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Cnv and a analysis strategies Cnv and a analysis strategies Presentation Transcript

  • Sample & Assay Technologies Copy Number Variation & Alteration Analysis Strategies Krishnan Allampallam PhD, MBA Global Marketing Manager PCR Array Technology The qBiomarker Copy Number PCR Arrays are intended for molecular biology applications. This product is not intended for the diagnosis, prevention or treatment of a disease
  • Sample & Assay Technologies Agenda Copy Number Variation – brief introduction Current methods to quantify Copy Number Reference Genome in Copy Number Assays – current practice ‘MRef’ - introduction and impact qBiomarker Copy Number PCR Arrays Introduction Product offering Data Analysis Application Testing gene dosage with Aneuploidy samples Copy Number Variations in Liposarcoma Resources Summary and Questions 2
  • Sample & Assay Technologies What is copy number? Copy Number changes are Genomic Structural Variations 3
  • Sample & Assay Technologies What is copy number? Copy Number changes are Genomic Structural Variations 4
  • Sample & Assay Technologies Facts about copy number variation Definition: DNA segment with 1 kb or larger variation in comparison to reference genome Copy Number Variation or Copy Number Polymorphism Frequent and occur semi-randomly throughout the genome Occurs in wide range of organisms Humans, Mice, Chimpanzees, Rhesus macaques, Cows, Chickens, Arabidopsis thaliana, Fruit flies, C.elegans, Saccharomyces cerevisiae 5
  • Sample & Assay Technologies Copy number variations Influences gene expression, phenotypic variation and adaptation by disrupting genes and altering dosage ■ Gene Expression ■ Wild type B+B+ Gastric cancer ■ Increase in CN # of 20q corresponds to frequent amplifications ■ Decrease in CN # in 9p corresponds to down regulation of 12 tumor suppressor genes1 ■ Phenotye Heterozygous Bar B+B ■ ■ ■ ■ Homozygous Bar BB Bar gene in Drosophila melanogaster2 1936 -First Association of CNV with phenotype X-linked duplication of Bar region As the # of bar increases, the size of the eyes become smaller and bar sharped ■ Human salivary amylase gene (AMY1) Chimpanzee: Has two diploid copies Humans: Ranges 6 – 15 Might be an adaptation to a high- starch diet Double bar Heterozygous B +B D 1-Fan et al, PLoSone, e29824, April 2012; 2-Bridges CB, Science 83, 210-211, 1936; 6
  • Sample & Assay Technologies Copy number variations - impact Pharmacogenomics Drug Efficacy CYP2D6 – Expressed in human liver – Critical in drug metabolism – Highly polymorphic - 75 alleles known – Affects wide range of drug classes – antidepressants, beta blocking agents and endocrine therapy – eg. Taxmoxifen Drug Toxicity CYP2D6 – affects Codeine Pharmacogenomics – Patient Stratification He Y et. al.,(2011) Trends Mol Med , 17 (5): 244-251. 7
  • Sample & Assay Technologies Agenda Copy Number Variation – brief introduction Current methods to quantify Copy Number Reference Genome in Copy Number Assays – current practice ‘Mref’ - introduction and impact qBiomarker Copy Number PCR Arrays Introduction Product offering Data Analysis Application Testing gene dosage with Aneuploidy samples Copy Number Variations in Liposarcoma Resources Summary and Questions 8
  • Sample & Assay Technologies Current methods of copy number analysis Different Methods for Different Experimental Questions Pre-Screen Discovery Validation •Array CGH •SNP Chips •NGS • • Diagnostic Test FISH qPCR 9
  • Sample & Assay Technologies Array CGH: hybridization based mapping Microarray : From image to copy number Increased intensity DNA hybridization Normal Affymetrix Mapping Greater DNA Copy number Tumor 250K Sty-I chip ~250K probe sets ~250K SNPs probe set (24 probes) CN=2 Deletion CN=2 CN=1 Deletion CN=0 CN=2 Amplification CN>2
