QIAGEN: Sample-to-Insight Success factors for NGS analysis and accurate data interpretation

Transcription

1 QIAGEN: Sample-to-Insight Success factors for NGS analysis and accurate data interpretation Dr. Anne Arens & Dr. Jens Winter QIAGEN GmbH Title, Location, Date 1

2 How to prevent false positives with NGS right from the beginning (sample) to the data interpretation (insight) How to get the most out of limited, small or low concentrated samples, like small biopsies, single cells or circulating DNA ( liquid biopsy) How to control the quality of your NGS process and to get the most out of samples with varying sample quality How to get the correct and relevant data out of your massive NGS data and how to interpret them based on the current scientific and clinical knowledge? Title, Location, Date 2

3 QIAGEN Work with any sample on any common platform Fresh tissue/ FFPE NGS Illumina & IonTorrent Blood/ Serum/ Plasma Liquid Biopsy QIAGEN Sample Prep & Automation QIAGEN Content NGS & qpcr Panels Gene Reader QIAGEN GeneReader qpcr ABI, BioRad etc. QIAGEN BioX Single Cell QIAGEN RG Q QIAGEN s integrated universal workflow allows you to go from sample to insight, regardless of the sample type and qpcr- or NGS-platform 3

4 Quality criteria DNA RNA Purity Yield Integrity % of DNA, which can be amplified Sequence artifacts Purity Yield Integrity % of RNA which can be reversed transcribed Gene expression level Title, Location, Date 4

5 Quality criteria DNA RNA Purity Yield Integrity % of DNA, which can be amplified Sequence artifacts Purity Yield Integrity % of RNA which can be reversed transcribed Gene expression level Title, Location, Date 5

6 QC is an essential step in all workflows Gene Expression Analysis real-time RT-PCR At least one QC step in each workflow Sample Collection RNA purification cdna synthesis Real-time PCR Epigenetics Pyrosequencing Sample Collection DNA purification Bisulfite conversion Clean-up PCR Amplif. sspcr amplicon Pyro sequencing Gene Expression Analysis Microarrays RNA purification cdna Synthesis cdna cleanup crna Synthesis crna cleanup Quantif. & Normaliz. Fragmenta tion Array Hyb. Array Scanning Next-generation Sequencing DNA purification Quantification Normalization Target Enrichm. Library Prep empcr NGS read Genotyping Sample Collection DNA purification Amplification PCR based analysis Sequencing analysis

7 QC But Which Method? QC Criteria Nanodrop Gels Qubit QIAxcel [CE] QIAxpert Protein contaminants (A260/280) Salts & other contaminants* (A260/230) Yield ( ) Degradation/ Sample integrity Size range Quantity of dsdna vs. other NA There is no one-for-all solution QIAxcel & QIAxpert will cover your needs! *i.e. - phenole, carbohydrates, EDTA, GuHCl, Trizol QIAGEN NGS Solutions

8 QIAxpert system facts & figures Small benchtop spectrophotometer WxDxH 23 x 30 x 28 cm; 9kg µ-fluidics based 1-16 samples in less than 2 min. 2µl sample input volume; LOD 1.5ng/µl Integrated touchscreen No repetitive drop & clean actions No cross-contamination Export results to USB, network or QR-code Full UV/VIS scan & quantification For QIAamp/RNeasy/QIAquick: unique spectral content profilint protocols to differentiate of molecules of interest detect other absorbing impurities quantify DNA or RNA fraction specifically check turbidity Accelerate micro-volume DNA, RNA and protein quantification and QC on every bench QIAxp ert Webin

9 Key Features Fully automated DNA and RNA analysis Ready-to-run gel cartridges Fast processing: 12 samples in 3 10 min Up to 96 samples per run (unattended processing of up to twenty 96-w.plates possible) Sample input amounts < 0.1µl Detection limit of 0.1 ng/µl High resolution of 3 5 bp up to 500 bp Digital data output 9

10 QIAGEN NGS Solutions QIAGEN Universal NGS workflow: From Title, Location, Date 10

11 QIAGEN Solutions for BRCA1 & BRCA2 NGS Analysis QIAGEN Universal NGS workflow: From Title, Location, Date 11

12 Pre-analytical Challenges Nucleic Acid degradation/ fragmentation RNA/DNA degradation/fragmentation low yields/detection issues Enzymatically in cells (e.g. RNases) E.g. paraffinization procedure Free-circulating DNA Deamination sequence artifacts FFPE procedure Chemical modification Cross-linking low yield: Formalin fixation Gene expression regulation Up- and down regulation of RNA changed information Non-stabilized tissue, cells, blood etc.

