Flexible Microarray Platform from Agilent Technologiestechcomm.lifescience.ntu.edu.tw/P1/TC1 071221.pdfMedaka Microarray. Annotation: Gene Information ZebraFish O. ... selected gene

FFlexible lexible Microarray Platform from Microarray Platform from Agilent TechnologiesAgilent Technologies

Welgene Biotech.YiYi--ShingShing Lin Ph.DLin Ph.D

2007/Dec2007/Dec

[email protected]@welgene.com.tw

Clinical LabOffice Area

microarray services

Authorized distributor

Welgene Biotech. Welgene Biotech. Distributor & Service ProviderDistributor & Service Provider

GeneExpressionmRNA LevelaCGH Chromosomal AberrationmiRNA post-transcriptional regulationChIP-on-chip epigenomics, transcription factor

HumanMouseMouse DevelopmentPig (Custom Array)RatArabidopsisC. elegansDog

Species M. griseaMonkey Rice Xenopus laevisYeastZebrafishCustom Build

Agilent Technologies60-mer Oligo Microarray

Application

InIn--situsitu Synthesis of Agilent ArraysSynthesis of Agilent Arrays

Nature Biotechnology Vol 19 April 2001 P342

A C T G

2.

3.

1.

6060--mer mer OligoOligo arrayarray

High Sensitivity in 60mer OligoHigh Sensitivity in 60mer Oligo

Average LLD*60mers: 0.004pM25mers: 0.032pM

*LLD:Lower Limit of Detection

Spike-in Concentration

BG

Sub

stra

cted

Sig

nal

Inkjet Technology for High DensityInkjet Technology for High Density

Back toPerformance

244,000

Accuracy drop volume drop placement jet spacing

Speed and throughput Reliability

Pin-spotting

Ink jet

Resistor Off

LiquidVaporizes

GasExpands

Resistor On Resistor Off

DropBreaks Off Reservoir

Refills

FillReservoir

< 1 msec

To Explore Gene RegulationTo Explore Gene Regulation

DNA level

RNA level

DNA copy number

uclesome / TFs binding

DNA methylation

Gene expression

microRNA regulation

CH3

Trend: More Microarray ApplicationsTrend: More Microarray Applications

Chromosome DNA Gene promoters mRNA mRNA variants microRNAs

aCGH SNPs miRNA

DNA RNA

SNPs ChIP/LA miRNAGene

ExpressionAlternateSplicing

Specificity

Sensitity

Key requirements

Informatics

Flexibility

Concept of ArrayConcept of Array--based CGHbased CGHReference DNA Test DNA

Log2

ratio

=0


Chromosomes DNA Gene promoters mRNA mRNA variants microRNAs

aCGH SNPs miRNA

DNA RNA



Specificity

Sensitity

Key requirements

Informatics

Flexibility

ChIPChIP--onon--chipchip(chromatin (chromatin immunoprecipitationimmunoprecipitation--onon--chip)chip)

To answer:

which genes are regulated by a known TF/DNA binding protein

High Accuracy of ChIPHigh Accuracy of ChIP--onon--chipchipStart with optimal probe designStart with optimal probe design

Methodology Tile 60-mers at 1-bp spacing across non-RepeatMasked genome (1.3B probes). Reduce 10-fold by thermodynamic scores (130M probes). Homology search against the genome using ProbeSpec (custom homology

search tool designed for probe matching). Reduce 10-fold using homology scores (13M probes). Re-score homology using MegaBlast (catches gapped alignments).

GENE XGENE YTF

Probe optimization criteria: Uniqueness (homology) Tm Self-structure

CpG island

CpG Island Array

95 bp

probe design target interval

probes

Array Design StrategyArray Design Strategy

Human and Mouse CpG island array available now!!!

