Illumina, Inc. · NASDAQ:ILMN
Illumina — Technical Presentation (November 2006)
39 slides from Illumina's technical presentation, Nov 28, 2006
Slide 1: High-Resolution Genomic Profiling of Chromosomal Aberrations Using Infinium Whole Genome Genotyping
making sense out of life High-Resolution Genomic Profiling of Chromosomal Aberrations Using Infinium® Whole Genome Genotyping Daniel Peiffer, Ph.D. Associate Product Manager November 28, 2006 © 2006 Illumina, Inc. Illumina, Sentrix, Array of Arrays, BeadArray, DASL, Infinium, GoldenGate, BeadXpress,
Slide 2: Overview
Overview Introduction to DNA copy number analysis Whole-genome genotyping as a tool for detecting aberrations Infinium data analysis and visualization of aberrations Advantages of the Infinium assay for copy number measurements Copy number applications in the analysis of: – Congenital disorders – Ca
Slide 3: History of Cytogenetics and DNA Copy Number Analysis
History of Cytogenetics and DNA Copy Number Analysis BIRTH OF CANCER Boveri CYTOGENETICS! Array-CGH implemented proposes that Hungerford discovers (BAC, PAC, YAC) chromosome abnormality on abnormalities chromosome 22 FISH cause cancer associated with CML technology High-density SNP used in genotypin
Slide 4: Copy Number Terminology Clarified
Copy Number Terminology Clarified SEGMENTAL STRUCTURAL COPY NUMBER COPY NUMBER DUPLICATION/LOW ABERRATION VARIANT POLYMORPHISM COPY REPEAT Present at a variable Segment of DNA that CNV that occurs in Most gross genomic copy number in occurs in 2+ copies per DEFINITION aberrations comparison with a m
Slide 5: What Causes Each Type of Common Aberration?
What Causes Each Type of Common Aberration? DUPLICATIONS/ • Section of DNA is accidentally amplified when a chromosome is copied AMPLIFICATIONS • Bloom and Rett Syndrome are caused by duplications (Increase in copy number) • Losses from translocation • Unequal crossing over DELETIONS • Breaking with
Slide 6: Array-CGH versus SNP Genotyping for Measuring Copy Number
Array-CGH (oligo and BAC) versus SNP Genotyping for Measuring Copy Number SNP GENOTYPING How relevant is genotyping MEASURES ALLELIC RATIO AND COPY NUMBER information? – Detection of copy neutral aberrations via LOH – Detection of uniparental disomy (UPD) and mitotic recombination or gene conversion
Slide 7: Infinium Whole-Genome Genotyping
Infinium Whole-Genome Genotyping 7
Slide 8: Genotyping Tools from Illumina
Genotyping Tools from Illumina # SNPs 650000 HumanHap650Y STANDARD CONTENT 558K-650K HumanHap550+ CUSTOM CONTENT 550,000 HumanHap550 325K–377K HumanHap300-Duo+ 317,000 HumanHap300-Duo 240,000 HumanHap240S 100,000 Human-1 iSelect™Technology 7,600-60,800 96 – 6,000 GoldenGate® Technology 1 2 12 96 # S
Slide 9: Applications of the Infinium Assay
Applications of the Infinium Assay WHOLE-GENOME ASSOCIATION STUDIES FINE-MAPPING STUDIES WHOLE-GENOME LOH/COPY NUMBER ANALYSIS DISEASE-ASSOCIATED CNV STUDIES 9
Slide 10: Effective Resolution for each BeadChip
Effective Resolution for each BeadChip MEDIAN EFFECTIVE BEADCHIP NUMBER OF SNPs SNP SPACING RESOLUTION 13kb Human-1 ~109k ~130kb in genes Hap300 ~317k 5kb ~50kb Hap550 ~555k 2.8kb ~28kb Hap650Y ~650k 2.5kb ~25kb *On average, a window size of 10 includes three to four heterozygous SNPs per window bas
Slide 11: The Infinium Whole Genome Genotyping Workflow
The Infinium Whole Genome Genotyping Workflow Advantages of the GENOMIC DNA (750 ng) Infinium assay: DAY 1 DAY 2 DAY 3 Easy workflow 50-mer hybridization 3 Fragment amplified DNA PCR-free protocol 1 Make amplified DNA High 4 Precipitate & resuspend reproducibility 2 Incubate amplified DNA (>99.9%) 8