13 Sample isolation and sample QC In formalin-fixed material up to one mutation artifact per 500 bases was recorded. The chance of such artificial mutations in formalin-fixed material was inversely correlated with the number of cells used in the PCR the fewer cells, the more artifacts. A total of 28 artificial mutations were recorded, of which 27 were C-T or G-A transitions. we perform whole genome sequencing of DNA isolated from two triple-negative breast cancer tumors archived for >11 years as 5 µm FFPE sections and matched germline DNA. The tumor samples show differing amounts of FFPE damaged DNA sequencing reads revealed as relatively high alignment mismatch rates enriched for C G > T A substitutions compared to germline samples. We show that DNA molecules amplified by PCR from DNA extracted from animal bones and teeth that vary in age between and over years carry C T and G A substitutions. These substitutions can reach high proportions among the molecules amplified and are due to the occurrence of modified deoxycytidine residues in the template DNA. If the template DNA is treated with uracil N-glycosylase, these substitutions are dramatically reduced. They are thus likely to result from deamination of deoxycytidine residues.

14 The FFPE-challenge for NGS All FFPE samples potentially carry such random C>T mutation artefacts A T A T C G T A T A A T Random deamination C turns to U A T A T U G T A T A A T Amplification of mismatch during PCR A T A T U G T A T A A T Produces two strands with different sequence Sequencing result is biased A T A T T A T A T A A T A T A T C G T A T A A T Cytosine bases within the DNA strand in FFPE samples are subject deamination This chemical reaction turns a Cytosine into a Uracil base It is believed to occur randomly during formalin fixation and with aging The reasons for this chemical reaction is still unknown In subsequent sequencing analysis this base appears as Thymine (SNP)

15 All-in one solution for minimizing sequencing artifacts from FFPE GeneRead DNA FFPE Kit Workflow Deparaffinizati Deparaffinization Lysis Artifact removal Bind Wash Elute 10 min 2 h 1 h 30 min Deparaffinization Solution included Improved Lysis Buffer for higher yields Enzymatic removal of C>T artifacts DNA isolation according to the proven QIAamp technology Automated on the QIAcube

16 QIAcube: the sample prep multi-talent More than 110 ready-to-use standard protocols available Genomic DNA from human samples QIAamp DNA Blood Mini Kit QIAamp DNA Blood Mini Kit QIAamp DNA Mini Kit QIAamp DNA Stool Mini Kit QIAamp DNA Micro Kit QIAamp MinElute Media Kit QIAamp DSP DNA Blood Mini kit [IVD] NEW QIAamp DSP DNA Mini kit [IVD] Forensic samples QIAamp DNA Investigator Kit Viral DNA and RNA purification QIAamp MinElute Virus Spin Kit QIAamp Viral RNA Mini Kit QIAamp DSP MinElute Virus Spin Kit [IVD] QIAamp DSP Virus Mini kit [IVD] QIAamp DSP Viral RNA Mini kit [IVD] Genomic DNA from animal samples DNeasy Blood & Tissue Kit Multiple analyte purification AllPrep DNA/RNA Kits AllPrep DNA/RNA FFPE Kit RNA purification from a range of samples RNeasy Mini Kit RNeasy MinElute Cleanup Kit RNeasy Plus Mini Kit RNeasy Micro Kit RNeasy Lipid Tissue Mini Kit RNeasy Fibrous Tissue Kit RNeasy Protect Bacteria Mini Kit RNeasy Protect Animal Blood Kit mirneasy Protect Animal Blood Kit RNeasy Protect Animal Blood Kit PAXgene Blood RNA Kit [IVD] PAXgene Blood mirna Kit QIAamp RNA Blood Mini Kit RNeasy Plus Universal Kit FFPE samples RNeasy Plus Universal Kit RNeasy FFPE Kit QIAamp DNA FFPE Kit Plant or food samples DNeasy Plant Mini Kit RNeasy Plant Mini Kit DNeasy mericon Food Kit Protein purification and serum depletion Ni-NTA Spin Kit Qproteome Albumin/IgG Depletion Kit Qproteome Murine Albumin Depletion Kit Qproteome Total Glycoprotein Kit Qproteome O-Glycan Glycoprotein Kit Bisulfite conversion and cleanup EpiTect Bisulfite Kit EpiTect Bisulfite Plus Kit Plasmid DNA purification QIAprep Spin Miniprep Kits DNA cleanup QIAquick PCR Purification Kit QIAquick Gel Extraction Kit QIAquick Nucleotide Removal Kit MinElute PCR Purification Kit MinElute Reaction Cleanup Kit MinElute Gel Extraction Kit NGS workflows GeneRead rrna Depletion Kit GeneRead Size Selection Kit GeneRead Library Prep Kit Achieve greater levels of consistency and standardization RS 16

