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High-Throughput Technologies for Human Genetics

Carsten Rosenow, PhD Associate Director Global Market

Development

HKU May 22st, 2012

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Our Vision Innovating for the Future of Genetic Analysis

To be the leading provider of integrated solutions that advance the understanding of genetics and health

To Targeted Validation and Beyond…

From Genome Wide Discovery…

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Next-Gen Sequencing

Custom Genotyping Arrays and Sequencing

Targeted resequencing

Next-gen GWAS Arrays

Sequencing and Arrays Leading the Next Wave of Discoveries

Array Design

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Continuum of Human Genetics Discovery From bench to bedside

Variant Identification: Cataloging the

complete picture of variation in humans

Whole-genome Applications: Agnostic scan of

the entire genome to narrow down

regions of interest 3 Billion BP à

a few MB

Targeted Applications: Identification of causal loci in

regions of association or

candidate genes A few MB à a few specific variants

Prioritization and Functional

Understanding: Combining

orthogonal data (expression, protein, drug

response, etc.)

Translational Genomics:

Moving research findings from the RUO space into

the clinic: assessing utility

and patient impact, cost

Diagnostics: Routine use of

genetic information in the

diagnosis, treatment and prevention of

disease

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Whole-Genome Applications

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Published Genome-Wide Associations through 3/2012, ˜700 published GWA at p < 5 x 10-8

NHGRI GWA Catalog www.genome.gov/GWAStudies

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Complex Diseases The GWAS Area

GWAS Publications, 2005 ‒ 6/2011

Tota

l Pub

licat

ions

951

Calendar Quarter

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Understanding Heritability

Sample Size

Rarer Variants

Epistasis Environment

Copy Number

Heritability Measures Understanding

Biology

Novel Variants

? Epigenetics

?

?

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Despite thousands of discoveries by GWAS, for any given disease, only a portion of the heritability has been explained.

Heritability Remains Nebulous

0%

20%

40%

60%

80%

100%

Huntingd

on's

Cystic

fibro

sis AMD

Crohn

'sLu

pus T2D

HDL cho

lester

ol

Height

Early M

I

Fasti

ng glc

Rare Common Disease\Traits

Adapted from Manolio et al 2009

Nature, 2008

Explained Heritability

Missing Heritability

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Sample Size

Rarer Variants

Epistasis Environment

Copy Number

Heritability Measures

Novel Variants

? Epigenetics

?

?

Understanding Heritability

Understanding Biology?

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Primary GWAS: 20,000 samples ~30 loci 50% implicated in plasma lipid Meta Analysis: 100,000 samples* 95 loci, 59 novel with links to plasma lipid *Derived from 46 GWAS studies from 137-22041 Individuals

Teslovich et al, (2010) Nature

Advances in Understanding Blood Lipids

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Functional Validation shows Biological Relevance All 95 Loci Identified in This Study Will Be Subject to Further Investigation

“We expect that future investigations of the new loci (for example, resequencing efforts to identify low-frequency and rare variants, or functional experiments in cells and animal models) will uncover additional important new genes.”

Targeted Resequencing

Functional Validation

eQTL Analysis

Functional Validation

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Identified a common, non-coding causal variant that creates a transcription binding site and alters expression of the SORT1 gene.

From Noncoding Variant to Phenotype Via SORT1 Musunuru et al, (2010) Nature

Tag SNP (P=1 x 10-170)

Expression in Liver

SORT1

CELSR2 PSRC1 MYBPHL SORT1

SNP Creates Enhancer Binding Site

Mobility shift assay

As noncoding DNA variants may alter gene expression, we previously used expression quantitative trait locus (eQTL) analyses to explore whether 1p13 SNPs are cis-acting regulators of nearby genes in human liver

eQTL analysis

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Overexpression of Sort1 à 46% decrease in cholesterol Knockdown of Sort1 à 70% increase in cholesterol

Sort1 Viral overexpression

RNAi knockdown

The Sort1 gene alters cholesterol levels in mice Musunuru et al, (2010) Nature

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Evolving Genomic Tools To explore the entire allele spectrum

Effe

ct s

ize

Sm

all

Larg

e

Allele Frequency Low High

Very Rare Variants Large Effect Size

Rare/Intermediate Variants

Common Variants

Small Effect Size

(Common Variants Large Effect Size)

(Rare Variants Minimal Effect Size)

LIN

KA

GE

N

GS

GW

AS

A

RR

AYS

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Illumina’s Whole-Genome Portfolio Explore the allelic spectrum with the Omni Family of Microarrays and WGS

Product   Omni Express   Omni2.5 Omni5 WGS: 5x WGS: 30x

Description

Highest-throughput common variant

array with industry-proven quality at an exceptional price.

