity High level of false positives is undesirable Should you add additional RNAs?


http://www.google.com/url?sa=i&rct=j&q=&esrc=s&frm=1&source=images&cd=&cad=rja&docid=TS1KlPU1Jl_V0M&tbnid=3u99ISWe9Kxz_M:&ved=0CAUQjRw&url=http://www.qiagen.com/Products/Catalog/Automated-Solutions/Sample-Prep/BioRobot-Universal-System&ei=TkQ7UtaYIZOx4APgrIDgCQ&bvm=bv.52288139,d.dmg&psig=AFQjCNELSkEcJc1F4GmHjuTihZk1aX6OXw&ust=1379702217477816

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Agenda




More from QIAGEN5


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Building a better biomarker – combining several RNA species


Can specificity for a biological process or disease be increased by combining several RNAs?

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Total RNA discovery starts with sample isolation


Simple workflow to capture all the RNA in a sample

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Workflow

• Samples: frozen thyroid tissue

o Benign: nodular hyperplasia (NH) and follicular adenoma (FA)

o Cancer: papillary thyroid cancer (PTC) and follicular variant of papillary thyroid carcinoma (FVPTC)

• Sample prep: miRNeasy Mini Kit

• Real-time PCR Assays:o mRNA and lncRNA: RT2 Expression System

o mRNA: Thyroid cancer-related mRNAs

o lncRNA: Cancer PathwayFinder

o miRNA: miScript PCR System

o miRNA: miFinder 384HC

• Data Analysis: GeneGlobe Data Analysis Center

• Interpretation: Ingenuity Pathway Analysis


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mRNA expression results

• mRNAs upregulated: 68o CHI3L1 (Jarzab et al., Cancer Res, 2005)

• mRNAs downregulated: 46o KIT (Marini et al., Journal of Thyroid

Research, 2011)

• A Custom RT2 PCR Array that features mRNAs potentially expressed in thyroid tissue is well suited for thyroid cancer expression profiling


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lncRNA expression results

• lncRNAs upregulated: 6 (3 significant)o PCA3 (oncogene)

o MIR31HG (114-fold upregulated)

• lncRNAs downregulated: 6 (3 significant)o LINC00261 and PTCSC1 (tumor-suppressor

genes)

• The lncRNA Cancer PathwayFinder Array is a great tool to assess lncRNA expression in cancer samples


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miRNA expression results

• miRNAs upregulated: 17 (11 significant)o hsa-miR-146b-5p and hsa-miR-221-3p

(Chen et al., Mod Pathol, 2008)

• hsa-miR-31-5p (136-fold) and hsa-miR-31-3p (75-fold) significantly up-regulatedo lncRNA MIR31HG is also robustly

upregulated (114-fold)

• miRNAs downregulated: 4 (1 significant)o hsa-miR-211-3p (downregulated in breast

cancer)

• The miFinder 384HC can uncover potentially high impact miRNAs associated with thyroid cancers


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Understanding miRNA regulation of mRNAs in thyroid carcinoma

• MicroRNA Target Filter: searched for mRNA targets that were differentially expressed in papillary thyroid carcinoma (PTC) and follicular variant of papillary thyroid carcinoma (FVPTC)o For example, seven miRNAs target seven mRNAs

• Filterso Inverse pairing between miRNA & mRNAo Relationships observed in Cancer and or Endocrine Disorderso Selected signaling pathwayso Thyroid Gland


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Seven miRNAs target seven mRNAs to collectively affect invasion of cells

Importance of miRNA regulation is shown here by targeting genes involved in invasion of cells necessary for tumor progression and metastasis


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miRNA / mRNA / lncRNA expression patterns affect metastasis progression

Using MAP (molecule activity predictor): metastasis is predicted to be increased based on expression pattern of miRNA, mRNA and lncRNA


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Total RNA discovery


QIAGEN has expertise in designing projects from Sample to Insight

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Typical project timelines and milestones