  • Sample & Assay Technologies NGS : paired – end mapping Next Generation Sequencing – difference between the two sequenced end points Deletion sample reference genome Sequenced end points and the distance between the two point is known If distance between two ends in reference is greater than sample : deletion If distance between two ends in reference is less than than sample : insertion Insertion sample reference genome Schrider and Hahn (2010) Proc. R. Soc. 277, 3213-3221 11
  • qPCR Sample & Assay Technologies 1. Determine the gene and reference genome 2. Design the primers , amplify the gene by PCR, determine the Ct value 3. As copy number increases Ct value decreases Sample 1 A B C D 1x Sample 2 A B B C D 2x Sample 3 A B B B C D 3x 12
  • Sample & Assay Technologies qPCR-based relative quantification of copy number How does it work? Key assumption Copy number of a reference gene (Ref) is consistent across different samples Copy Number of Gene of Interest (GOI) is normalized to reference gene by ∆∆CT method – – – – CN of reference gene remains unchanged with CN of GOI(s) Assay for GOI(s) Control sample(s) with known copy number of GOI(s) Sample of interest with unknown copy number for GOI(s) Calculation of copy number gain or loss in sample of interest 13
  • Sample & Assay Technologies Agenda Copy Number Variation – brief introduction Current methods to quantify Copy Number Reference Genome in Copy Number Assays – current practice ‘MRef’ - introduction and impact qBiomarker Copy Number PCR Arrays Introduction Product offering Data Analysis Application Testing gene dosage with Aneuploidy samples Copy Number Variations in Liposarcoma Resources Summary and Questions 14
  • Sample & Assay Technologies Reference genome – current practice ■ Single copy reference genes RNase P TERT (Telomerase Reverse Transcriptase) ■ General recommendation Quantifying the amount of DNA in your samples by testing these genes against your samples before your GOI….. ■ Potential pitfalls Copy Number Events SNP–related effects on qPCR efficiency Unstable Genomes
  • Sample & Assay Technologies Reference genome – current practice Variations don’t discriminate based on you experiment. TERT - not a reliable reference genome 16
  • Sample & Assay Technologies Reference genome – current practice RNase P also has documented Structural Variations RNaseP - not a reliable reference genome 17
  • Sample & Assay Technologies Inefficiencies associated with single gene reference assays SNPs may effect qPCR efficiency and lead to altered CT values Genomic map of RNAse P (RPPH1) has at least 17 documented SNPs over 341 bases 18
  • Sample & Assay Technologies Reference genome – current practice ■ Single copy reference genes RNase P TERT (Telomerase Reverse Transcriptase) ■ General recommendation Quantifying the amount of DNA in your samples by testing these genes against your samples before your GOI….. ■ Potential pitfalls Copy Number Events SNP–related effects on qPCR efficiency Unstable Genomes
  • Reference gene copy number has dramatic effects Sample & Assay Technologies Reference Assay Copy Number of Reference Gene CT change (reference assay) GOI copy number (real) GOI copy number (calculated) Copy Number Call RNase P 2 0 2 2.00 No Change 2+1 -0.58 2 1.33 Loss 2–1 +1 2 4.00 Gain 20
  • Sample & Assay Technologies Agenda Copy Number Variation – brief introduction Current methods to quantify Copy Number Reference Genome in Copy Number Assays – current practice ‘MRef’ - introduction and impact qBiomarker Copy Number PCR Arrays Introduction Product offering Data Analysis Application Testing gene dosage with Aneuploidy samples Copy Number Variations in Liposarcoma Resources Summary and Questions 21
  • Sample & Assay Technologies Multicopy reference assay (MRef) The ideal reference assay should fulfill the following criteria: ■ Not be affected by a local change in the genome ‒ ■ Copy number: ‒ ■ Copy Number or SNP >20 copies in a diploid genome Location distribution: ‒ ‒ ≤ 10% copies concentrated on a single chromosome ‒ ■ Located on different chromosomes For copies on the same chromosome, preferably on different arms Sequence: ‒ Sequence stable in human genome Benefit: Superior Assay For Input Normalization 22