Mid-level microarray with coverage of both common and rare

SNP content

Flagship microarray with industry leading

coverage of common and rare

variation

Low-depth NGS dataset that leverages

informatics to fill in missing data

Ultimate whole-genome dataset

Samples per array / flowcell 12 8 4 ~18 ~3

Markers ~730K + up to 200K semi-custom

~2.3M + up to 200K semi-custom

~4.3M + up to 500K semi-custom

~20M w/ imputation ~2.85 Billion

Target MAF >5% >2.5% >1% Varies Varies Time to run 1000

samples* 1 week 1 week 3 weeks 9 months 4.5 years

*Assumes 1 scanner/sequencer and standard automation

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! Intelligently selected Tag SNPs provide industry best coverage of Chinese populations

–  Higher coverage for Chinese populations compared to OmniExpress & competing Chinese whole-genome array

! Proven Infinium Assay –  Industry leading call-rates and reproducibility

! 8x1 Sample format provides exceptional throughput for large-scale GWAS studies

–  Up 960 spls/week with our standard configuration §  1 iScan, 1 Autoloader, 1 tecan, 5 day week

The Human OmniZhongHua-8 The First Chinese Population-Specific Whole Genome Array from Illumina

The ZhongHua delivers coverage of both Hapmap and Thousand genomes variants, & exceptional throughput at an enabling price.

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The HumanExome BeadPool Leveraging microarrays for validation, to boost power

! The HumanExome BeadChip –  Collaborative effort among genetics key

opinion leaders –  A tool for assaying function exonic SNPs

in large sample sets to drive statistical power

Over 1 Million Samples Sold

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Exome SNPs Are Distributed Across Gene Regions

Feature Type Number of Markers

Total Markers ≈250K

SNPs within RefSeq ≈248K

SNPs in coding regions 246K

SNPs within 10Kb of RefSeq 260K

Non-Synonymous 232K

Promoter 7K

SNPs in splice sites 67K

Other Regions: Ancestry; IBD; GWAS; ADME/MHC; X/Y/Mito; Indels

5’ UTR 3’ UTR exon

Access ˜260K exonic markers from diverse populations

intron 10kb US

10kb DS

Splice site

Promoter

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Targeted   Whole  Genome   Variant  Discovery  /  Targeted  

Exome BeadChip   OmniExpress Exome Omni2.5 Exome Omni5 Exome TruSeq

Exome Seq

The most comprehensive set of functional exonic variants for quick,

economical screening

The most economical common variant GWAS with enhanced coverage

of the exome

Whole-genome coverage down to

2.5% MAF with enhanced coverage

of the exome

The ultimate whole-genome array with

coverage down to 1% MAF with the most

exonic coverage of any microarray

Industry leading NG exome sequencing protocol – variant

discovery and targeted screening

~250K functionally relevant variants

~730K common tagSNPs + ~250K exonic variants

~2.3M tagSNPs down to 2.5% MAF

+ ~250K exonic variants

~5.3M tagSNPs down to 1% MAF + ~250K exonic variants

Capture and sequence >94% of refseq coding

exons

Illumina’s Exome Family From variant discovery to variant screening and back again Infinium Exome Content TruSeq Exome

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Product   VeraCode   GoldenGate iSelectHD 3k-90k

iSelectHD 90k-250k

iSelectHD 250k-1m Semi-Custom

Product  Descrip.on  

The power of the GoldenGate

assay on a platform and at

a price accessible to all

labs.  

