Functional genomics
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Functional genomics uses genome-wide experimental approaches to assess gene function on a large scale. It analyzes gene expression through techniques like transcriptomics and proteomics. Transcriptomics analyzes gene expression profiles through RNA sequencing or microarray analysis. Microarray analysis involves hybridizing fluorescently-labeled cDNA or cRNA to microarrays containing DNA probes to measure gene expression levels across thousands of genes simultaneously. Functional genomics provides a global understanding of gene function and molecular interactions through integrated omics approaches.
Functional genomics
- 1. Functional Genomics Presented by: Pawan Kumar (1101034) Sumit Dahiya (1101020) Arpan Pandey (1101058) PK 1 Understand Gene function Bhaskaracharya College of Applied Sciences (University of Delhi) 06March 2014
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- 3. Levels of Genome Research PK 3
- 4. Definition (1)- Hieter & Boguski 1997 The development & application of global • Genome-wide or • System-wide experimental approaches to assess gene function by making use of the information & reagents provides by structural genomics. It is characterized by high-throughput or large scale experimental methodology. • Combined with statistical or computational analysis of the results. PK 4
- 5. Definition (2) – UC Davis Genome Center A means of assessing phenotype differs from more classical approaches primarily with respect to The scale & automation of biological investigations A classical investigation works only fxal genomics can examine On a single gene. Over 1k-10k genes PK 5
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- 8. TranscriptomicsAnalysis PK 8 • Study of transcriptomes (i.e. complete set of RNA produced by genome at any one time) • Provides gene expression profile. • Gene expression identification.
- 10. Exploratory studies Which genes are differentially expressed? Which genes are co-expressed? Which genes interact Which genes show alternative splicing? PK 10
- 11. Prognostic studies Which cancer has Patient X? Is my drug safe for PatientY? Which treatment is the most efficient? PK 11
- 12. An integrated system is needed for functional genomics Profiles Transcript Genotype Proteome Metabolome Interactions DNA : Protein Protein : Protein Phenotypes High Throughput Screening Tissue Microarray Loss / Gain of Function Quantitative Trait Loci Bioinformatics
- 13. Requirements: Database backed storage. Specialized signal processing algorithms. Dedicated analysis environment. Additional knowledge about genes and transcripts PK 13
- 14. Techniques… PCR based methods. DNA microarray analysis. PK 14
- 15. Polymerase Chain Reaction • Developed in 1983 by Kary Mullis ,a biochemical technology in molecular biology used to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. PK 15
- 16. Uses • DNA cloning for sequencing. • DNA-based phylogeny, or functional analysis of genes. • Diagnosis of hereditary diseases. • Identification of genetic fingerprints. • PCR can be extensively modified to perform a wide array of genetic manipulations & hence may replace gene cloning completely PK 16
- 17. DNA MICROARRAY • A DNA microarray (aka DNA chip or biochip) is a collection of microscopic DNA spots attached to a solid surface. • can be used to detect DNA or RNA that may or may not be translated into proteins. • Expression Analysis/expression profiling which is a process of measuring gene expression via cDNA PK 17
- 18. MIAME ? • internationally adopted standard for the Minimal Information About a Microarray Experiment. • The result of an MGED (www.mged.org) driven effort to codify the description of a microarray experiment. • Ultimately, it tries to specify the collection of information that would be needed to allow somebody to completely reproduce an experiment that was performed elsewhere. PK 18
- 19. Six Parts of MIAME 1. Experimental design: the set of hybridization experiments as a whole 2. Array design: each array used and each element (spot, feature) on the array 3. Samples: samples used, extract preparation and labeling 4. Hybridizations: procedures and parameters 5. Measurements: images, quantification and specifications 6. Normalization controls: types, values and specifications PK 19