Slide 12: Illumina's Bead Technology and the Infinium II Assay
Illumina’s Bead Technology and the Infinium II Assay BEADCHIP INFINIUM II SINGLE BASE EXTENSION Utilizes a single probe sequence (50mer) designed to hybridize immediately adjacent to the SNP query site 12
Slide 13: From Raw Data to Transformed Data: Genotype Clustering
From Raw Data to Transformed Data: Genotype Clustering A B C Raw – Rect. Normalized – Rect. Normalized – Polar 1.4 2.0 10000 1.8 NormIntensity (B) 1.2 1.6 AA Intensity (B) 8000 1.0 1.4 NormR 0.8 1.2 6000 AB 1.0 0.6 0.8 4000 BB 0.4 0.6 2000 0.4 males 0.2 0.2 0 0 0 -0.2 -0.2 55 20 45 12000 14000 10000
Slide 14: Calculating log R ratios and B allele frequency
Calculating log R ratios and B allele frequency A B LOG R RATIO log2(Robserved/Rexpected) Data Point of interest AA B ALLELE FREQUENCY Transforms theta (allelic intensity ratio) Genotype BB Clusters Example data point falls 1/3 of the way from the AB to BB cluster at 0.76. AB Allele frequency = 0.5+
Slide 15: DNA Copy Number Analysis
DNA Copy Number Analysis illumina: 15 ry
Slide 16: Allelic Ratios and Log Ratios of Normalized Intensities
Allelic Ratios and Log Ratios of Normalized Intensities LOG RATIOS OF NORMALIZED INTENSITIES (USED IN COPY NUMBER ANALYSIS) BB AB AA ALLELIC RATIOS (USED IN LOH ANALYSIS) A 20Mb HEMIZYGOUS DELETION DETECTED IN A COLON TUMOR SAMPLE USING A HUMANHAP300 BEADCHIP 16
Slide 17: Allelic Ratios and Log Ratios of Normalized Intensities
Allelic Ratios and Log Ratios of Normalized Intensities A 12Mb DUPLICATION DETECTED IN HL60 USING THE HUMANHAP550 BEADCHIP 17
Slide 18: Why is the Allelic Ratio Unique and Significant?
Why is the Allelic Ratio Unique and Significant? ALLELIC RATIO/ALLELIC FRACTION Extremely precise and robust measurement High signal to noise ratios (~9.0) Low noise (stdev ~0.04) Allows estimation of percentage contamination with normal cells 18
Slide 19: Detecting Single Copy Changes With X-Copy Cell Lines
Detecting Single Copy Changes With X-Copy Cell Lines A D B E 0.8 0.6 Dose Response 0.4 LOG R RATIO 0.2 0.0 C -0.2 -0.4 Chr 10 (Human-1) Chr X (Human-1) -0.6 Chr 10 (Hap300) Chr X (Hap300) -0.8 1 copy 2 copies 3 copies 4 copies (male) (female) (XXX) (XXXX) X COPY NUMBER 19 Peiffer et al., Genome Rese
Slide 20: Signal to Noise Ratios Using Different Window Sizes
Signal to Noise Ratios Using Different Window Sizes SIGNAL TO NOISE RATIOS 10 Log R Ratio Moving Average (N=1) 9 Log R Ratio Moving Average (N=10) 8 B Allele Frequency Moving Average (N=1) 7 6 5 4 3 2 1 0 Deletion Duplication Amplification 20
Slide 21: Comparison of Copy Number Metrics from Different Platforms
Comparison of Copy Number Metrics from Different Platforms “PROBE” ARRAY PLATFORM METRIC AVERAGE STDEV LENGTH Agilent CGH* 60mer Log R ratio 0.64 Affymetrix Mapping 25mer Log R ratio 0.40 100k# In-house printed 150-250kb Log R ratio 0.22 BAC array* Illumina 50mer Log R ratio 0.22 HumanHap300% UCSF (
Slide 22: A Wide Spectrum of Aberrations Can Be Detected
A Wide Spectrum of Aberrations Can Be Detected A B C D 22
Slide 23: Confirmation of Aberrations with BAC Array-CGH and FISH
Confirmation of Aberrations with BAC Array-CGH and FISH A B C Clone Location Combined T-stat RP11-528G9 2:q13 0.236 8.649 F5 22:q11.2 -0.292 -0.637 RP11-186O8 22:q11.2 -0.491 -5.962 Medical Genetics Laboratories Department of Molecular and Human Genetics – Houston, TX RP11-316L10 22:q11.2 -0.641 -8.