Pre-optimized design of custom content using the

most trusted low-plex assay with the highest

quality data in the industry

Validate genomic

discoveries with the most robust

data and flexible content

design

Enable more genomic

discoveries with the most robust data and flexible content design

Enable more genomic

discoveries with the most robust data and flexible content design

Combine custom content with

Illumina’s GWAS or focused genotyping arrays to maximize

your association study

Samples  per  array   96 32 24 12 4 Varies

Markers   1 - 384 96 – 3,072 3,072 – 90K 90K – 250K 250K – 1M Varies List  Price  

    $ $ $-$$ $$ $$ Varies

Custom Genotyping Family of Options Driving the cycle of discovery, validation and powering statistical significance

Increasing Plexity

Increasing Price

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Illumina’s DNA Technology Portfolio Complementary Technologies Working Together for Discovery

STRENGTHS

Throughput

Price

Ease of use

Discovery technology

Capture more variation

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RNA-Seq: Analysis of Severe Acute Respiratory Syndrome (SARS) -infected Mouse RNA Samples

The SARS Virus: SARS-CoV [Coronavirus; RNA virus; 29,727 bases ]

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RNA-Seq: Using Counts instead of Intensity

VS

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Even distribution of a transcript 100 Kb mRNA

Normal polyA select mRNA-seq

Total RNA

Poly-A + DSN

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Experimental strategy

! Infect mouse with SARS Coronavirus (positive-strand, enveloped RNA virus)

! Extract RNA from lung tissue –  Mixture of host and pathogen RNA

! Input 1 µg Total RNA into standard Illumina mRNA-seq assay inc. poly A –  Abilities demonstrated here compatible with all Illumina RNA-seq assays

including the Total RNA-seq –  Now input is as low as 100ng

! Align reads against Mouse Genome

! Align reads against SARS Genome

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Global Mouse Gene Expression Changes

SARS infected mouse lung tissue

Uni

nfec

ted

mou

se lu

ng ti

ssue

19,013 RefSeq Genes

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Largest Fold Change increase & decrease in mouse after SARS exposure

Genes SARS-MF1 Control-MF2 Fold-ChangeCcl2 2588 5 517.6Serpina3h 3590 7 512.8571429Rsad2 11663 34 343.0294118Cxcl10 6613 20 330.65EG667977 1762 7 251.7142857Cxcl9 1849 10 184.9Slfn4 5302 29 182.8275862Isg15 7780 54 144.0740741Oasl1 3127 26 120.2692308Oas3 3555 30 118.5Ifi202b 2422 22 110.0909091Irf7 22871 212 107.8820755Slfn8 1604 15 106.9333333Zbp1 4405 42 104.8809524Mx2 2758 27 102.1481481Mx1 4146 45 92.13333333Oas2 2485 29 85.68965517H2-Q2 1455 18 80.83333333Ifi44 5551 77 72.09090909Phf11 1067 16 66.6875H2-Q10 1360 22 61.81818182Ifit1 10147 179 56.68715084Apod 7419 137 54.15328467Oas1a 1567 30 52.23333333Mnda 412 8 51.5

Genes SARS-MF1 Control-MF2 RatioCar3 16 1878 0.00852Igfbp2 15 1268 0.01183Psca 5 366 0.013661Cfd 29 1818 0.015952Cntn1 8 471 0.016985Scn3a 8 359 0.022284Gdpd3 32 1370 0.023358Ptma 12 409 0.02934H2-Eb1 109 3204 0.03402Rps18 8 228 0.035088Lrrc17 5 141 0.035461Cyp4f16 8 218 0.036697Aox3 70 1764 0.039683Cidec 25 577 0.043328Cidea 7 161 0.043478Cyp1a1 89 2046 0.0435Tnn 19 426 0.044601Glp1r 15 325 0.046154A930038C07Rik 55 1179 0.04665D430041D05Rik 8 171 0.046784Thrsp 39 819 0.047619Adrb3 15 306 0.04902Mamdc2 98 1988 0.049296Ociad2 8 162 0.049383Pcolce2 178 3604 0.04939

Increased Expression Decreased Expression

Chemokine (C-C motif) ligand 2 (CCL2): recruits memory T-Cells to sites of infection

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Transcripts that are not detected in one sample or the other…

Genes SARS-MF1 Control-MF2H2-Ea 1036 0Cyp2b13 168 0Olfr56 145 0Cngb3 100 0Csf3 96 0Saa2 70 0Ccl20 53 0Klri2 46 0Rhcg 42 0Ubd 40 0Trim12 33 0LOC547349 32 04930503B20Rik 24 0BC049730 24 0Il27 22 0Trim69 21 01700009N14Rik 20 0Klri1 20 0

Genes SARS-MF1 Control-MF2Ucp1 0 276Ear2 0 181Tmem45b 0 102Klk1b22 0 85Fabp1 0 82Glrb 0 50Ces5 0 49Slc15a1 0 44Tmem16e 0 38Chrm1 0 37Hes2 0 37Hepacam2 0 35Kcna1 0 35Rbp7 0 34Clca3 0 33Prss35 0 32St6galnac1 0 27Tpsb2 0 25

