Post on 23-Jan-2017
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Automation of ccfDNA isolation using the QIAsymphonyDr. Marco Polidori
Global Product Manager
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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
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Technical Services or your local distributor.
Automation of ccfDNA isolation using the QIAsymphony
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Agenda
Introduction
Challenges of ccDNA isolation
ccDNA isolation methods
Validation data
Application data
Summary
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Automation of ccfDNA isolation using the QIAsymphony
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Free circulating Nucleic acids• DNA• mRNA• miRNA
Exosomes• Total RNA• DNA• Protein
Circulating Tumor Cells• Enumeration• Genotyping• Gene expression
The main areas of Liquid Biopsy
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Liquid Biopsy: Game changing potential
Automation of ccfDNA isolation using the QIAsymphony
From sequencing an entire fetal genome using maternal plasma…
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Liquid Biopsy: Game changing potential
Automation of ccfDNA isolation using the QIAsymphony
…to groundbreaking oncology studies
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Application areas
Automation of ccfDNA isolation using the QIAsymphony
New paradigm: Less invasive
bio3400.nicerweb.com
Zeit.de
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QIAGEN Sample to Insight Solutions for ccfDNA
Automation of ccfDNA isolation using the QIAsymphony
QIAamp Circulating Nuecleic Acid Kit
QIAsymphony free circulating DNA Kit
GeneRead DNAseq cancer panel v2
CLC cancer workbench
Ingenuity Variant Analysis
QIAseq Ultra Low Input Library prep Kits
Any sequencerPAXgeneccfDNA Tubes (in development)
Sample Collection
Sample Isolation Amplification Library
preparation SequencingData analysis
& interpretation
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9Automation of ccfDNA isolation using the QIAsymphony
Blood draw (venipunctur
e)Separate plasma
(Logistics)
Extract circulating nucleic acids:
QIAamp Circulating NA Kit,QIAsymphony Circulating NA Ki
Real-time PCR digital PCR
therascreen assays
Next-generation sequencing
SamplePre-
analytical workflow
Analytical workflow Results
Circulating nucleic acids: Analysis workflow
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Critical points along the workflow
Automation of ccfDNA isolation using the QIAsymphony
Real-time PCR digital PCR
Sequencing library prep
Next-generation sequencing
• gDNA background due to hemolysis Stabilization
• Very low concentration of ccfDNA Efficient large volume prep
• Low concentration efficient and sensitive downstream processing
Optional DNA modification (e.g., bisulfite treatment)
Blood draw (venipunctur
e)
Extract circulating nucleic acids:QIAamp Circulating NA Kit,QIAsymphony ccfDNA Kit
Separate plasma
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Sample handling: An example
Automation of ccfDNA isolation using the QIAsymphony
Mutant allele frequency variation and concentration example
Chetan Bettegowda et al. 2014 doi:10.1126/scitranslmed.3007094
Amount of mutated fragments vary strongly from <100 to >100,000 per 5 ml.
Amount of ctDNA also depends on cancer type and tumor burden!
Effectively, between 10 and 20 ml of blood should be taken for ccfDNA studies
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Store at -80 °CThaw plasma
Extraction of cfDNA
Draw whole blood in Cell-Free DNA BCT (Streck)
Spin @ 300g for plasma separation
Spin @ 16,000g
Supernatant: plasma w/o cell debris and
reduced gDNA background
Carefully save supernatant
Spin @ 1900g for plasma separation
Draw EDTA whole blood
1-2h 14 days
Blood sample processing
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Sample stabilization: PAXgene project
Automation of ccfDNA isolation using the QIAsymphony
No elevated levels of free DNA using PAXgene ccfDNA Tubes*
t1 t3 t6 t8 t10 t1 t3 t6 t8 t10EDTA PAXccfDNA
0
50
100
150
200
250
300
350 18S rDNA qPCR assay
66 bp amplicon
500 bp amplicon
ratio
tx/t0
*product under development
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EDTA t0 PAXccfDNA t0 EDTA t10 PAXccfDNA t10
35 100 200 300 400 500 700 2000 10380 [bp]
Sample stabilization: PAXgene project
Efficient long-term stabilization of cells and extraction of ccfDNA
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In development (automated):QIAsymphony Circulating DNA KitUsing new beads and chemistry 2 or 4 ml input | plasma from EDTA and Streck tubes 96 samples | 6 hours (hands-off) | IVD use
Automation of ccfDNA isolation using the QIAsymphony
Current solution (manual): QIAamp Circulating NA Kit≤5 ml plasma input | 24 samples | 3 hours
2009
MBA 2015
IVD 2016
Evolution of cfDNA extraction
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QIAsymphony SP instrument: Workflow
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Binding buffer
Proteinase K
Plasma
Magnetic beads
Binding at RT
Bead separation
Elution in 60 µl
Wash step 1
Wash step 2
Binding at RT
Bead separation
Wash step 3
2 ml protocol
4 ml protocol
QIAsymphony SP instrument: Workflow
