Sample to Insight
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QIAGEN Service Solutions for Biomarker Research
QIAGEN Service Solutions for Biomarker Research
Samuel J. Rulli, Ph.D. Global Product [email protected]
Sample to Insight
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.
QIAGEN Service Solutions for Biomarker Research
Sample to Insight
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
QIAGEN Service Solutions for Biomarker Research
Sample to Insight
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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
QIAGEN Service Solutions for Biomarker Research
Sample to Insight
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QIAGEN's service core teams
QIAGEN Service Solutions for Biomarker Research
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?
QIAGEN Service Solutions for Biomarker Research
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
QIAGEN Service Solutions for Biomarker Research
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
QIAGEN Service Solutions for Biomarker Research
• 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
QIAGEN Service Solutions for Biomarker Research
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
Preclinical biomarker generation
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
Preclinical biomarker generation
Topic selection &
sample collection
Sample collection
& assay
ModelModel--basedbaseddata data
analysisanalysis
Preclinical biomarker generation
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
QIAGEN Service Solutions for Biomarker Research
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 Service Solutions for Biomarker Research
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
Sample to Insight
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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
QIAGEN Service Solutions for Biomarker Research
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Case study I: cancer biomarker
QIAGEN Service Solutions for Biomarker Research
• 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
QIAGEN Service Solutions for Biomarker Research
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
QIAGEN Service Solutions for Biomarker Research
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
QIAGEN Service Solutions for Biomarker Research
Researcher microarray or RNA-seq dataWhole-genome screening data selection and quality control
Custom pathway enrichment / feature ranking
• 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
Custom pathway enrichment / feature ranking
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Cancer biomarker
QIAGEN Service Solutions for Biomarker Research
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 Service Solutions for Biomarker Research
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
QIAGEN Service Solutions for Biomarker Research
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
Sample to Insight
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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
Sample to Insight
21QIAGEN Service Solutions for Biomarker Research
Cancer biomarker
Whole-genome screening data selection and quality controlLiterature-mining – gene-pathway-disease network
Sample to Insight
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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
QIAGEN Service Solutions for Biomarker Research
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
Sample to Insight
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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
QIAGEN Service Solutions for Biomarker Research
Sample to Insight
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Case study II: miRNA as a non-invasive biomarker for acetaminophen poisoning
QIAGEN Service Solutions for Biomarker Research
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
QIAGEN Service Solutions for Biomarker Research
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Stable extracellular miRNAs in blood
QIAGEN Service Solutions for Biomarker Research
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 Service Solutions for Biomarker Research
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?
QIAGEN Service Solutions for Biomarker Research
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
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microRNA biomarker strategy
QIAGEN Service Solutions for Biomarker Research
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Phase one: determine expressed miRNAs
QIAGEN Service Solutions for Biomarker Research
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
QIAGEN Service Solutions for Biomarker Research
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
QIAGEN Service Solutions for Biomarker Research
• 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?
QIAGEN Service Solutions for Biomarker Research
Sample to Insight
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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
QIAGEN Service Solutions for Biomarker Research
Sample to Insight
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Building a better biomarker – combining several RNA species
QIAGEN Service Solutions for Biomarker Research
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
QIAGEN Service Solutions for Biomarker Research
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
QIAGEN Service Solutions for Biomarker Research
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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
QIAGEN Service Solutions for Biomarker Research
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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
QIAGEN Service Solutions for Biomarker Research
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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
QIAGEN Service Solutions for Biomarker Research
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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
QIAGEN Service Solutions for Biomarker Research
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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
QIAGEN Service Solutions for Biomarker Research
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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
QIAGEN Service Solutions for Biomarker Research
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Total RNA discovery
QIAGEN Service Solutions for Biomarker Research
QIAGEN has expertise in designing projects from Sample to Insight
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Typical project timelines and milestones
QIAGEN Service Solutions for Biomarker Research
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
QIAGEN Service Solutions for Biomarker Research
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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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
QIAGEN Service Solutions for Biomarker Research
Sample to Insight
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
QIAGEN Service Solutions for Biomarker Research
Sample to Insight
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
QIAGEN Service Solutions for Biomarker Research
Sample to Insight
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
QIAGEN Service Solutions for Biomarker Research
Sample to Insight
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
QIAGEN Service Solutions for Biomarker Research
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DNA variant validation and assay development
QIAGEN Service Solutions for Biomarker Research
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
QIAGEN Service Solutions for Biomarker Research
• 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
QIAGEN Service Solutions for Biomarker Research
• 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
QIAGEN Service Solutions for Biomarker Research
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
QIAGEN Service Solutions for Biomarker Research
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
Sample to Insight
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Preserving limited DNA / RNA samples by WGA / WTA
QIAGEN Service Solutions for Biomarker Research
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
Sample to Insight
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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
Sample to Insight
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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
Sample to Insight
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Report for WGA service projects – validation of WGA process
QIAGEN Service Solutions for Biomarker Research
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
Sample to Insight
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Sanger sequencing – the gold standard in validation
QIAGEN Service Solutions for Biomarker Research
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
Sample to Insight
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Verification sequencing
QIAGEN Service Solutions for Biomarker Research
• 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
Sample to Insight
Verification sequencing – report
QIAGEN Service Solutions for Biomarker Research
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Sample to Insight
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Microbiome detection and quantification
QIAGEN Service Solutions for Biomarker Research
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)
Sample to Insight
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Microbial panels by micobiota or application
QIAGEN Service Solutions for Biomarker Research
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?
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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.
QIAGEN Service Solutions for Biomarker Research
Samuel J. Rulli, Ph.D. Global Product [email protected]