BUDDING TECHNOLOGIES AND BUDDING YEAST 2012 HHMI Summer Workshop for High School Science Teachers

The Genomics of S.cerevisiae

GOALS  Introduction to the Genomics of Yeast  Sequencing Technologies and how they are evolving  Introduction to Systems Biology and modern Yeast Genetics

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

Basic Yeast Statistics  16 chromosomes

Genomic Organization & Nomenclature  16 Chromosomes.  Range from 230kbp – 1.5Mbp

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

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

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

Where to learn more:  Saccharomyces Genome Database

Where to learn more: Browser  Saccharomyces Genome Database

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

More about Yeast  About75% of yeast genes have something known about them.

More about Yeast  About75% of yeast genes have known functions.  Many genes serve to regulate other genes.

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.

GOALS  Introduction to the Genomics of Yeast  Sequencing Technologies and how they are evolving  Introduction to Systems Biology and modern Yeast Genetics

Sequencing the First Eukaryote 600 Scientists >100 labs World wide effort

Sanger Sequencing

So… How do you sequence a Genome?  Walking

So… How do you sequence a Genome?  Walking

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.coli kb YACYeast100 – 3,000 kb

So… How do you sequence a Genome?  Walking  Shotgunning ~1-2kb Randomly fragment Completely sequence Reassemble

 Walking  Shotgunning  Mixed Approach  Prescaffolding So… How do you sequence a Genome? markers Large vectors

So… How do you sequence a Genome?  Walking  Shotgunning  Mixed Approach  Prescaffolding  Shotgunning the fragments markers Large vectors Small plasmids

Yeast to Human….

A new revolution  454  Solexa  ABI

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

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

How NGS works  Primer then binds to the sequence.  Bases are flowed over the cluster and nucleotides are read.

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.

A new revolution  Sequencing costs are plummeting.

A new revolution  Sequencing costs are plummeting.  Cut in half every year.

A new revolution  Sequencing costs are plummeting.  Cut in half every year.  Yields are sky rocketing.

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

Applications: Genetics Mutation in alk in 224A/+ R>H D>N homozygous

GOALS  Introduction to the Genomics of Yeast  Sequencing Technologies and how they are evolving  Introduction to Systems Biology and modern Yeast Genetics

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.)

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

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.

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.

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.

The Modern Yeast Toolkit  Two-Hybrid  Knockout library  GFP Fusion library  Overexpression library  High Copy  Low Copy  GST fusion library

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

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

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

The Future – Synthetic Biology  Key limitations of current toolset  Have to create each strain separately.  Finite number of mutations being created.

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

The End  Introduction to the Genomics of Yeast  Sequencing Technologies and how they are evolving  Introduction to Systems Biology and modern Yeast Genetics