17 Removal of false positive cancer mutations GeneRead DNA FFPE removes false positive cancer mutations Chrom Pos COSMIC ID dbsnp ID Gene Name Ref Var Common FFPE Prep GeneRead FFPE chr COSN rs PARP1 C G chr COSM rs ALK T C chr COSM MSH6 C T chr COSM rs TGFBR2 T A chr COSM rs SETD2 G A chr COSM FGFR3 C T chr COSM22413 rs PDGFRA C T chr COSM12708 rs KIT C T chr COSM rs IL7R T C chr COSM rs IL7R G A chr COSN rs IL7R A T chr COSN rs FLT4 C G chr COSM42978 rs EGFR C T chr COSM14251 rs11515 CDKN2A C G chr COSM33747 rs10521 NOTCH1 G A chr COSM rs MEN1 T C chr COSM HNF1A G A chr COSM CREBBP C T chr COSM rs16942 BRCA1 T C Table 1 shows COSMIC mutations found in a 15 year old carcinoma from liver tissue. The tissue was processed in parallel using a standard FFPE kit and the new GeneRead kit with artifact reduction. Both samples were amplified with the GeneRead DNAseq Comprehensive Cancer Panel (Sabiosciences), and sequenced with massivley parallel sequencing. The frequency of the mutation is found in the last two columns. Four mutations found when using the standard kit were not found in the tissue processed using the GeneRead kit. These were the lowest frequency mutations, and all of them were C>T G>A transitions, thus likely artifacts resulting from formalin fixation and storage. All other mutations were found in both tissues, with very similar frequencies, indicating that the new kit specifically targets artifactual mutations while leaving true mutations unchanged.

18 QIAGEN Solutions for BRCA1 & BRCA2 NGS Analysis QIAGEN Universal NGS workflow: From Quality and Quantity of gdna are the most important determinants of successful NGS analysis. DNA to be analyzed has to be amenable to PCR amplification Title, Location, Date 18

19 Quality control of input DNA Requirements for the analysis of low-quality DNA FFPE DNA Quantify DNA that is amenable to PCR amplification Qualify DNA for variant calling Optimize DNA input for targeted enrichment Number of PCR cycles needed for targeted enrichment NGS RS 19

20 GeneRead DNA QuantiMIZE kits Overcome the challenges of DNA quantification & qualification Control and sample gdna Multicopy PCR assay + MasterMix Innovative multicopy qpcr assays to increase accuracy Two qpcr assays to query 40 genomic loci that are randomly distributed in the genome Quantify only PCR-amplifiable DNA Provide guidance to rescue low quality DNA sample Amount of input DNA and number of target enrichment cycles Appropriate amount of input DNA Number of targeted enrichment cycles Ensure reproducible high post-amplification yield RS Title, Location, Date 20

