生物信息学课件英文原版课件 (110)

Microarrays for Gene Expression Analysis,Questions: What genes are expressed in this tissue under these conditions?What genes are expressed in my treated cells versus the control?What genes are expressed during the phases of the cell cycle?What genes are expressed in diseased tissue versus normal tissue?,Microarrays other uses,Questions: What point mutations exist and what bases are located at the substitution positions?What bases are substituted where there are multiple mutations very close together?Which allele of this gene do we have?Is this the mutant or wildtype?,Goals,Finding Co-Regulated GenesUnderstanding Gene Regulatory Networks,Expressed Genes = mRNA,DNA,messenger RNA,protein,Expressed Genes = Currently Transcribed,mRNA,Extract RNA,mRNA,mRNA,mRNA,mRNA,Isolate mRNAs,Affymetrix Oriented,Fluorescently tagged cRNA One chip per sample One for control One for each experiment Other methods include two dyes/one chip Red dye Green dye Control and experiment on same chip,Creating Targets,PCR Amplificationof DNA,In Vitro transcription to create cRNA,RNA-DNA Hybridization,probe setsDNA(25 base oligonucleotides of known sequence),TargetsRNA,Non-Hybridized Targets are Washed Away,“probe sets” (oligos),Targets(fluorescently tagged),Non-bound ones are washed away,Picture of Gene Chip,Handling Chip,Argon laser488nm,570nm,Scanner based on epifluorescence confocal microscopy,Custom Chips vs Affy Chips,Affy chips contains thousands of gene probes Genes selected from sources such as GenBank Custom chips can be designed for individual investigators Few genes, but more copies of each Done on microscope slide,Example Affy Chips,Rat Toxicology Chip - 850 genes CYP450s, Heat Shock proteins Drug transporters Stress-activated kinases Rat Neurobiology chip - 1,200 genes Synuclein 1, prion protein, Huntingtons disease Syntaxin, Neurexin, neurotransmitters,Example Affy Chips,Arabidopsis Genome Chip Murine Genome Chip - 36,000 genes E. coli Genome Chip - 4,200 ORFs Drosophila Genome Chip - 13,500 sequences Yeast Genome Chip - 6,400 ORFs Human Genome Chip - 60,000 human genes,Definitions,Probe a single-stranded DNA oligonucleotide complementary to a specific sequence. Each probe cell consists of millions of probe molecules.Probe Array a collection of probes sets.Probe Set a set of probes designed to detect one transcript. 16-20 probe pairs. A 20 probe pair set is made up of 20 PM and 20 MM for a total of 40 probe cells.Probe Pair Two probe cells, a PM and its corresponding MM.Perfect Match(PM) probes that are designed to be complementary to the reference sequence.MisMatch(PM) probes that are designed to be complementary to the reference sequence except for 1 base.Target sequence from your sample.,GeneChip Hierarchy,Probe Array = ChipProbe Set 16-20 probe pairs(to detect particular gene)Probe Pair Probe Cell (MisMatch) 20Probe Cell (Perfect Match) 20Probes MM intensityNegative Probe Pair = MM intensity PM intensityCalculate Positive FractionCalculate Pos/Neg RatioCalculate Log Average Ratio & Avg Difference,Absolute Analysis Parameters,Probe Set Name Positive - number of pairs scored positive Negative number of pairs scored negative Pairs number of probe pairs for a probe set Pairs Used those not masked for some reason PairsInAvg excludes those with extremely intense or weak scores,Absolute Analysis Parameters,PM Excess have exceeded limit for intensity MM Excess have exceeded limit for intensity Avg Diff average difference of fluorescence intensity between the PM and MM cells. Log Avg Ratio a measure of the hybridization performance Higher = better Log Avg = 0 indicates random cross hybridization Pos/Neg ratio of positive probe pairs to negative probe pairs Positive Fraction positive probe pairs/probe pairs Abs Call Present, Absent or Marginal. Is this gene present in this sample?,Raw Data to Cooked Data,Positive Fraction Pos/Neg Ratio Log Avg Ratio,Decision Matrix,Absolute Call(Present, Absent, Marginal),Data Analysis,Absolute Analysis used to determine whether transcripts represented on the probe array are detected or not within one sample(uses data from one probe array experiment).Comparison Analysis used to determine the relative change in abundance for each transcript between a baseline and an experimental sample(uses data from two probe array experiments). Intensities for each experiment are compared to a baseline/control.,Approaches,What genes are Present/Absent in my tissue? What genes are Present/Absent in the experiment vs control? Which genes have increased/decreased expression in experiment vs control? Which genes have biological significance based on my knowledge of the biological system under investigation?,Approaches to Data Analysis,Database Queries,Graphical AnalysisStatistical Analysis,Biological Knowledge,Set Filter Parameters,Query,Pivot,Scatter/Fold Graph,Select Points,Identify interestingrelationships,Add probe sets to filter,Bar Graph,Adjust filter parameters,Data Analysis,Absolute Analysis used to determine whether transcripts represented on the probe array are detected or not within one sample(uses data from one probe array experiment).Comparison Analysis used to determine the relative change in abundance for each transcript between a baseline and an experimental sample(uses data from two probe array experiments). Intensities for each experiment are compared to a baseline/control.,Comparison Analysis Parameters,Inc number of probe pairs that increasedDec number of probe pairs that decreased Inc Ratio Dec Ratio Max Inc & Dec Ratio Pos Change Neg Change Inc/Dec DPos-DNeg Ratio Log Avg Ratio Change Diff Call did this gene increase or decrease? Increase, Marginal Increase, Decrease, Marginal Decrease, No Change,Avg Diff Change how much did the difference between PM and MM change from the control to the treated?(Avg Dif Exp Avg Dif Control) B=A was this gene present in the control? Fold Change how many times more expression did the treated have compared to the control? (positive or negative) Sort Score a ranking based fold change and avg diff change,Comparison Analysis Parameters(continued),Data Analysis,Filter/Query:Select those oligos which have shown a real,significant change.,Filter & Sort to Find Real Changes,Avg. Difference Change = 200 andFoldChange 3 andINC = 70% and DEC = 0%,Query Results,Query Results,Probe1Probe2Probe3Exp1 1 4 9Exp2 3 6 8,Pivot the Query Results,Experiments = columns Genes = Rows Shows how genes change across experiments,Pivot Results,Exp1Exp2Probe1 1 3Probe2 4 6Probe3 9 8,Pivoted DataCan be sorted by any parameter. Sort in descending order to show greatest differences.,Description of gene,Link to NCBI,Graphical Analyses,Scatter Plot Graph requires Control vs Experiment,How many times did the expression of this gene change in the treated tissue versus the control?,Fold Change Graphs,Statistical Techniques,Statistical Techniques,Self-Organizing MapsCorrelation Coefficient ClusteringAnalysis FunctionMatrix,Self-Organizing Maps,ClusteringAutomatically discovering classes,Self-Organizing Maps,Self-Organizing Maps,Genes whose expression level rise/fall togetherunder the same conditions, cluster togetherCo-regulated ?,Statistical Techniques,Average Standard Deviation Median T-test parametric comparison of means. assumes a normal distribution Mann-Whitney for the comparison of two samples non-parametric