Increased Expression Decreased Expression

H2-Ea: major histocompatibility complex, class II, high homology in humans: candidate susceptibility gene for pulmonary fibrosis

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GenomeStudio View of IL8RB Expression Changes

547 Counts

30 Counts

+ SARS

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GenomeStudio View of RSAD2 Expression Changes

11,663 Counts

34 Counts

+ SARS

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Coverage of reads on SARS Genome in MF1 and MF2

MF2, 9 reads

MF1 ~250K reads

a. Total length of the SARS sequence.

b. First half of the sequence

MF1

+ SARS

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SNPs called by CASAVA and displayed in GenomeStudio in SARS

12 SNPs

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An Instrument for Every Need. Every Budget. Every Lab

Two proven technologies. One powerful platform.

HiScanSQ

The most widely cited platform, now at

half the price

GAIIx MiSeq

My Samples. My Study. MiSeq

Powerful. Flexible. Scalable.

HiSeq 1000/1500

Redefining the trajectory of sequencing.

HiSeq 2000/2500

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A Solution for Every Application Illumina Sequencing Platform

Application Optimal Platform MiSeq HiSeq

Targeted Resequencing

Amplicons (Nextera, Tailed PCR) √√√ TruSeq Custom Amplicon (up to 100 kb / 50 genes) √√√ TruSeq Custom Enrichment (up to 2 Mb / 500 genes) √√√ √√√ TruSeq Custom Enrichment (up to 20 Mb / 5K genes) √√√ TruSeq Exome (62 Mb; >20 K genes) √√√

RNA-Seq

Small RNA √√√ √√ Microbial RNA-Seq √√√ √√ Human RNA-Seq √ √√√

Whole Genome Resequencing De Novo Sequencing

Large complex genomes (eg. Human) √√√ Microbial genomes √√ √√√

Regulation ChIP-Seq √ √√√ WG Methylation √√√

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TruSeq Exome Enrichment Targets = 100,000s

TruSeq Custom Enrichment Targets = 1000s

TruSeq Custom Amplicon Targets = 100s

TruSeq Targeted Resequencing The simplest and most scalable targeted resequencing solutions

Nextera PCR Amplicons Targets = 10s

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TruSeq Custom Amplicon The fastest and easiest multiplexed amplicon assay optimized for MiSeq

! Rapid & economical –  Up to 384 amplicons per sample –  Up to 96 samples per plate –  Plate-based processing –  <8 hrs from gDNA to sequencing-ready

library –  Utilizes standard lab equipment –  No quant needed before sequencing

! Fully customized target probes & capture –  DesignStudio for interactive design and

ordering –  Personalized and easy to use –  Proven extension and ligation-based assay –  Rapid design turnaround

! Pre-configured, automated data analysis

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BaseSpace The Best Place to Store Your NGS Data

Reads and qualities Sample and experiment descriptions Analysis results

variants contigs metagenomes coverage statistics miRNA counts more…

! Eliminates need for onsite storage and compute

! Results available anywhere, anytime

! Browse the results via web-based graphical environment

! Access to a growing suite of analysis tools

! Tools for collaboration and sharing

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Targeted Cancer Sequencing Lipson et al. Nature Genetics 2012

! Targeted  cancer  panel  

! Deep  sequencing  

! FFPE  samples  

! Fusion  genes  detected  by  exome  sequencing  

! Informa.ve  graphics  

Nat Med, 2012 e-pub ahead of print

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Experimental Design Overview

! Panel  of  145  cancer-­‐relevant  genes  with  2574  coding  exons  

! 40  colorectal  cancer  and  24  non–small  cell  lung  cancer  

!  DNA  isolated  from  40  microns  of  formalin  fixed  paraffin  embedded  (FFPE)  tumor.  

! For  all  specimens  ≥25%  of  the  nuclear  area  was  malignant  tumor  cells  so  no  micro/macro  dissec.on  .ssue  enrichment  was  performed.    