21 GeneRead DNA QuantiMIZE kits Generate libraries from low-quality FFPE samples Isolate FFPE DNA from 4 FFPE samples Quantify and Qualify FFPE DNA by QuantiMIZE (1) or Spectrophotometry (2) Samples T3 T7 T13 N Targeted enrichment (Prostate cancer V2) Library construction Library analysis by BioAnalyzer QuantiMIZE ensures good library yields MiSeq sequencing RS 21

22 Sample QC: GeneRead DNA QuantiMIZE kits Quantify only amplifiable functional DNA Input DNA Output Quantity Quality Optimized PCR cycles RS Title, Location, Date 22

23 Sample Isolation and Sample QC GeneRead DNA QuantiMIZE Kit for easy sample QC Challenge: Due to degradation, inaccurate quantification of input DNA can lead to poor target enrichment and NGS library yield, eventually wastage of your precious sample and time. Solution: GeneRead QuantiMIZE system: Quantification of functional genomic DNA Quality score for DNA on the basis of degradation status and contamination Optimized number of PCR cycles and DNA input for NGS target enrichment to rescue low-quality DNA 23

24 Liquid Biopsy: Non-Invasive Approaches to Research & Diagnostics A liquid biopsy is a liquid biomarker that can be isolated from body fluids, such as blood, saliva, urine, ascites, or pleural effusion. Like a tissue biopsy, it is a representative of the tissue from which it has spread. Liquid biopsies have become more clinically useful in recent years due to the ability to pair tests on circulating tumor cells with genomic tests. Diaz, Jr., L.A. and Bardelli, A. (2014) Liquid biopsies: genotyping circulating tumor DNA. Am. Soc. Clin. Oncol. 32,

25 Liquid Biopsy Liquid Biopsy has the potential to transform healthcare and holds benefits for Title, Location, Date 25

26 Liquid Biopsy: The currently main analyte domains/soures Tumors shed both intact cells (resulting in circulating tumor cells) as well as cellular components, such as nucleic acids (resulting in cell-free DNA or RNA). Liquid biopsies CTCs (circulating tumor cells) ctna (circulating tumor nucleic acids) Exosomes Cancer cells released from primary tumor mass into the bloodstream ctdna (circulating tumor DNA), mirnas, mrna, & long non-coding RNA Small membrane-derived vesicles ( nm) contain various molecules such as signal proteins, micrornas, mrnas, lipids, and exodna. Samples: blood, serum/plasma, urine, CSF, saliva Diaz, Jr., L.A. and Bardelli, A. (2014) Liquid biopsies: genotyping circulating tumor DNA. Am. Soc. Clin. Oncol. 32,

27 Liquid Biopsy: The currently main analyte domains/soures Tumors shed both intact cells (resulting in circulating tumor cells) as well as cellular components, such as nucleic acids (resulting in cell-free DNA or RNA). Liquid biopsies CTCs (circulating tumor cells) ctna (circulating tumor nucleic acids) Exosomes Cancer cells released from primary tumor mass into the bloodstream ctdna (circulating tumor DNA), mirnas, mrna, & long non-coding RNA Small membrane-derived vesicles ( nm) contain various molecules such as signal proteins, micrornas, mrnas, lipids, and exodna. Samples: blood, serum/plasma, urine, CSF, saliva Diaz, Jr., L.A. and Bardelli, A. (2014) Liquid biopsies: genotyping circulating tumor DNA. Am. Soc. Clin. Oncol. 32,

28 Exosomes as Circulating Biomarkers Exosomes: small membrane vesicles ( nm), secreted by most cell types into the bloodstream. Functional biomolecules: DNA fragments (exosomal DNA, exodna) Proteins and/or peptides mrna/lncrna microrna (mirna) Lipids Exosomes play a central role in cell-to-cell communication. The majority of DNA associated with tumor exosomes is double-stranded, representing whole genomic DNA. Biological molecules (protein, RNA, and mirna) contained in exosomes are well protected by a lipid bilayer membrane that confers a high degree of stability. Rolfo, C. et al. (2014) Liquid biopsies in lung cancer: the new ambrosia of researchers. Biochimica et Biophysica Acta 1846, 539. Klevebring, D. et. al. (2014) Evaluation of exome sequencing to estimate tumor burden in plasma. PLOS One 9, e

