Liquid biopsy: Overcome Challenges of Circulating DNA with Automated and Standardized Extraction Processes
•
2 likes•3,004 views
Circulating cell-free DNA (ccfDNA) originating from malignant tumors, a developing fetus and also from inflammatory tissues, is present in the cell-free nucleic acids in plasma, serum and other body fluids and is considered a “liquid biopsy”. Access to ccfDNA for analysis allows for specific detection of certain disease states based on a simple blood sample. Circulating cell-free DNA shows distinctive properties – it is present mostly as shorter fragments of less than 500 bp and the concentration of ccfDNA in a plasma or serum sample is low (approximately 1–100 ng/ml) compared to cellular materials and varies considerably between different individuals. Because of their fragmented nature and low concentration, ccfDNA presents a particular challenge for efficient extraction / purification and quantification, such as by qPCR. We present data on solutions for the following critical problems concerning the purification of ccfDNA for research and molecular diagnostic applications: • Pre-analytical workflow (blood processing) for analyzing ccfDNA • Optimization of ccfDNA extraction from plasma samples: low target concentrations require efficient ccfDNA enrichment from larger sample volumes • Novel automated extraction of ccfDNA using the QIAsymphony SP instrument for liquid biopsy diagnostic applications.
Recommended
Recommended
More Related Content
What's hot
What's hot (20)
Viewers also liked
Viewers also liked (20)
Similar to Liquid biopsy: Overcome Challenges of Circulating DNA with Automated and Standardized Extraction Processes
Similar to Liquid biopsy: Overcome Challenges of Circulating DNA with Automated and Standardized Extraction Processes (20)
More from QIAGEN
More from QIAGEN (20)
Recently uploaded
Recently uploaded (20)
Liquid biopsy: Overcome Challenges of Circulating DNA with Automated and Standardized Extraction Processes
- 1. Sample to Insight Automation of ccfDNA isolation using the QIAsymphony Dr. Marco Polidori Global Product Manager 1
- 2. Sample to Insight Legal disclaimer 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. Automation of ccfDNA isolation usingthe QIAsymphony
- 3. Sample to Insight Agenda 3 Introduction Challenges of ccDNAisolation ccDNAisolation methods Validation data Application data Summary 1 2 3 Automation of ccfDNA isolation usingthe QIAsymphony 4 5 6
- 4. Sample to Insight Automation of ccfDNA isolation usingthe QIAsymphony 4 FreecirculatingNucleicacids • DNA • mRNA • miRNA Exosomes • Total RNA • DNA • Protein CirculatingTumorCells • Enumeration • Genotyping • Gene expression The main areas of Liquid Biopsy
- 5. Sample to Insight Liquid Biopsy: Game changing potential Automation of ccfDNA isolation usingthe QIAsymphony 5 From sequencing an entire fetal genome using maternal plasma…
- 6. Sample to Insight Liquid Biopsy: Game changing potential Automation of ccfDNA isolation usingthe QIAsymphony 6 …to groundbreaking oncology studies
- 7. Sample to Insight Application areas Automation of ccfDNA isolation usingthe QIAsymphony 7 New paradigm: Less invasive bio3400.nicerweb.com Zeit.de
- 8. Sample to Insight QIAGEN Sample to Insight Solutions for ccfDNA Automation of ccfDNA isolation usingthe QIAsymphony 8 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 sequencerPAXgene ccfDNA Tubes (in development) Sample Collection Sample Isolation Amplification Library preparation Sequencing Data analysis & interpretation
- 9. Sample to Insight 9Automation of ccfDNA isolation usingthe QIAsymphony Blood draw (venipuncture ) Separate plasma (Logistics) Extract circulating nucleic acids: QIAampCirculating NA Kit, QIAsymphony CirculatingNA Ki Real-time PCR digital PCR therascreen assays Next-generation sequencing Sample Pre- analytical workflow Analytical workflow Results Circulating nucleic acids: Analysis workflow
- 10. Sample to Insight Critical points along the workflow Automation of ccfDNA isolation usingthe QIAsymphony 10 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 (venipuncture ) Extract circulating nucleic acids: QIAampCirculating NA Kit, QIAsymphony ccfDNA Kit Separate plasma
- 11. Sample to Insight Sample handling: An example Automation of ccfDNA isolation usingthe QIAsymphony 11 Mutant allele frequency variation and concentration example Chetan Bettegow da 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
- 12. Sample to Insight Automation of ccfDNA isolation usingthe QIAsymphony 12 Store at -80 °C Thaw 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