  • Genomic changes effect single gene references Sample & Assay Technologies Reference Assay GOI copy number (real) GOI copy number (calculated) Copy Number Call 0 2 2.00 No Change 2+1 - 0.58 2 1.33 Loss 2–1 Multicopy Reference Assay CT change (reference assay) 2 RNase P Copy Number of Reference Gene +1 2 4.00 Gain 40 0 2 2.00 No Change 40 + 1 -0.035 2 1.96 No Change 40 -1 + 0.035 2 2.04 No Change 23
  • Sample & Assay Technologies Multicopy reference assay yields increased accuracy CNA Experimental Setup Genomic DNA Samples Wildtype (Calibrator sample) SKBR3 cells Gene Of Interest (GOI) GRB7 (qBiomarker Assay) Reference Genome RNaseP or Multicopy Reference Assay 24
  • Sample & Assay Technologies Multicopy reference assay yields increased accuracy CNA Experimental Setup Genomic DNA Samples Wildtype (Calibrator sample) SKBR3 cells Gene Of Interest (GOI) GRB7 (qBiomarker Assay) Single Copy Gene as a Reference (RNase P) Reference Genome RNaseP or Multicopy Reference Assay Result Calculated CNV ~ 2-fold different Validation Experiment Blend DNA samples at various ratios Multicopy Reference Assay 25
  • Sample & Assay Technologies Multicopy reference assay yields increased accuracy Using a Single Copy Gene as a Reference (RNase P) Genomic DNA Samples Wildtype (Calibrator sample) SKBR3 cells Gene Of Interest (GOI) GRB7 (qBiomarker Assay) Reference Genome RNaseP or Multicopy reference assay Using Multicopy Reference Assay Result Calculated CNV ~ 2-fold different Validation Experiment Blend DNA samples at various ratios 26
  • Sample & Assay Technologies Multicopy reference assay yields increased accuracy Using a Single Copy Gene as a Reference (RNase P) Typical CNA Experimental Setup: Genomic DNA Samples Wildtype (Calibrator sample) SKBR3 cells Measure GRB7 (qBiomarker Assay) and either RNase P or Multicopy Reference Assay Result: Calculated Copy Number are almost 2-fold different Validation Experiment: Using Multicopy Reference Assay Blend DNA samples at various ratios Calculate Expected Copy Number Measure GRB7 (qBiomarker Assay) and either RNase P or Multicopy Reference Assay Compare Expected Copy Number to Observed Copy Number 27
  • Sample & Assay Technologies Why RNase P led to an overestimate of GRB7? Above experiment uses MRef for Normalization of DNA Input and tests RNase P as the GOI With cancer cells having increased genomic content (sometime 80 or more chromosomes), single copy genes “appear” like deletions because they are diluted. Since RNase P, was the denominator in the last experiment the Copy Number value appears artificially high, while the multicopy reference assay better mirrrors the amount of DNA input. 28
  • Sample & Assay Technologies Multicopy reference assay summary Superior Assay For Input Normalization Multicopy Reference yields stable CT values in spite of local changes SNPs do not significantly effect CT values of Multicopy Reference Assay Large amplifications or deletions of DNA are more consistently normalized Stable Normalization Assay across human populations Relative ∆∆CT calculation yields calculated Copy Number 29
  • Sample & Assay Technologies Agenda Copy Number Variation – brief introduction Current methods to quantify Copy Number Reference Genome in Copy Number Assays – current practice ‘Mref’ (Multi Copy Reference)- introduction and impact qBiomarker Copy Number PCR Arrays Introduction Product offering Data Analysis Application Testing gene dosage with Aneuploidy samples Copy Number Variations in Liposarcoma Resources Summary and Questions 30
  • Sample & Assay Technologies qBiomarker copy number assays Tiling the human genome at 200 base pair resolution Assay design 1. “Virtually-Cut” the Genome into 200 bp tiles • Yielded ~11.6 million designable tiles Gene A Gene B 2. Design a Primer assay within the tile 200 base pair Tile 3. Lab-test the Primer assay for performance 4. Available as single assays or array content 31