29 Exosomes shedding from cells What are exosomes? Scanning EM of exosomes shedding into the blood from a GBM brain cancer cell published in Nature Cell Biology, Dec 2008, Skog et al. Transmission EM of Multi Vesicular Bodies in Kidney, courtesy of Leileata Russo

30 Exosomal RNA as biomarker for disease Snapshot of the transcriptomes Exosomes are found in all biofluids (blood, urine, CSF, saliva, etc.) Exosomes contain RNA and protect it from the RNases present in biofluids The RNA in exosomes provides a snapshot of the transcriptomes in e.g. the primary tumor, metastases and surrounding microenvironment in real-time #1 #2 RNA levels; Disease fingerprints (mrna, mirna, non-coding RNA) Disease specific mutations (KRAS, BRAF, EGFR, IDH1, PI3K etc)

31 Exosomal RNA extraction - Considerations Background from cellular RNA Plasma and serum often contain residual cells or cell fragments which result in undesired background Solution: additional up-front filtration or centrifugation step Also important for cell culture supernantant Specificity for vesicular RNA Ultracentrifugation - or precipitation-based methods are known to recover large protein complexes together with vesicles Solution: affinity-based binding of vesicles RNA Purity Serum or plasma contain high concentrations of contaminants that can interfere with RNA isolation and assays Solution: enrichment for vesicles prior to RNA isolation Title, Location, Date 31

32 Exosomal RNA extraction - Considerations RNA integrity High levels of RNase in serum and plasma Solution: remove during vesicle isolation How much serum/plasma? Many transcripts (esp. mrna) are present at low copy numbers Solution: Start with large sample volumes ( up to 4ml) to detect low-abundance RNAs with high confidence Blood stabilization agents? Do NOT use heparin as anticoagulant as it can interfere in RT-PCR Serum or plasma? Microvesicles from filtered serum or plasma Be consistent in collection and handling Title, Location, Date 32

33 Vesicular RNA from cell culture medium Effect of freeze / thaw cycles on vesicle integrity (RNA Yield) 2 ml HeLa culture medium per prep; exorneasy started from fresh cultured supernatant, or after 1 or 5 freeze/thaw cycles; negative control sample used 0.5% SDS to lyse vesicles no difference in RNA recovery even after 5 freeze/thaw cycles

34 Influence of blood collection device on RNA

35 exorneasy exosome RNA analysis made simple exorneasy Serum/Plasma Kit Quickly isolates purified total RNA from microvesicles Enables use of high input volumes for sensitive detection of low abundance transcripts Efficiently isolates mrna and mirna from plasma or serum Specifically enriches for RNA contained in vesicles Processes multiple parallel samples with a convenient spin-column procedure

36 exorneasy Serum/Plasma Kit Sample from microvesicles to total RNA, in just one hour Separate Serum/Plasma Isolate Exosomes Isolate RNA Microvesicle isolation 20 minutes RNA isolation 35 minutes

37 Intact vesicles are eluted from the exoeasy column Scanning EM (20000x magnification) reveals higher purity with exoeasy Ultracentrifugation (UC) Eluate from exoeasy Both preparations contain vesicle-shaped structures within an expected size range UC: Many smaller, unidentified structures/particles that do not match the expected size exoeasy: Intact vesicles with higher purity 37

38 exorneasy isolates small and large RNAs from EV Bioanalyzer sizing, 2 ml plasma was pre-filtered (0.8 µm) to exclude larger particles. Both methods purify RNA of similar size and yield

39 exorneasy captures all mrna & vesicle-specific mirnas mrna exclusively within vesicles near 100% bound mirna in vesicular and non-vesicular fractions (e.g. free Ago2 complexes) * Arroyo, J.D. et al. (2011) Argonaute2 complexes carry a population of circulating micrornas independent of vesicles in human plasma. Proc. Natl. Acad. Sci. USA 108, 5003.