Stage II: five weeks

Stage III: fifteen weeks

Stage V: clinical

Stage I: five weeks

Assay development

Data mining and biomarker ranking

QIAGEN qPCR biomarker assay development in ~26 weeks

Biomarker optimization

Cell-based phenotypic assays

Clinical development

Sample collection qPCR assays Signature selection

Variable

Stage IV: four weeks

Biomarker finalization

No. of Targets

No. of Samples

~ 10000s ~100s 10~20

>100s10~100s10~100s

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QIAGEN – providing solutions through the continuum


Biomarker profiling and

validationFinal diagnostic – QIAGEN CDx

QIAGEN’s expertise in biomarker assay development, services and

interpretation can be transferred to later diagnostic development

Seamless assay transfer keeping target region consistent and optimization for point of care testing

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Agenda




More from QIAGEN5


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Targeted DNAseq for validation or discovery Minimal DNA input

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Actionable Mutations

Clinically-relevant

Tissue-specific

Comprehensive

Breast Cancer

Colorectal Cancer

Myeloid Ceoplasms

Liver Cancer

Lung Cancer

Ovarian Cancer

Prostate Cancer

Gastric Cancer

Cardiomyopathy

Cancer

Carrier testing

Cancer predisposition

Only 40 ng DNA needed per panel(except Actionable Mutations Panel which needs a total of 20 ng)

Largest collection of predesigned panels suitable for a wide range of needs


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GeneRead DNAseq Targeted Panels V2

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Application Panel name #genes

Target region(bases)

Coverage(%)

Specificity(%)

Uniformity(%)

Solid tumorsTumor Actionable Mutations 8 7,104 100.0 98.2 91

Clinically Relevant Tumor 24 39,603 98.1 95.3 90Hematologic 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


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Customize a GeneRead panel for your research goals

Minimal DNA input

Actionable Mutations

Clinically-relevant

Tissue-specific

Comprehensive

Breast Cancer

Colorectal Cancer

Myeloid Neoplasms

Liver Cancer

Lung Cancer

Ovarian Cancer

Prostate Cancer

Gastric Cancer

Cardiomyopathy

Mix and Match – Select from 570 verified gene designs to make your own panel


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Build your unique panel – and let us verify it for you

GeneRead DNAseq Custom Panel

Customization tools to fit your requirements:

Increase breadth of coverageAdjust amplicon length

Allow denser tilingAdd QC primers

Adjust exon paddingAdd UTRs to CDS

Gene(s) or Genomic region(s) of interest (GROI)

Fully customized panel

Choose any gene from the human

genome OR almost any genomic region

Build a custom panel with a

turnaround time of two weeks

Pick your genomic loci and validate your panel with QIAGEN service core – or just pick your genes and send us your samples


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DNA variant validation and assay development


Allele-specific amplification using ARMS – Amplification Refractory Mutation System

• With the 3’-residue matching at the exact spot of the mutation, your mutant template will amplify

• Oligos with a mismatched 3’-residue will not function as primers in a PCR reaction

F Q

F Q

No amplification

Positive call for mutation

Common probe

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Assay sensitivity – p53 R280K mutation detection


• Test mixed genomic DNA samples of wild type and mutant

• Mutant Source: MDA-MB-231

• Wild type Source: GM00131

• DNA samples contain 4, 21, 106, 528, 2640 and 13200 copies of mutant DNA

• Mutation detection limit for this assay is determined to be 0.03%.

• Sensitivity is ample enough for DNAseq validation and screening

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qBiomarker Somatic Mutation Assay Coverage


• ABL1 (14)• AKT1 (2)• AKT3 (1)• ALK (10)• APC (63)• ATM (6)• BRAF (16)• CBL (4)• CDH1 (5)• CDKN2A (19)• CEBPA (10)• CRLF2 (1)• CSF1R (1)• CTNNB1 (51)• EGFR (35)• ERBB2 (15)• EZH2 (3)• FBXW7 (7)• FGFR1 (2)• FGFR2 (2)• FGFR3 (12)

• FLT3 (20)• FOXL2 (1)• GATA1 (4)• GATA2 (1)• GNAQ (3)• GNAS (6)• HNF1A (2)• HRAS (14)• IDH1 (5)• IDH2 (4)• JAK2 (7)• KIT (32)• KRAS (24)• MEK1 (4)• MET (9)• MPL (3)• NF1 (14)• NOTCH1 (8)• NOTCH2 (1)• NPM1 (7)• NRAS (17)