  • Sample & Assay Technologies qBiomarker copy number array layout ■ qPCR Primer Assays are pre-plated ■ 1 sample per PCR plate/ring ■ 23 or 95-genes/loci tested per array ■ each # in a well represents a different gene ■ 1 qBiomarker Copy Number Assay / gene ■ Assay design ■ Assays cover exons ■ Assay position : close to center of a gene ■ 1 qBiomarker Multicopy reference assay (MRef) ■ Assay in technical quadruplicate for accuracy 32
  • Sample & Assay Technologies qBiomarker copy number arrays Profile disease or pathway-focused copy number profiling ■ Platform-independent ■ Compatible with almost any qPCR instrument ■ Choose appropriate mastermix for instrument type ■ 23 or 95-genes/loci tested per array ■ 1 sample per PCR plate/ring ■ Gene Selection ■ Arrays by disease ■ Arrays by pathway ■ Arrays by functional gene classes 33
  • Sample & Assay Technologies Search for qBiomarker copy number assays Users can search by 1.Gene Symbol TP53 2.Refseq/Transcript: NM_001126116 3.NCBI Gene ID: 7157 4.DGV ID: Database like COSMIC 5.Chromosome Position: Chr. 17 7571801-7572001 6.Assay ID: VPH117-1234567A 34
  • Sample & Assay Technologies qBiomarker copy number arrays Diseases/Disorders Diseases/Disorders Pathway-Focused Associated with Associated with CNA CNV High Content Arrays Breast Cancer Birth Defects Kinases & Phosphatases Oncogenes & TSGs Lung Cancer Intellectual Disability WNT signaling Pathway Ovarian Cancer Prostate Cancer Gastric Cancer Glioma Pancreatic Cancer Custom Copy Number Arrays are also available 35
  • Sample & Assay Technologies Example qBiomarker copy number PCR array Description for the content of each array 36
  • Sample & Assay Technologies Example qBiomarker copy number PCR array Technical Assay Details for each array are provided in Gene Table 37
  • Sample & Assay Technologies Make it your own… with custom PCR arrays 38
  • Sample & Assay Technologies qBiomarker copy number arrays Profile disease or pathway-focused copy number profiling Isolate genomic DNA from fresh, frozen or FFPE samples using QIAamp or DNeasy kits recommended in the handbook. 2 Add qBiomarker SYBR mastermix to genomic DNA • 400 – 1000 ng fresh/frozen DNA • 800 – 1200 ng FFPE DNA 3 1 sample goes on 1 plate Assays in technical quadruplicate 4 Standard 40 cycle PCR on most real-time Thermocyclers. 5 Upload CT values to Data Analysis Webportal 39
  • Sample & Assay Technologies Included data analysis
  • Sample & Assay Technologies Agenda Copy Number Variation – brief introduction Current methods to quantify Copy Number Reference Genome in Copy Number Assays – current practice ‘Mref’ - introduction and impact qBiomarker Copy Number PCR Arrays Introduction Product offering Data Analysis Application Testing gene dosage with Aneuploidy samples Copy Number Variations in Liposarcoma Resources Summary and Questions 41
  • Sample & Assay Technologies Validated copy number assays yield accurate results Testing Gene Dosage with Aneuploidy Samples Aneuploidy samples that have different numbers of X chromosomes Samples acquired from Coriell Cell Repositories Cell line Copy number Coriell NA 13619 (XY) 1 Coriell NA 0192 (XX) 2 Coriell NA 03623 (XXX) 3 Coriell NA11226 (XXXX) 4 Use qBiomarker Copy Number Assays for Androgren Receptor (AR) and Methyl CpG binding Protein 2 (MECP2) that are single copy genes on the X chromosome Use qBiomarker Multicopy Reference Assay to normalize for DNA Input Use ∆∆CT to calculate relative copy number changes compared to the XX sample 42
  • Sample & Assay Technologies Agenda Copy Number Variation – brief introduction Current methods to quantify Copy Number Reference Genome in Copy Number Assays – current practice ‘Mref’ - introduction and impact qBiomarker Copy Number PCR Arrays Introduction Product offering Data Analysis Application Testing gene dosage with Aneuploidy samples Copy Number Variations in Liposarcoma Resources Summary and Questions 43