40 Detection of low-abundance RNAs exorneasy can be scaled up to 4 ml of plasma Not detected Input Volume 46% CT Value (90 mrnas, pre-amplified) mrnas from known oncogenes are readily detected in high volumes of plasma

41 Exosomal RNA qpcr workflow Sample collection & Analyte enrichment Sample Isolation Amplification PCR Data Interpretation RNA exorneasy Serum/Plasma Kit RT² PreAMP cdna Kit RT 2 PCR System Ingenuity Pathway Analysis mirna exorneasy Serum/Plasma Kit miscript PreAMP PCR Kit miscript PCR System Ingenuity Pathway Analysis 41

42 Comparison of mirna populations in plasma Abundance of mirnas inside and outside of extracellular vesicles Mainly inside vesicles Mainly outside vesicles

43 Isolated exrnas are suitable for NGS applications Detection of somatic mutations from CRC in exrnas Patient with KRAS G12D positive colorectal cancer (CRC) 2 ml pre-filtered plasma exrna isolation using the exorneasy Serum/Plasma Maxi Kit Targeted re-sequencing on an Illumina MiSeq No. of Reads % 5000 MUT WT 10% KRAS G12D 20% Frequency wildtype KRAS No. of reads single variants Over 10% of all reads that matched to the KRAS gene carry the c.35 G>A / p.g12d mutation previously identified in the primary tumor

44 THANK YOU! exorneasy Serum/Plasma Maxi Kit (50) exorneasy Serum/Plasma Midi Kit (50) exorneasy Maxi Kit up to 4 ml #77064 to detect low abundance RNA biomarkers from MVs with high confidence Biomarker Discovery from Serum, Plasma, CSF & Cell Culture Supernatant Downstream applications that require certain amounts of RNA o NGS o RNA/miRNA profiling exorneasy Midi Kit up to 1 ml for known RNA biomarkers from MVs with high abundance #77044 When working with known biomarker and high copy numbers Downstream applications that require lower amounts of RNA o Smaller panels exorneasy Serum/Plasma Starter Kit (20) exorneasy Starter Kit (20) contains 10 Midi and 10 Maxi columns to evaluate starting amount #77023 Getting started with exosome research Enabling analysis at varied volumes to asses optimal column size When abundance of targets are unknown exoeasy Serum/Plasma Maxi Kit (20) ExoEasy Maxi Kit (20) contains 20 exoeasy Maxi columns for isolation of intact exosomes & microvesicles For functional studies in exosome signaling Targeted drug delivery research May

45 Liquid Biopsy: The currently main analyte domains/soures Tumors shed both intact cells (resulting in circulating tumor cells) as well as cellular components, such as nucleic acids (resulting in cell-free DNA or RNA). Liquid biopsies CTCs (circulating tumor cells) ctna (circulating tumor nucleic acids) Exosomes Cancer cells released from primary tumor mass into the bloodstream ctdna (circulating tumor DNA), mirnas, mrna, & long non-coding RNA Small membrane-derived vesicles ( nm) contain various molecules such as signal proteins, micrornas, mrnas, lipids, and exodna. Samples: blood, serum/plasma, urine, CSF, saliva Diaz, Jr., L.A. and Bardelli, A. (2014) Liquid biopsies: genotyping circulating tumor DNA. Am. Soc. Clin. Oncol. 32,

46 Circulating nucleic acids: workflow Blood draw (venipuncture) Separate plasma <1 hour Avoid release of cellular nucleic acids Extract circulating nucleic acids Highly efficient large-volume nucleic acid extraction Optional DNA modification (e.g., bisulfite treatment) Sequencing library prep Maximize recovery improve sensitivity Real-time PCR digital PCR therasceen assays Next-generation sequencing Reduce interference of non-target ( normal ) nucleic acids

47 QIAGEN s solutions for cfdna extraction Current solution (manual): QIAamp DSP Circulating NA Kit 5 ml plasma input 24 samples 3 hours for IVD use In development (automated): QIAsymphony DSP Circulating DNA Kit Using new beads and chemistry 1-4 ml input plasma from EDTA and Streck tubes 96 samples 6 hours (hands-off) IVD use (2016) 47