- 13. Sample to Insight Sample stabilization: PAXgene project Automation of ccfDNA isolation usingthe QIAsymphony 13 No elevated levels of free DNA using PAXgene ccfDNA Tubes* 0 100 200 300 400 500 600 t1 t3 t6 t8 t10 t1 t3 t6 t8 t10 EDTA PAXccfDNA ratiotx/t0 18S rDNA qPCR assay 66 bp amplicon 500 bp amplicon *product under development
- 14. Sample to Insight Automation of ccfDNA isolation usingthe QIAsymphony 14 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
- 15. Sample to Insight In development (automated): QIAsymphony Circulating DNAKit Using 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 usingthe QIAsymphony 15 Current solution (manual): QIAamp Circulating NAKit ≤5 ml plasma input | 24 samples | 3 hours Evolution of cfDNA extraction
- 16. Sample to Insight Automation of ccfDNA isolation usingthe QIAsymphony 16 QIAsymphony SP instrument: Workflow
- 17. Sample to Insight Automation of ccfDNA isolation usingthe QIAsymphony 17 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
- 18. Sample to Insight Size distribution of extracted ccfDNA from plasma Automation of ccfDNA isolation usingthe QIAsymphony 18 Set-up of experiment: • ccfDNAextracted 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 DNAKit (5-500 pg/μl) Set-up of experiment: • ccfDNAextracted 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
- 19. Sample to Insight Automation of ccfDNA isolation usingthe QIAsymphony 19 • 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: Compatibilityof ccfDNA eluates in library prep and NGS End repair A-addition Adapter ligation Cleanup and size selection HiFi library amplification (optional)
- 20. Sample to Insight Automation of ccfDNA isolation usingthe QIAsymphony 20 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™
- 21. Sample to Insight Automation of ccfDNA isolation usingthe QIAsymphony 21 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
- 22. Sample to Insight Automation of ccfDNA isolation usingthe QIAsymphony 22 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
- 23. Sample to Insight Automation of ccfDNA isolation usingthe QIAsymphony 23 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 37 20 Results: Sensitivity for low copy recovery
- 24. Sample to Insight Automation of ccfDNA isolation usingthe QIAsymphony 24 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
- 25. Sample to Insight Automation of ccfDNA isolation usingthe QIAsymphony 25 cut-off 4 ml plasma QS Circulating DNA Kit QIAamp Circulating NA Kit QuantYfeX® assay PrenaTest® Early Access Kit: Customer feedback
- 26. Sample to Insight Automation of ccfDNA isolation usingthe QIAsymphony 26 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
- 27. Sample to Insight Summary Automation of ccfDNA isolation usingthe QIAsymphony 27 • 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
- 28. Sample to Insight Visit QIAGEN blogs: Biomarker Insights Automation of ccfDNA isolation usingthe QIAsymphony 28 http://biomarkerinsights.qiagen.com/ http://biomarkerinsights.qiagen.com/category/liquid-biopsy/ Sign up here:
- 29. Sample to Insight Automation of ccfDNA isolation usingthe QIAsymphony 29 Katharina Beller Martin Horlitz Thorsten Voss Manuel Frietsch Kevin Matthaei Stephan Rachwal Agata Stoltmann Rita Kist Annabelle Schubert Annette Nocon Sandra Hammerschmidt Nicole Hoffmann Stephanie Angenendt Patricia Weide Dagmar Herold Yun Kyung Lee Andrea Klose Gaby Brockerhoff Silke Sonnwald Nan Fang Holger Wedler Many thanks to…
- 30. Sample to Insight Marco Polidori, Ph.D. Marco.Polidori@qiagen.com Tel: +49 2103 29 11441 Questions? Thank you for attending Contact QIAGEN Technical Service Call: 1-800-426-8157 for US Call: +49 2103-29-12400 for EU Email: techservice-na@QIAGEN.com techservice-eu@QIAGEN.com QIAwebinars@QIAGEN.com Automation of ccfDNA isolation usingthe QIAsymphony 30
Editor's Notes
- Whole blood from 6 healthy donors was drawn in EDTA BCTs. After draw, blood in EDTA tubes was either stored or directly transferred into tubes containing PAXgene stabilization reagent (PAXgene). Stabilized blood was immediately processed (t0) or stored for up to 10 days at room temperature (t10) until plasma was generated. ccfDNA from 2.4 ml plasma was extracted using the PAXgene Blood ccfDNA QIAsymphony protocol and ccfDNA yield was quantified by Real-Time PCR (18S rDNA gene, 66 bp/500 bp amplicon).
- Whole blood from 2 healthy donors was drawn in EDTA BCTs. After draw, blood in EDTA tubes was either stored or directly transferred into tubes containing PAXgene Blood ccfDNA stabilization reagent (PAXccfDNA). Stabilized blood was immediately processed (t0) or stored for up to 10 days at room temperature (t10). Plasma was generated by double centrifugation after 10 days of storage and ccfDNA was extracted from 2.4 ml plasma using the PAXgene Blood ccfDNA QIAsymphony protocol. 1 μl eluate was subjected to Agilent High Sensitivity DNA Kit.