Slide 24: Detection of a Deletion in FOXP2 Involved in Language Impairment
Detection of a Deletion in FOXP2 Involved in Language Impairment using the HumanHap300 A Medical Genetics Laboratories Department of Molecular and Human Genetics – Houston, TX B C D E 24 American Journal of Medical Genetics (in press)
Slide 25: DNA Copy Number Analysis in Cancer Biology
DNA Copy Number Analysis in Cancer Biology 25
Slide 26: Detection of Various Deletions in a Cancer Cell Line (MCF7)
Detection of Various Deletions in a Cancer Cell Line (MCF7) HEMIZYGOUS LOG R RATIO HOMOZYGOUS 150 kb 83 kb MTAP IS HOMOZYGOUSLY DELETED CDKN2A (p16) AND CDKN2B (p15) HEMIZYGOUSLY DELETED *USED TO DIAGNOSE ACUTE LYMPHOBLASTIC LEUKEMIA *Bertin et al., 2003, Genes Chromosomes Cancer, 44-57. 26
Slide 27: Sensitivity Levels is ~50% in Heterogeneous Tumor Samples
Sensitivity Levels is ~50% in Heterogeneous Tumor Samples A B C D E F 27
Slide 28: Analyzing Large Tumor Sets for Trends: Chromosome in Breast Cancer
Analyzing Large Tumor Sets for Trends: Chromosome 1 in Breast Cancer 1 120 BREAST TUMOR SAMPLES DNA samples were courtesy of Vessela Kristensen, 28 from Department of Genetics Institute for Cancer Research the Norwegian Radium Hospital in Oslo, Norway
Slide 29: The HumanHap300-Duo Allows Paired-Sample Analysis on the Same Array
The HumanHap300-Duo Allows Paired-Sample Analysis on the Same Array Features/advantages of the HumanHap300-Duo High-genomic coverage Ability to run paired (matched) samples on the same array SAMPLE A SAMPLE B – Such as a Normal/Tumor pair – Or, a Blood/Sample of interest pair – Software allows for p
Slide 30: Paired-sample Analysis Reduces Log R Ratio Noise in Non-Standard DNA Samples
Paired-sample Analysis Reduces Log R Ratio Noise in Non-Standard DNA Samples A B 30
Slide 31: Copy Number Variants (CNV's)
Copy Number Variants (CNV’s) 31
Slide 32: Copy Number Variant (CNV) Genomic Regions
Copy Number Variant (CNV) Genomic Regions About a year ago……. Present at a variable copy number in 4,284 SNPs from 495 regions specifically included in comparison with a reference genome HumanHap550 1 kb or larger (50kb or larger for large-scale) Regions were identified in 3 CNV publications – Conra
Slide 33: Analysis of a Copy Number Variant Region on Chromosome 15 with the HumanHap550
Analysis of a Copy Number Variant Region on Chromosome 15 with the HumanHap550 •Size: ~6kb •Starting at 32417874 •Ending at 32575858 •Contains 11 SNPs •23/120 (20%) HapMap samples show reduction in copy number •Reported as “Locus1673” in CNV database Flanking CNV Flanking •Log R ratio data shown 33
Slide 34: Percentage of CNV Regions Covered
Reported CNV regions are well-covered with Illumina’s current product offerings and better than randomly selected SNPs 100 PERCENTAGE OF CNV REGIONS COVERED 90 80 TOTAL SNPs COVERING THE TORONTO CNV REGIONS* 70 60 Random Hap300 Hap550 Hap650Y 500k Not Covered 50 1+ SNP 40 Coverage 24729 46952 54752
Slide 35: High Coverage of Reported CNV Regions with the HumanHap550
High Coverage of Reported CNV Regions with the HumanHap550 500 1000 450 213 Total Number of CNV Regions NUMBER OF CNV REGIONS 400 CNV REGION (log scale) NUMBER OF SNPS PER HumanHap550 regions with 1+ SNP 94 350 Average Number of SNPs per region 74 100 300 51 34 250 16 200 8 10 150 100 3 50 0 1 0 00
Slide 36: Median SNP Spacing in Toronto CNV Regions
Reported CNV regions are densely covered with Illumina’s current product offerings MEDIAN SNP SPACING IN TORONTO CNV REGIONS (kb) 6 MEDIAN SNP SPACING (kb) 5 4 3 2 1 0 Hap300 Hap550 Hap650Y 36
Slide 37: BeadStudio Features Many Different Copy Number Analysis Tools
BeadStudio Features Many Different Copy Number Analysis Tools Ability to analyze paired samples (matched normal/tumor pair) Illumina Genome Viewer – Plot log ratios of normalized intensities and allelic ratios – View genome-wide profiles Illumina Chromosome Browser – View data at high resolution – V
Slide 38: Concluding Remarks
Concluding Remarks THE INFINIUM ASSAY DEMONSTRATES INTRINSIC FLEXIBILITY AND EXCEPTIONALLY HIGH DATA QUALITY INFINIUM ASSAY AND COPY NUMBER APPLICATIONS GENOMIC COVERAGE ANALYSIS • Infinium assay enables • Allelic ratio measurement is • Identify copy number trends in unconstrained SNP selection robu
Slide 39: Join our next webinar!
Join our next webinar! Monday, February 12th, 2007 at 11:00 AM (PST) Dr. Peter Laird of USC Topic: High-throughput DNA Methylation Profiling Register at http://www.illumina.com 39