1ClassClass InformationInformation• http://www.biostat.jhsph.edu/~ririzarr/stanford• Download and install R 2.3.1• Download and install Bioconductor 1.8• M and T we talk about general methods• Th we introduce a problem and analyzerelated data using R• If you can, please bring a laptop on Th• If taking for grade final project requires dataanalysis otherwise literature review• Class shaped as we go alongIntroduction to Genome BiologyIntroduction to Genome Biologyand and Microarray Microarray TechnologyTechnologyLecture 1Credit for some of today’s materials:Terry Speed, Sandrine Dudoit,Victor Jongeneel, Giovanni ParmigianiWhatWhat we can learnwe can learn• Deal with background noise•Normalize across arrays• The probe effect• Find differentially expressed genes• Enrichment analysis• The multiple comparison problem• Experimental design• Clustering and classification• Time series experiments• Annotation• Using gene information• New applications: SNP chips, tiling arrays, Epigenomics, etc…2TodayToday1. Basics of Transcription2. Basics of Hybridization Theory3. How Microarrays WorkCells and the genomeCells and the genome• Each cell contains a complete copy of anorganism’s genome, or blueprint for allcellular structures and activities.• The genome is distributed alongchromosomes, which are made of compressedand entwined DNA.• Cells are of many different types (e.g. blood,skin, nerve cells), but all can be traced backto a single cell, the fertilized egg.Why are cells different?Why are cells different?3Gene expression experiments measure theamount of mRNA to see which genes are beingexpressed in (used by) the cell.Measuring protein levels directly is also possible,but is currently harder.DNADNA• A deoxyribonucleic acidor DNA molecule is adouble-stranded polymercomposed of four basicmolecular units callednucleotides.• Each nucleotidecomprises a phosphategroup, a deoxyribosesugar, and one of fournitrogen bases: adenine(A), guanine (G),cytosine (C), andthymine (T).• The two chains are heldtogether by hydrogenbonds between nitrogenbases.• Base-pairing occursaccording to thefollowing rule: G pairswith C, and A pairs withT.Cells and the genomeCells and the genome• A (protein-coding) gene is a segment ofchromosomal DNA that directs the synthesisof a protein• An intermediate step is the gene beingtranscribed or expressed• Most microarray experiments measure geneexpression4TranscriptionTranscriptionG T A A T C C T C | | | | | | | | |C A T T A G G A GDNAG U A A U C CRNApolymerasemRNAFrom DNA to mRNAReverse transcriptionReverse transcriptionClone cDNA strands, complementary to the mRNAG U A A U C C U CReversetranscriptasemRNAcDNA C A T T A G G A G C A T T A G G A G C A T T A G G A G C A T T A G G A GT T A G G A G C A T T A G G A G C A T T A G G A G C A T T A G G A G C A T T A G G A G C A T T A G G A GWhat are we measuring?What are we measuring?We call what we want to measure the target• The amount of RNA transcripts– Expression arrays– RT-PCR• The existence or abundance of a DNAsequence– SNP chips, Tiling arrays• Yeast mutant representation– With TAG arraysNotice all of them are Nucleic Acid moleculesuniquely defined by a sequence of bases5Nucleic acid hybridizationNucleic acid hybridizationMicroarrays: the game planMicroarrays: the game plan• Use hybridization to measureabundance of target molecule• Fix probes to a solid support and createfeatures• Hybridize labeled target to probes andwash to get rid of non-hybridizedmaterial• Use labels to measure feature intensityHybridizationHybridizationTarget (RNA)CATGAT…CGAT6HybridizationHybridizationFeatures or ProbesLabeled TargetCATGAT…CGATGTACTA…GCTATechnology OverviewTechnology OverviewVarious platforms:• Probes can be sequenced or cloned• Features can be high-density orcircles in a grid• One or two samples hybridized toarraySequenced (High density)7Before LabelingBefore LabelingArray 1Array 2Sample 1Sample 2Before Hybridization: One ChannelBefore Hybridization: One ChannelArray 1Array 2Sample 1Sample 2After HybridizationAfter HybridizationArray 1Array 28Scanner ImageScanner ImageArray 1Array 2QuantificationQuantificationArray 1Array 24 2 0 3 0 4 0 3Two colorTwo color9Microarray ImageMicroarray ImageBefore Labeling: Two ChannelBefore Labeling: Two ChannelArray 1Sample 1Sample 2Before HybridizationBefore HybridizationArray 1Sample 1Sample 210After HybridizationAfter HybridizationArray 1Scanner ImageScanner ImageArray 1QuantificationQuantificationArray 14,0 2,4 0,0 3,311Microarray ImageMicroarray ImageMore on Spotted ArraysMore on Spotted Arrays384 wellplateContains cDNAprobesGlass SlideArray of bound cDNA probes4x4 blocks = 16 print-tip groupsPrint-tipgroup 6cDNA clonesSpotted in duplicatePrint-tipgroup 1Pins collect cDNAfrom wells12Image analysisImage analysis• With the images in place, we have datafor first time• First step is image analysis: determinewhich pixels are part of features andwhich are not• We leave this to the company engineersalthough some academics haveattacked the problemsNomenclature ReviewNomenclature Review• Target - what we want to measure. Canbe RNA, trated RNA, DNA, treated DNA,DNA Barcodes• Probes - Molecules used to measuretarget. Can be synthesized or cloned• Features - contiguous region on thearray with same probe. We usuallyobtain one intensity reading from eachfeatureFeature Level DataFeature Level Data• Image analysis software produces featurelevel data• This is where we starts• First step is to get a hold of the files with thisdata and parse them• Currently most files are CEL (Affymetrix), XYS(Nimblegen), and GPR (Two color platformsread with genepix scanner). But others exists!• We also need to match each feature with atarget molecule of interest. This is sometimesdone in another