• PDGFRA (27)• PIK3CA (59)• PIK3R1 (3)• PIK3R5 (1)• PTCH1 (5)• PTEN (42)• PTPN11 (16)• RB1 (5)• RET (10)• RUNX1 (10)• SMAD4 (1)• SMARCB1 (7)• SMO (1)• SRC (1)• STK11 (17)• TET2 (1)• TP53 (113)• TSHR (15)• VHL (16)• WT1 (4)

Total Entries in COSMIC

105929

Our >1200 assays cover over 77819 of these occurrences

75% coverage

Custom Assay Development and Validation Service Available

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Copy number detection


qPCR is an economical method for detection CNV in 100s of samples

1 x

2x

3 x

Sample 1

Sample 2

Sample 3

A B C D

A B C DB

A B C DB B

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200 base pair Tile

qBiomarker copy number assays


Tiling the human genome at 200 base pair resolution

Gene BGene A

CNV Assay design

• “Virtually cut” the genome into 200 bp tiles

• Yielded ~11.6 million designable tiles

• Design a Primer assay within the tile

• Lab-test the Primer assay for performance

Multicopy Reference Assay

• Recognizes a stable sequence present in the human genome over 60 times

• Not affected by local genomic changes

• More accurate than TERT or RNAase P normalization

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Preserving limited DNA / RNA samples by WGA / WTA


Consistent and accurate Whole-genome amplification

• Highly uniform whole-genome amplification from small or precious samples

• Based on Multiple Displacement Amplification (MDA) technology

• 100 µg or 500 µg genomic DNA from as little as 10 ng starting material

• Starting materials include genomic DNA, whole blood, dried blood spots, buccal swabs, tissue and cells

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Next-generation sequencing of WGA-amplified gDNA

Copy number changes in a bladder tumor sample compared to normal tissue for purified gDNA and WGA-amplified gDNA

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Normal tissue sample

Cancer tissue sample

Next-generation sequencing of WGA-amplified gDNASomatic mutations as analyzed by the CLC Cancer Research Workbench with and without WGA

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Report for WGA service projects – validation of WGA process


The results of the WGA reaction are provided as a Microsoft Excel worksheet and include:

• A report on yields and assessment of the quality of the amplification results

• A worksheet with sample names

• A worksheet with results in a tabular format

Data delivery – download from FTP server

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Sanger sequencing – the gold standard in validation


Single-read services

Routine sequencing of: • Plasmid inserts • PCR products Both 96-well / 384-wellformats

Verification services

Comparison with reference sequences

Speciality services

• Validation of variants from NGS• De Novo Sequencing• siRNA Hairpin Run• MLST (Multi Locus Sequence Typing) • Direct Genomic DNA sequencing

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Verification sequencing


• Verification compared to reference sequence• Includes cultivation and template preparation (if necessary)• Includes primer selection and resourcing based on reference

sequence (primer walking strategy)

• Manual editing of sequencing data• Data delivery comprises final report plus raw data

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Verification sequencing – report


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Microbiome detection and quantification


QIAGEN pipeline for microbial detection

• Bacteria identification assay (>300)

• Fungi identification assay (8)

• Protist identification assay (1)

• Antibiotic resistance genes (87)

• Virulence factor genes (87)

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Microbial panels by micobiota or application


Sixteen pre-cataloged arrays• Antibiotic resistance genes• Bacterial vaginosis• Biodefense• Food testing:

o Dairyo Meato Poultryo Seafoodo Vegetable

• Intestinal infections• Respiratory infections• Sepsis • Urinary tract infections• Vaginal flora • Water analysis• Customizable Arrays

o Pick from over 500 assayso Service core design and validates

your targets

Sample to Insight

Contact QIAGENCall: 1-800-426-8157

Email: [email protected]

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Thank you for attending today’s webinar!

Questions?

Thank you for attending!

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.


Samuel J. Rulli, Ph.D. Global Product [email protected]






Healthcare

Accelerate Your Discovery with QIAGEN Service Solutions for Biomarker Research: qPCR Technology Webinar Series Part 4