~250 bp

Probe ProbeProbeProbe

gene

Transcription Start Site

8 kb promoter region 5.5 kb upstream, 2.5 kb downstreamPromoter Array

ChIPChIP--onon--chip processchip process

ChIP ChIP from A Local Vantage Pointfrom A Local Vantage PointGENE XTF

Chromatin broken intosmall fragments

Genomic Region:

TF

TF

TF

TF

TF

TF

TF

TF

TF

TFTF

Antibody-coatedMagnetic Bead

UNTREATED Anti-TF TREATED

IP-enriched DNA fragments

GENE XGENE YTF

ChIPChIP--enriched DNA enriched DNA vs.vs. total DNA inputtotal DNA input

Total DNA input (WCE)

Enriched DNA (IP)

Note: chromatin DNA fragments

are ~100 - 500 bp

244K randomized 60-mer

Richard A. Young, PhDWhitehead Institute

MIT

Details

Application: ChIP-on-chip

Comprehensive and genome-wide localization of 18,000 putative binding events

Co-occupancy OCT4, SOX2, NANOG help understand the mechanisms of pluripotency

Identification of autoregulatory(feed-forward) circuits

Stem Cell Transcriptional Regulation Stem Cell Transcriptional Regulation NetworksNetworks



aCGH SNPs miRNA

DNA RNA



Specificity

Sensitity

Key requirements

Informatics

Flexibility

miRNAmiRNAScientific Background and Scientific Background and ImportanceImportance

Cancer Genomics: Small RNAs with big impactsfrom Nature 435: 745-746 (9 June 2005)

*Source: NIH CRISP database at http://crisp.cit.nih.gov/

Definition: 19-30 nucleotide long single-stranded RNAs

that post-transcriptionally regulate gene expression

Key regulators of gene expression, development, proliferation, differentiation, and apoptosis

May regulate >30% of human genes Current Sanger miRBASE Release 9.1 has

4361 entries, 474 identified in humans Projected $100M to be awarded by the NIH for

miRNA-related research in 2008*Discovery of new miRNAs is ongoing

Challenges in Challenges in miRNAmiRNA ProfilingProfiling Small size High sequence homology Expressed with large dynamic range Growing & changing database

http://www.ipmc.cnrs.fr/images/equipes/honore/mirna_steps.jpg

Add T-tilt to increase miRNA hybridization accessibility

G-addition to stabilize the hyb. duplex

Adjust Tm by shortening the probe at the 5end (5-end is conserved among miRNA, I.e less specifific)

Add 5-hat to repellthe longer RNA, such as mRNA

Probe design of Probe design of miRNAmiRNA arrayarray

Figure 2bmiRNAs

hsa-let-7ahsa-let-7bhsa-let-7chsa-let-7dhsa-let-7ehsa-let-7fhsa-let-7ghsa-let-7i

UGAGGUAGUAGGUUGUAUAGUUUGAGGUAGUAGGUUGUGUGGUUUGAGGUAGUAGGUUGUAUGGUUAGAGGUAGUAGGUUGCAUAGUUGAGGUAGGAGGUUGUAUAGUUGAGGUAGUAGAUUGUAUAGUUUGAGGUAGUAGUUUGUACAGUUGAGGUAGUAGUUUGUGCUGU

2222222121222121

miRNA Sequence Length (nts)

0

85 - 100%70 - 84%55 - 69%40 - 54%25 - 39%10 - 24%5 - 9%0 - 4% Grey Number0

0000000

1

0000

11

4

33

2

00

00

00

00

12

75

5

30

1

1

1

1

11

11 00

0

1 24

3

6

Data ComparisonData Comparison

32 (58%)32 (70%)Overlap number (%)

5546 Down-regulated miRNAs (3-fold)

47 (70%)47 (78%)Overlap number (%)

6760Up-regulated miRNAs(3-fold)

203 (90%)313 (67%)Detectable miRNAs (%)

225470Total miRNAs

qRT-PCRMicroarray



aCGH SNPs miRNA

DNA RNA



Specificity

Sensitity

Key requirements

Informatics

Flexibility

Agilent Complete Design FlexibilityAgilent Complete Design Flexibility

105K

105K

244K

44K 44K 44K 44K

For genome wide design

Lower Cost with high resolution

Lower cost per experiment

15K 15K 15K 15K

15K 15K 15K 15K

High-throughput sampling

Data Driven

Hypothesis Driven

Probe cost by home-brew spotting:244000 x 60 x 8 = 117,120,000

105000 x 60 x 8 = 50,400,000

44000 x 60 x 8 = 21,120,000

15000 x 60 x 8 = 7,200,000

Agilent Custom Array ProgramAgilent Custom Array Program

Create Probe

Groups

Upload Probes

Search Agilent Probes

Create Microarray Designs

Submit to Manufactu

ring

Order Arrays

Upload genome

sequence

Custom Probe Design

Computational Probe SelectionComputational Probe Selection

Rosetta/Agilent probe selection algorithms

Validated selection criteriai. Vector maskingii. Repeat maskingiii. Tmiv. Base compositionv. BLAST for specificityvi. Folding characteristics