  • Sample & Assay Technologies Liposarcoma collaboration Initial Screen for Copy Number Changes Thirty (30) liposarcoma samples were tested by aCGH for copy number events Results from one of those samples (T50) is shown • Analysis with Partek software • Deletions on Chromosome 11 • Amplifications on Chromosome 12 Initial screen yielded a list of 23 genes with copy number changes. All samples were re-tested using Custom Copy Number PCR Array Data courtesy of Kara Pascarelli and Dominique Broccoli, Memorial University Medical Center, Savannah, GA, USA; and Lesley Ann Hawthorne, Medical College of Georgia, Georgia Health Sciences University, Augusta, GA, USA) 44
  • Copy number assay in liposarcoma Sample & Assay Technologies Copy Number PCR Array Data for Sample T50 35 Copy Number 30 * 25 20 15 * 10 * * * * * 5 * * * * * 11 1 1 * * 0 Chr 9 9 9 9 12 12 12 12 12 12 12 11 11 11 1 1 1 1 1 19 aCGH qBiomarker Data courtesy of Kara Pascarelli and Dominique Broccoli, Memorial University Medical Center, Savannah, GA, USA; and Lesley Ann Hawthorne, Medical College of Georgia, Georgia Health Sciences University, Augusta, GA, USA)
  • Sample & Assay Technologies Agenda Copy Number Variation – brief introduction Current methods to quantify Copy Number Reference Genome in Copy Number Assays – current practice ‘Mref’ - introduction and impact qBiomarker Copy Number PCR Arrays Introduction Product offering Data Analysis Application Testing gene dosage with Aneuploidy samples Copy Number Variations in Liposarcoma Resources Summary and Questions 46
  • Sample & Assay Technologies Protocol & product overview What do you need to measure copy number by qPCR? DNA Isolation - QIAamp DNA Mini Kit (51304 or 51306) - DNeasy Blood & Tissue Kit (69504 or 69506) - QIAamp DNA FFPE Tissue Kit (56404) Whole Genome Amplfication (optional) - Repli-G Individual Assays - qBiomarker Copy Number PCR Assays - qBiomarker Multicopy Reference PCR Assay - qBiomarker SYBR Green Mastermix (based on instrument) Copy Number Profiling - qBiomarker Copy Number PCR Arrays - Custom qBiomarker Copy Number PCR Arrays - qBiomarker SYBR Green Mastermix (based on instrument) qBiomarker Data Analysis
  • Sample & Assay Technologies Search for qBiomarker copy number assays Use SABiosciences.com 48
  • Sample & Assay Technologies Agenda Copy Number Variation – brief introduction Current methods to quantify Copy Number Reference Genome in Copy Number Assays – current practice ‘Mref’ - introduction and impact qBiomarker Copy Number PCR Arrays Introduction Product offering Data Analysis Application Testing gene dosage with Aneuploidy samples Copy Number Variations in Liposarcoma Resources Summary and Questions 49
  • Sample & Assay Technologies Summary From Discovery to Validation Copy Number Alterations/Variations are important biological changes with ramifications for human health Experimental Solutions for Copy Number Determination Discovery experiments - Arrays, beadchips, etc. Validation / Pre-Screen / Hypothesis-driven experiments - qPCR Better normalization assays yield better data - Multicopy reference assay vs. single gene Bench-validated assays at highest resolution Copy Number Profiling - Pathway-focused - Custom designs 50
  • Sample & Assay Technologies Special offer for Webinar Attendees Try any qBiomarker Copy Number Assay Get 20% off + QIAGEN Watch Promo Code: FDK-WN20WA3 Experience PCR Array Performance Try them today! Questions? Contact Technical Support: 9 AM – 6 PM M – F ET Contact: 1-800-742-4368 OR support@SABiosciences.com Thousands of scientists have discovered the power of PCR Arrays Join them on the road to success!
  • Sample & Assay Technologies Questions Contact Technical Support 9 AM – 6 PM Eastern M – F Telephone: 888-503-3187 Email: support@SABiosciences.com Thank you! Krishnan Allampallam krishnan.allampallam@qiagen.com QIAWEBINARS@QIAGEN.COM 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