48 Liquid Biopsy: The currently main analyte domains/soures Tumors shed both intact cells (resulting in circulating tumor cells) as well as cellular components, such as nucleic acids (resulting in cell-free DNA or RNA). Liquid biopsies CTCs (circulating tumor cells) ctna (circulating tumor nucleic acids) Exosomes Cancer cells released from primary tumor mass into the bloodstream ctdna (circulating tumor DNA), mirnas, mrna, & long non-coding RNA Small membrane-derived vesicles ( nm) contain various molecules such as signal proteins, micrornas, mrnas, lipids, and exodna. Samples: blood, serum/plasma, urine, CSF, saliva Diaz, Jr., L.A. and Bardelli, A. (2014) Liquid biopsies: genotyping circulating tumor DNA. Am. Soc. Clin. Oncol. 32,

49 Liquid Biopsy CTC Products CTC isolation made easy Reliable detection and quantification of CTCs from blood using COCP Proven combination of tumor associated markers at mrna level provides required specificity and sensitivity High specificity and reduced bias due to limited marker range Product range: AdnaTest ColonCancer AdnaTest BreastCancer AdnaTest ProstateCancer AdnaTest OvarianCancer AdnaTest Melanoma AdnaTest EMT StemCell 49

50 Liquid Biopsy CTC Products Each AdnaTest uses a unique combination of different antibodies and targets 2-Step procedure: 5 hours Blood collection (5ml) Enrichment of CTCs using antibody coated beads Cell lysis Multiplex RT-PCR Result Selective enrichment of CTCs using magnetic beads conjugated with an optimized capturing antibody mix Multiplex RT-PCR detection based on a combination of tumor associated marker Easily to be integrated into clinical laboratory routines

51 Single cell analysis Challenges and solutions Challenges Insufficient DNA/RNA recovered from low-input amounts Existing amplification methods introduce bias Solution: REPLI-g Single Cell Kit Whole genome/transcriptome amplification by Multiple Displacement Amplification Totally clean kit components sequence target DNA only Create representative genome libraries, even from single cells High-fidelity amplification for high-quality DNA and minimal bias QIAGEN NGS Solutions

52 One Cell - Versatile downstream analysis REPLI-g: No DNA & RNA limitations due to MDA-Technology Unlimited DNA and RNA amounts Negligible sequence bias enabling even single cell NGS analysis

53 Multiple displacement amplification for single cell NGS Representative amplification with Repli-G Single Cell Kit Alkaline Denaturation Hexamer Random Primers Phi29 Polymerase strand displacement (30 C) Gentle denaturation results in intact DNA-template Even priming events covering the whole genome accurate data High fidelity amplification minimal error rate 1000 fold more accurate than Taq No effect of secondary structure minimal bias µg high-molecular-weight DNA from single cells or purified DNA ( kb) Very simple three-step workflow Multiple Displacement Events efficient amplification of DNA

54 QIAGEN Solutions for BRCA1 & BRCA2 NGS Analysis QIAGEN Universal NGS workflow: From Title, Location, Date 54

55 Why choose multiplex PCR-based targeted enrichment? It delivers unmatched specificity and uniformity (compared to capture-based methods) Features Offers specificity that beats capture-based approaches Offers uniformity that beats capture-based approaches Benefits Use sequencing capacity on regions targeted by the panel, with minimal offtarget sequencing Achieve more uniform enrichment for more sequencing efficiency RS

56 GeneRead DNAseq Panels V2 Multiplex PCR-based enrichment of gene(s) or genomic region(s) Builds on our 10-year experience in designing PCR assays GeneRead DNAseq Panel V2 GeneRead DNAseq PCR Kit V2 Collection of primer pairs dispended into primer pools/tubes. Primers correspond to the targeted region(s) (Gene 1 and Gene 2, as example) RS Primer pair; amplicon

57 GeneRead DNAseq Panels V2 Multiplex PCR-based enrichment of gene(s) or genomic region(s) Gene 1 Gene 2 Primer design algorithm PCR Chemistry Primer separation algorithm GeneRead DNAseq Panel V2 (sets of primer pools/tubes) GeneRead DNAseq PCR Kit V2 RS