Probe locationi. Adjacent to 3-endii. Evenly distributed

Gene

Transcript 1

Transcript 2

Transcript 3

Transcript 4

Merged into oneconsensus region

Cant merge

I II III

Merged ConsensusRegion

CandidateProbes

Real Design Case by WelgeneReal Design Case by Welgene

Custom Array DesignCustom Array Design

Probe content ofProbe content of MagnaportheMagnaporthe griseagriseaMicroarrayMicroarray

15,170 predicted genes by the Fungal Genomics Laboratory at North Carolina State University

Rice ESTs were derived from cDNA generated from uninfected as well as infected rice tissues

Array Design fromArray Design from dede--novonovo SequencingSequencing

cDNA Libraries Construction

ESTs Sequencing

Sequences Assemble

Gene ClusterGene ClusterCustom Array Design

Annotation

Example:Medaka Microarray

Annotation: Gene InformationAnnotation: Gene Information

ZebraFish O

Total 12,429 clusters->8,091 clusters to Agilent custom array

Leveraging Leveraging AgilentAgilentss Flexible Printing PlatformFlexible Printing Platform

We will manufacture: What you want When you want it In the quantity you need With no or low set up fees With or without professional

bioinformatics support Customers dont have to: Adapt your research needs to standard

product designs Wait for complex setups or oligo sets Amortize large design or oligo

investments over large volumes

is the engine that drives Agilents microarray

End of Section I.End of Section I.

Any Question?Any Question?

Glass

Substrate

Microarray

Quarantine

Process

Dice/Package

Not for sale!

IndustrialIndustrial--Scale InkScale Ink--jet Printingjet PrintingQuality Control and Process MonitoringQuality Control and Process Monitoring

Microarray Manufacture WorkflowMicroarray Manufacture Workflow

Define Genetic Content

Probe Design and Selection(Computational & Empirical)

Design File Creation

Substrate Applied to Glass Wafers

Nucleic Acids & ReagentsPrepared for Printing

In-situ Chemical Synthesis via Inkjet Printing Technology

Quality Assessment ofRepresentative Microarrays

Microarrays Packaged for Shipping

On-line eArray Design Microarray Print

QC

QC

QC

QC

QC

Introduction of Microarray Data AnalysisIntroduction of Microarray Data Analysis--using using GeneSpringGeneSpring as an exampleas an example

Welgene Biotech.YiYi--ShingShing Lin Ph.DLin Ph.D

2007/Dec2007/Dec

[email protected]@welgene.com.tw

Information storage

Data storage

execution

Idea: measure the amount of mRNA to see which genes are being expressed in (used by) the cell.

Measuring protein directly might be better, but is currently harder.

Microarray principle (two color system)Microarray principle (two color system)Sample A

(cells,tissue,etc.)Extract total RNA

Label withfluorescent Cy5

in RT rxn

AAAAAAAAAA

AAAAAAAAAA

Sample B(cells,tissue,etc.)

AAAAAAAAAA

AAAAAAAAAA


in RT rxn

Pool

Hyb target - spotted DNA

Competitive hybridization

Microarray

Laser scan forCy5

Laser scan forCy3

Cy5 signalCy3 signal

ExpressionRatio

DiseaseNormal

1 hourt=0

TreatedUntreated

Examples:

Microarray principleMicroarray principle(single color system)(single color system)

Sample A(cells,tissue,etc.)

Extract total RNA


in RT rxn

AAAAAAAAAA

AAAAAAAAAA

Sample B(cells,tissue,etc.)

AAAAAAAAAA

AAAAAAAAAA


in RT rxn

Hyb target - spotted DNA

hybridization

Microarray (A)

hybridization

Microarray (B)

Laser scan forCy3

Laser scan forCy3

Cy3 signal(A)Cy3 signal(B)

ExpressionRatio

Microarray (A) Microarray (B)

From Raw Expression Data to Biological Knowledge

Go from this

prostaglandinsynthesisIn TGF-b treatment

...To this!

