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BUDDING TECHNOLOGIES AND BUDDING YEAST 2012 HHMI Summer Workshop for High School Science Teachers
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The Genomics of S.cerevisiae
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GOALS Introduction to the Genomics of Yeast Sequencing Technologies and how they are evolving Introduction to Systems Biology and modern Yeast Genetics
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Genetics and Genomics GENETICS is the science of genes, heredity and variation. Genetic studies typically focus on a single gene. Experiments typically involve mutation of the model organism, then looking to figure out what went wrong. GENOMICS is a discipline of systems biology that focuses on the genome. Genomic studies typically study all genes at once
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Basic Yeast Statistics 16 chromosomes
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Genomic Organization & Nomenclature 16 Chromosomes. Range from 230kbp – 1.5Mbp
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Basic Yeast Statistics 16 chromosomes 13.1 Mbp of sequence Yeast: 13.1 Mbp Zebrafish: 1.2 Gbp Drosophila: 122 Mbp Human: 3.3 Gbp E.coli: 4.6 Mbp
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Basic Yeast Statistics 16 chromosomes 13.1 Mbp of sequence 6,183 open reading frames 73% of the genome codes for genes Yeast: 6,183 Zebrafish: 15,800 Drosophila: 17,000 Human: 23,000 E.coli: 4,377
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Basic Yeast Statistics 16 chromosomes 13.1 Mbp of sequence 6,183 open reading frames 73% of the genome codes for genes Genes are named by position. Y A L 014 C Chromosome I 14 th gene from the centromere Left arm Crick Strand
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Where to learn more: Saccharomyces Genome Database
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Where to learn more: Browser Saccharomyces Genome Database
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Yeast as a Model System Yeast share most basic systems with human. - Polymerases - Nucleosomes - Translation - Splicing - Stress response - DNA damage response - Cell Cycle - Mitotic mechanisms - Meiosis
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More about Yeast About75% of yeast genes have something known about them.
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More about Yeast About75% of yeast genes have known functions. Many genes serve to regulate other genes.
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More about Yeast About75% of yeast genes have known functions. Many genes serve to regulate other genes. About 1/3 of proteins are in the nucleus.
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GOALS Introduction to the Genomics of Yeast Sequencing Technologies and how they are evolving Introduction to Systems Biology and modern Yeast Genetics
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Sequencing the First Eukaryote 600 Scientists >100 labs World wide effort
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Sanger Sequencing
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So… How do you sequence a Genome? Walking
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So… How do you sequence a Genome? Walking
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So… How do you sequence a Genome? Walking Types of vectors TypeHostAmount of DNA plasmidE.Coli1-20 kb cosmidE.Coli / phage37-52 kb fosmidE.Coli – F’ element40 kb 1/cell BACE.coli150-350 kb YACYeast100 – 3,000 kb
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So… How do you sequence a Genome? Walking Shotgunning ~1-2kb Randomly fragment Completely sequence Reassemble
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Walking Shotgunning Mixed Approach Prescaffolding So… How do you sequence a Genome? markers Large vectors
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So… How do you sequence a Genome? Walking Shotgunning Mixed Approach Prescaffolding Shotgunning the fragments markers Large vectors Small plasmids
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Yeast to Human….
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A new revolution 454 Solexa ABI
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How NGS works Fundamentally different from Sanger Detect each base individually, then extend Watch as polymerase moves along the chain Each molecule is read multiple times
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How NGS works Illumina Sequencing uses “Sequencing by Synthesis Adaptors added to DNA to make them bind the flowcell. In situ, the DNA is amplified into a cluster
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How NGS works Primer then binds to the sequence. Bases are flowed over the cluster and nucleotides are read.
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How NGS works Primer then binds to the sequence. Bases are flowed over the cluster and nucleotides are read. Billions of reads are happening at once.
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A new revolution Sequencing costs are plummeting.
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A new revolution Sequencing costs are plummeting. Cut in half every year.
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A new revolution Sequencing costs are plummeting. Cut in half every year. Yields are sky rocketing.
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Applications gDNA mRNA miRNA IP Re-Sequencing De Novo Sequencing SNP Discovery Transcript Discovery Expression Analysis miRNA Analysis Allelic Expression ChIP-Seq Nuclear run-on … and more Copy Number Variation
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Applications: Genetics Mutation in alk in 224A/+ R>H D>N homozygous
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GOALS Introduction to the Genomics of Yeast Sequencing Technologies and how they are evolving Introduction to Systems Biology and modern Yeast Genetics
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Systems Biology Most molecular biology has been carried out with a reductionist point of view Look at one gene or one protein or a class of genes Systems Biology attempts to look at organisms holistically “OMICS” (genomics, proteomics, metabolomics, transcriptomics, etc.)
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Systems Biology: Beginnings First whole genome experiments were done with microarrays. Surface of the microarray is spotted with DNA reflecting every gene in the genome Total RNA is hybridized to the surface Amount of material can be measured by intensity
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Forward Genetics v Reverse Genetics Forward genetics is the classical method for doing screens. 1) Find a phenotype. 2) Find out why it happens. Reverse genetics mutates a gene, then sees what it does. This defined genetic alteration makes it amenable to systems biology approaches.
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Functional Screen: Two-Hybrid Screen genome wide for protein interaction partners. A “prey” library requires every protein to be fused to a transcription activation domain. Screen with a bait protein that binds to the DNA.
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Functional Screen: Two-Hybrid Screen genome wide for protein interaction partners. A “prey” library requires every protein to be fused to a transcription activation domain. Screen with a bait protein that binds to the DNA. Create large networks.
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The Modern Yeast Toolkit Two-Hybrid Knockout library GFP Fusion library Overexpression library High Copy Low Copy GST fusion library
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Screening GFP Libraries Control -factor HU Protein: RNR4 GFP Library STRESS Cntl -factor HU MMS FIX and STAIN IMAGE Quantify changes in intensity and location Data from Samson Lab
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Knockout Library and “BARseq” Knock out strains have unique molecular barcodes that act as finger prints. By pooling all the strains together, frequency of each strain can be determined by the frequency of the barcode in NGS experiments
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Knockout Library and “BARseq” Experiments can be done by looking at the variations in frequency of the pool after changing the environment of the library. ALL STRAINS RICH MEDIAMINIMALMINIMAL + AAs SEQUENCE AND LOOK FOR CHANGES IN FREQUENCY
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The Future – Synthetic Biology Key limitations of current toolset Have to create each strain separately. Finite number of mutations being created.
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The Future – Synthetic Biology Assembly of chromosomes in vitro. Can add any mutation anywhere by replacing a segment and reintroducing. Can create designer chromosomes with complex and unusual traits Do not require “carrier markers” Craig Venter, 2010
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The End Introduction to the Genomics of Yeast Sequencing Technologies and how they are evolving Introduction to Systems Biology and modern Yeast Genetics
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