!  Sequencing  on  the  HiSeq2000  instrument  (Illumina)  was  with  36  bp  paired  reads  to  average  depth  of  229X  

! Base  subs.tu.on:  >10%  mutant  allele  frequency  with  >99%  sensi.vity  

! Indels:  >20%  mutant  allele    frequency  with  >95%  sensi.vity  

! False  discovery  rate  <1%  

! Found  at  least  one  clinically  relevant  genomic  altera.on  in  59%  of  the  samples  

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40 CRC FFPE specimens

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24 NSCLC FFPE specimens

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Validation of the RET fusion genes

! Further screening of 561 lung adenocarcinomas identified 11 additional tumors with KIF5B-RET gene fusions

mRNA-­‐Seq  

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TruSeq®  Amplicon  –  Cancer  Panel  Hundreds of loci. Rapid prep. FFPE-ready.

! Comprehensive Content –  >35 kb total including oncogenes

such as BRAF, KRAS & EGFR –  212 amplicons in one tube; 48 genes

! Unrivaled Multiplexing –  Up to 96 sample pooling on MiSeq –  >90% specificity and uniformity –  Detect low frequency variants (<5%)

! Unparalleled Workflow –  FFPE-enabled with sample QC Kit –  No qPCR quant needed for

normalization –  Automated paired end sequencing

with MiSeq –  Pre-configured, automated data

analysis For research use only

ABL1   EGFR   GNAS   MLH1   RET  AKT1   ERBB2   HNF1A   MPL   SMAD4  ALK   ERBB4   HRAS   NOTCH1   SMARCB1  APC   FBXW7   IDH1   NPM1   SMO  ATM   FGFR1   JAK2   NRAS   SRC  BRAF   FGFR2   JAK3   PDGFRA   STK11  CDH1   FGFR3   KDR   PIK3CA   TP53  

CDKN2A   FLT3   KIT   PTEN   VHL  CSF1R   GNA11   KRAS   PTPN11  

CTNNB1   GNAQ   MET   RB1  

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Where is the Missing Heritability?

Sample Size

Rarer Variants

Epistasis Environment

Copy Number

Heritability Measures What is the biology?

Novel Variants

? Epigenetics

?

?

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Interest in Epigenetics Is On The Rise

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The Infinium HumanMethylation450 includes every content category requested by an expert consortium

Feature Included on array

Total # sites 485,553

RefSeq genes 99%

CpG islands 96%

island shores 92%

island shelves 86%

HMM islands >63K

FANTOM 4 promoters >12K

Informatically-predicted enhancers >102K

DNAse hypersensitive sites >60K

MHC sites >12K

Non-CpG loci >3K

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Methylation Sites Are Distributed Across Gene Regions

Feature Type Genes Mapped

Percent Genes Covered

Number of Loci on Array

NM_TSS200 14895 0.79 2.56

NM_TS1500 17820 0.94 3.41

NM_5'UTR 13865 0.78 3.34

NM_1stExon 15127 0.80 1.62

NM_3'UTR 13042 0.72 1.02

NM_GeneBody 17071 0.97 8.97

5’ UTR 3’ UTR TSS1500 TSS200 1st exon Gene body

Overall global average of 17 sites / RefSeq gene region

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CpG Islands, Shores Are Covered With Similar Strategy

Feature Type Islands Mapped Percent Islands Covered

Average Number of Loci on Array

Island 26153 0.94 5.08 N_Shore 25770 0.93 2.74 S_Shore 25614 0.92 2.66 N_Shelf 23896 0.86 1.97 S_Shelf 23968 0.86 1.94

N Shelf N Shore S Shore S Shelf CpG Island

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Compared methylation profiles of HTC-116 colorectal cancer cell line with normal colon mucosa

Identified distributions of hypermethylated (left) vs. hypomethylated (right) loci across region categories (tumor vs. normal)

Demonstrates ability of HumanMethylation450 to detect differential methylation across gene, CpG island regions

Recent study validates importance of HM450 gene region coverage, suitability for EWAS

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! 12 samples / array format

! Scan time 1 hr / BeadChip

! Manual or automated workflow

! Process up to 96 samples in parallel

! 4 days from DNA to data

! LIMS (Laboratory Information Management System) now available

–  Enables positive sample tracking –  Quality assurance in large sample size

studies

High-Throughput For Large Sample Size Study Designs

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CNV Detection: SNPs provide maximal CNV information Applicable for all Infinium Arrays

Normal (diploid)

Deletion (loss of one copy)

Duplication (gain of one copy)

Intensity Genotypes

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“Now this is not the end. It is not even the beginning of the end. But it is,

perhaps, the end of the beginning.”

-- Winston Churchill