Agilent GeneSpring GXAgilent GeneSpring GX

Start with the Raw

expression values

Load the data into

GeneSpring

Use powerful but easy to use

statistics to filter the list

Find biological relevance of the genes

GenomeGenome

Annotation updateAnnotation update

Analysis StepsAnalysis Steps

Untreated Treated with ILbeta

Data normalization

Quality filtering

Find DE genes

Find GO category & Gene function of DE genes

Data QC

Analysis stepsAnalysis steps


Data normalization

Quality filtering

Find DE genes


Data QC

Scatter Plot Scatter Plot

Check the Distribution of your Check the Distribution of your Signals with Box PlotsSignals with Box Plots



Data normalization

Quality filtering

Find DE genes


Data QC

Global (Per Chip) NormalizationGlobal (Per Chip) NormalizationNo Normalization Global Normalization

IntensityIntensity--dependent LOWESS dependent LOWESS NormalizationNormalization

Reality

Plot of log intensity Cy3 vs. log intensity Cy5

Ideal scenario

Cy3

Cy5



Data normalization

Quality filtering

Find DE genes


Data QC

Quality Quality FFilterilter

Data Filtering Data Filtering by Flagby Flag

Array Signal DistributionArray Signal Distribution

Highly expressed

moderately expressed

Low expressed

No expressed



Data normalization

Quality filtering

Find DE genes


Data QC

Defining parameter setupDefining parameter setup


Enter the parameters for replicate samples

Find Differentially Expressed Genes with Find Differentially Expressed Genes with ANOVAANOVA

Finding The Biological Significance Finding The Biological Significance

Function Category Gene Ontology

Molecular function Biological process Cellular component

Pathway Analysis KEGG Biocarta GeneMap

Full Gene Ontology BrowserFull Gene Ontology Browser

Allows you to more easily determine the function of your genes, by calculating the enrichment of your gene list with genes from a particular GO category

GO Ontology Browser: TableGO Ontology Browser: Table Genes in Category total

number of genes in the genome that have been assigned to this category

% of Genes in Category percentage of the total genome that has been assigned to this category.

Genes in List in Category the total number of genes that are both in the selected gene list and in this category

% of Genes in List in Category percentage of the selected gene list that falls into this category.

p-value the hypergeometric p-value (without multiple testing corrections). A measure of the statistical significance of the overlap between the selected gene list and this category.

KEGGKEGG

KEGG: Kyoto Encyclopedia of Genes and Genomes

http://www.genome.jp/kegg/

A grand challenge in the post-genomic era is a complete computer representation of the cell and the organism, which will enable computational prediction of higher-level complexity of cellular processes and organism behaviors from genomic information. Towards this end we have been developing a bioinformatics resource named KEGG, Kyoto Encyclopedia of Genes and Genomes, as part of the research projects in the KanehisaLaboratory of Kyoto University Bioinformatics Center.

Pathway ListPathway List

Example: ApoptosisExample: Apoptosis

BiocartaBiocarta

www.biocarta.com

Pathway Information in GeneSpring GXPathway Information in GeneSpring GX

From Raw Expression Data to From Raw Expression Data to Biological KnowledgeBiological Knowledge

Go from this

prostaglandinsynthesisIn TGF-b treatment

...To this!

Integrate with Ingenuity Pathway Integrate with Ingenuity Pathway Analysis (IPA)Analysis (IPA)

Run the analysis in IPA

Gene List from IPA to Gene List from IPA to GeneSpringGeneSpring

Save selected

My List for GeneSpring

Genes from IPA analysis.zip

Drag & Drop Gene List from IPA to Drag & Drop Gene List from IPA to GeneSpringGeneSpring

Genes from IPA analysis.zip

Thank You for Your ListeningThank You for Your ListeningHave a nice day and Goodbye!

Agilent platform NTU 2007m DecGeneSpring GX introduction 2007 NTU

Documents

Flexible Microarray Platform from Agilent Technologiestechcomm.lifescience.ntu.edu.tw/P1/TC1 071221.pdfMedaka Microarray. Annotation: Gene Information ZebraFish O. ... selected gene