58 Clinically Relevant Panels Largest collection of wet-bench verified gene panels Reference databases: The Cancer Genome Atlas National Comprehensive Cancer Network COSMIC Cancer Genome Census OMIM ClinVar (NCBI) Type Solid tumor Hematologic malignancies Tissue-specific Panel name Clinically Relevant Tumor Tumor Actionable Mutations Myeloid Neoplasms Breast Cancer Colorectal Cancer Liver Cancer Lung Cancer Ovarian Cancer Prostate Cancer Gastric Cancer Cardiomyopathy Cancer Predisposition Comprehensive Comprehensive Cancer Carrier Testing Custom Online custom builder Gene focused BRCA1 and BRCA2 58

59 GeneRead DNAseq BRCA1 and BRCA2 panel Design and specifications Overlapping amplicon design Experimentally-verified 100% coverage 100% coverage Regions targeted Coding regions + 20 bp intron-exon junctions # of bases targeted 21,472 Average amplicon length 150 bp Primer primer pools 4 Total input DNA 40 ng Number of amplicons 250 Specificity 99% Uniformity (0.2x mean) 97% % of bases callable 100% 59

60 Application-specific amplicon design Builds on >10 years in assay development Primers G12 Amplicons Deep (redundant) tiling amplicon design Dense overlap amplicon design Sparse overlap amplicon design RS 60

61 Why choose GeneRead panels workflow for targeted enrichment? Achieve better variant calling accuracy to reduce false negatives RS 61

62 Customized panels Custom and Mix-n-Match GeneRead Mix-n-Match Access to 570 bench-tested gene designs 1 Fixed specifications Turnaround time = 3 days 2 GeneRead Custom Bioinformatically target any gene(s) or genomic region(s) within the human genome Flexible specifications Turnaround time = 14 days 2 What is the list of your targets? RS Title, Location, Date 62

63 The power of Mix-n-Match pool of validated genes Consistent performance Experimental Coverage Uniformity 100,0% 90,0% 80,0% 70,0% 60,0% 50,0% 40,0% 30,0% 20,0% 10,0% 0,0% MET NRAS PDGFRA PIK3CA PTEN RB1 RET TP53 RS 63

64 A Universal Panel Work with any sample on any sequencing platform 64

65 QIAGEN Solutions for BRCA1 & BRCA2 NGS Analysis QIAGEN Universal NGS workflow: From Title, Location, Date 65

66 Library Construction with GeneRead Library Kit Fast and highly reproducible workflow For Ion Torrent and Illumina platforms 5 steps 2 tubes for 5 steps 130 minutes High yield Tube Cleaning Amplification BRCA 1/2 mutation testing 66

67 QIAGEN Solutions for BRCA1 & BRCA2 NGS Analysis QIAGEN Universal NGS workflow: From Title, Location, Date 67

68 Library QC with GeneRead DNAseq Library QC Kits Assess target enrichment and sample quality by qpcr Ratio of Target DNA : Control DNA Quality of DNA Input to NGS (= Conserved regions) QC Score QC Result 1-4 PASS 4 8 MARGINAL > 8 FAIL 68

69 GeneRead DNA Library Prep Product Preview New! Cell-free DNA Library Prep Protocol for GeneRead Robust results with limited material (1-100 ng) DNA Purification QIAamp Circulating Nucleic Acid Kit Streamlined workflow with reduced hands-on-time High Conversion Efficiency / Low Duplicate Rates 10 µl eluate (max µl) ~ ng Single-tube Workflow w/ 10min Hand-on Time Library Prep GeneRead DNA Library I Prep Kits Sequencing Title, Location, Date 69

70 QIAGEN Solutions for BRCA1 & BRCA2 NGS Analysis QIAGEN Universal NGS workflow: From Title, Location, Date 70

71 First Fastq-to-insight solution from QIAGEN Identify (somatic) cancer driver variants with one click! Sample Any NGS sequencing machine Ready-to-Analyze Workflows GeneRead DNAseq Panel plug-in CLC Cancer Research Workbench Identify (somatic) variants Validate variants in context of sequencing reads Direct transfer via plug-in* Identify causal variants/find cancer drivers Visualize variants with links to literature and phenotype * License needed for Ingenuity variant analysis 71