- 20. Principle • hybridization between two DNA strands. • A high number of complementary base pairs in a nucleotide sequence means tighter non-covalent bonding between the two strands. • Fluorescently labelled target sequences are used that bind to a probe sequence generate a signal that depends on the hybridization conditions, and washing after hybridization. • Total strength of the signal, from a spot, depends upon the amount of target sample binding to the probes present on that spot. • Microarrays use relative quantitation in which the intensity of a feature is compared to the intensity of the same feature under a different condition, and the identity of the feature is known by its position. PK 20
- 21. Platforms… • In standard microarrays, the probes are synthesized and then attached via surface engineering to a solid surface by a covalent bond to a chemical matrix. • The solid surface can be glass or a silicon chip, in which case they are colloquially known as an Affy-chip when an Affymetrix chip is used. • Other microarray platforms, such as Illumina, use microscopic beads, instead of the large solid support. PK 21
- 23. +1 AAAAAA TTTTTT - t7 promoter Preparing target TTTTTT - t7 promoter reverse transcription second strand synthesis in vitro transcription
- 24. HowTo Do…. PK 24
- 25. Scanning of arrays • Laser scanners • Excellent spatial resolution • Good sensitivity, but can bleach fluorochromes • Although slow • Charged couple device scanners • Spatial resolution can be a problem • Sensitivity easily adjustable (exposure time) • Faster and cheaper than lasers PK 25
- 26. Expression array • Cell growth in different environments, treatments etc. • Isolate RNA cDNAs • Measure expression using array technology • Create database of expression information • Data Analysis • Display information in an easy to-use format • Show ratio of expression under • Different conditions Affymetrix® food chip PK 26
- 27. • microRNA detection • Comparative Genomic Hybridization (CGH) detects deletions or amplifications of genomic sequence • ChIP on chip chromatin immunoprecipitation • Single Nucleotide Polymorphism screening (SNP) measures an individual’s genotype at known sites of variance • Cell Arrays • Protein Arrays • Tissue Arrays Other microarray-based assays
- 28. Image Analysis… • Image analysis.. • Translate the scan into expression numbers. • Queries... • Check for defects. • Quality metrics provided by scanner. PK 28
- 29. Bioinformatics of Microarrays • Array design: choice of sequences to be used as probes • Analysis of scanned images • Spot detection, normalization, quantitation PK 29
- 30. Primary analysis of hybridization data • Basic statistics, reproducibility, data scattering, etc. • Comparison of multiple samples • Clustering, SOMs (Self-Organizing Maps (a subtype of artificial neural network, low-dimensional views of high-dimensional data) • Unsupervised learning • Sample tracking and data basing of results. PK 30
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- 32. Benefits of using a data repository PK 32 Facilitates data sharing Catalogued / Backed-up Pervasive advertisement for your work End users/Researchers Access to data for analysis and algorithm development Improves search capabilities Encourages development of more capable software for annotation, analysis and submission Bioinformaticians/Developers
- 33. DATAWarehousing • The sheer volume of data, specialized formats (such as MIAME), and curtain efforts associated with the datasets require specialized databases to store the data. • Makes it easy to compare 2 Data files. • Easy to access. • User friendly. • Worldwide avalabilility PK 33
- 34. Microarray Data on the Web • Many groups have made their raw data available, but in many formats • Some groups have created searchable databases • There are several initiatives to create “unified” databases • EBI: Array Express • NCBI: Gene Expression Omnibus Companies are beginning to sell microarray expression data (e.g. Incyte) PK 34
- 35. Software forTranscriptomic Analysis • Bio-Linux • maxdView • GeneSpring • R/BioConductor PK 35
- 36. Which software should I use?? Commercial vs. Open Source GeneSpring maxdView R/BioConductor Ease of Use GeneSpring > maxdView > R/BioConductor Fine tuned control R/BioConductor > maxdView > GeneSpring PK 36