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Size distribution of extracted ccfDNA from plasma
Automation of ccfDNA isolation using the QIAsymphony
Set-up of experiment:• ccfDNA extracted from 4 ml
maternal plasma using:• QIAamp Circulating NA kit as
manual reference method (red)
• QIAsymphony® Circulating DNA Chemistry (blue)
• 1 μl eluate subjected to Agilent High Sensitivity DNA Kit (5-500 pg/μl)
Set-up of experiment:• ccfDNA extracted from 4 ml
plasma using:• QIAamp Circulating NA kit as
manual reference method (red)
• QIAsymphony® Circulating DNA Chemistry (blue)
• 5 ng eluate subjected to library prep (ThruPLEX-FD Prep Kit; Rubicon Genomics)
• 1 μl purified eluate from library prep subjected to Agilent DNA 7500 Kit
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•Qubit™ dsDNA HS Assay Kit: 0.23-0.56 ng/µl
•GeneRead Library Prep I Core Kit: Input 10 µl (2.3-5.6 ng DNA)
•Quantification of ccfDNA by qPCR; 10 nM required for Illumina Sequencing applications (green)
•MiSeq NGS run using 5 nM (5 µl) Calculation of mapped reads distributed on chromosomes
Results: Compatibility of ccfDNA eluates in library prep and NGS
End repair A-addition Adapter ligation
Cleanup and size selection
HiFi library amplification (optional)
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4 ml plasma
QS Circulating DNA Kit
QIAamp Circulating NA Kit
RT-PCR: 18S-66 bp
Qubit dsDNA HS Assay Kit
Results: Detection of ccfDNA comparing RT-PCR & Qubit™
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4 ml plasma
QS Circulating DNA Kit
RT-PCR: 18S-66 bp (∑20 µl) 2 µl - 4 µl - 8 µl
2 ml plasma 6 ml plasma
Results: Linearity for 2-6 ml plasma input
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Fill up to 2 ml 2 ml transferred
Liquid Level Detection (LLD)
FIX (no LLD)
Transfer of 1-2 ml plasma (dead volume required)
Results: Variations in sample input volume
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LoD
LoB
Set-up of experiment:
• 4 ml female plasma spiked with male plasma from 2-1000 µl
• DNA extracted from 4 ml plasma using the QS Circulating DNA Kit
• Male DNA yield determined by real-time PCR (DYS-14, SRY1) using the QIAGEN QuantiTect® Multiplex PCR Kit
• Results for SRY1 (upper figure) and DYS14 (lower figure) are depicted as copies per ml plasma
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Results: Sensitivity for low copy recovery
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Set-up of experiment: • Urine samples from 10 healthy donors; 3-4 ml urine as sample input• Circulating DNA yield determined by real-time PCR (18S coding
sequence, 66 bp amplicon) using the QIAGEN QuantiTect® Multiplex PCR Kit
• Results were calculated as target copies per ml plasma and compared to the results obtained with the QIAamp® Circulating Nucleic Acid Kit
Extraction of ccfDNA from 4 ml urine
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cut-off
4 ml plasma
QS Circulating DNA Kit
QIAamp Circulating NA Kit
QuantYfeX® assay PrenaTest®
Early Access Kit: Customer feedback
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Set-up of experiment: • plasma samples from 9 clinical donors; 3.0-3.5 ml
plasma as sample input• Circulating DNA yield determined by Qubit™ dsDNA HS
Assay Kit used with the Qubit® 2.0 Fluorometer: Results were calculated as ng DNA per µl eluate from 3.0-3.5 ml plasma input and compared to the results obtained with the QIAamp® Circulating Nucleic Acid Kit
• 12 µl eluate was subjected to library prep (Ion AmpliSeq™Library Kit 2.0, Thermo Fisher)
• Subsequently 3 nM of each ccfDNA was transferred to the pool which was diluted to 12 pM for targeted NGS. Calculated mutation frequency was compared to the results obtained with the QIAamp® Circulating Nucleic Acid Kit
1:w/o, 2:TP53 exon5, 3:KRAS exon2, 4.1:EGFR exon19, 4.2:EGFR exon20, 5:w/o, 6:EGFR exon19, 7:w/o, 8.1:EGFR exon21, 8.2:EGFR exon20, 8.3:TP53 exon3, 9: w/o
Detection of caner ccfDNA from clinical samples
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Summary
Automation of ccfDNA isolation using the QIAsymphony
• Proper tube choice and handling can minimize gDNA background
• Use at least 2-5 ml plasma (or other biofluids) whenever possible to maximize sensitivity
• The QIAsymphony Circulating DNA Kit isolates ccfDNA with the same or higher efficiency than QIAamp Circulating NA reference
• The QIAsymphony handles 96 samples in about 6 hours hands free starting directly with plasma/serum
• Input volumes for the QIAsymphpny Circulating DNA Kit can be ramped up to 6 ml
• Expected launch of IVD certified version in October 2016, already available as MBA (non-IVD)
Some take home points
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Visit QIAGEN blogs: Biomarker Insights
Automation of ccfDNA isolation using the QIAsymphony
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Katharina BellerMartin HorlitzThorsten VossManuel FrietschKevin MatthaeiStephan RachwalAgata StoltmannRita KistAnnabelle SchubertAnnette NoconSandra HammerschmidtNicole HoffmannStephanie AngenendtPatricia WeideDagmar HeroldYun Kyung LeeAndrea KloseGaby BrockerhoffSilke SonnwaldNan FangHolger Wedler
Many thanks to…
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Marco Polidori, Ph.D.Marco.Polidori@qiagen.com
Tel: +49 2103 29 11441
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