- 37. GeNet maxDLoad2 R/BioConductor ArrayExpress Raw Data Expression measures (not normalised) Proprietary software (e.g. Affymetrix) GeneSpring maxDViewR/BioConductor Quality Control Normalisation Analysis Presentation Other analysis programs MIAME/Env Annotation PK 37
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- 39. Overview of Microarray Analysis Steps PK 39 Load Data Apply Filters Normalise Analyse Quality Control Step 1 Text,GPR file, etc… Step 2 Step 3 Step 4 Step 5
- 40. Gene Chips PK 40
- 41. Study of the proteome. The proteome is the complete complement of proteins found in a complete genome or specific tissue. PK 41
- 42. Proteomics and genomics are inter-dependent Genome Sequence mRNA Primary Protein products Functional protein products Determination of gene Genomics Proteomics Proteomics Protein Fractionation 2-D Electrophoresis Protein Identification Post-Translational Modification PK 42
- 43. Aims of Proteomics • Detects the different proteins expressed by tissue, cell culture, or organism using 2-Dimensional Gel Electrophoresis • Stores the information in a database • Compares expression profiles between a healthy cell vs a diseased cell • The data comparison can then be used for testing and rational drug design. PK 43
- 44. Gel Electrophoresis • Motion of charged molecules in an electric field. • Polyacrylamide gel provides a porous matrix • (PAGE – Polyacrylamide Gel Electrophoresis) • Sample is stained with comassie blue to make it visible in the gel. • Sample placed in wells on the gel. PK 44
- 45. 2D – Separation is based on size and charge • First step is to separate based on charge or isoelectric point, called isoelectric focusing. • Then separate based on size (SDS-PAGE). PK 45
- 46. SDS-PAGE • Second Dimension. • Separation by size. • Run perpendicular to Isoelectric focusing. • The only unresolved proteins after the first and second dimensions are those proteins with the same size and same charge – rare! PK 46
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- 48. 2D-PAGE Analysis Software • 2D-PAGE technology has been in use for over 20 years, and potentially provides a vast amount of information about a protein sample. • However, due to difficulties with data analysis, it remains only partially exploited. PK 48
- 49. List of 2-D GEL DATABASES One can find an extensive list of such databases by following these links. We would discuss a few “Interesting ones”. •World 2-D PAGE •NCIFCRF •DEAMBULUM-Protein Databases •Ludwig Institute of Cancer Research •Phoretix PK 49
- 52. Background • Mass spectrometry (Mass Spec or MS) uses high energy electrons to break a molecule into fragments. • Separation and analysis of the fragments provides information about: • Molecular weight • Structure PK 52
- 53. Fragmentation Patterns • Mass spectrum of 2-methylpentane PK 53
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- 55. References….. • Concept of genetics –Klug & Cummings, 10th edition • Perou, C.M., Sorlie,T., Eisen, M.B., van de Rijn, M., Jeffrey, S.S., Rees, C.A., Pollack, J.R., Ross, D.T., Johnsen, H., Akslen, L.A., Fluge, O., Pergamenschikov,A., Williams, C., Zhu, S.X., Lonning, P.E., Borresen-Dale, A.L., Brown, P.O., Bolstein, D. 2000. Molecular portraits of human breast tumors. Nature 406(6797):747-752. • http://www.bioinformatics.org/wiki/Microarray_analysis • http://www.bing.com/images/search?q=Microarray+Assay&FORM=RESTAB#a • http://www.nature.com/nrn/journal/v5/n10/images/nrn1518-f2.jpg • http://farm4.staticflickr.com/3045/2516660289_a92fd6dae7_z.jpg?zz=1 • http://www.bio.Davidson.edu/courses/genomics/chip/chip.html • http://Onlinelibrary.wiley.com/journal/10.1002/(ISSN)1615-9861 • http://www.genomics.aglent.com/article.jsp?pageld=287 • http://www.genome.gov/10000533 • http://en.Wikipedia.org/wiki/DNA_microarray PK 55
- 56. Questions….. • What is functional genomics? • What are the implications of functional genomics? • DefineTranscriptomics? • Name diff. tech. involved in transcriptome analysis? • Explain exploitation of DNA microarray in hybridization tech.? • How microarray database is different from microarray analysis? • What are the different tools for microarray analysis? • What is the need of microarray analysis? • Define Proteomics? • Explain various tech. involved in protein separation? PK 56