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 14th 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

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

Type Host Amount of DNA

plasmid E.Coli 1-20 kb

cosmid E.Coli / phage 37-52 kb

fosmid E.Coli – F’ element

40 kb 1/cell

BAC E.coli 150-350 kb

YAC Yeast 100 – 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

Smallplasmids

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-SequencingDe Novo SequencingSNP Discovery

Transcript DiscoveryExpression AnalysismiRNA AnalysisAllelic ExpressionChIP-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

STRESSCntla-factor HU MMS

FIX and STAIN

IMAGEQuantify 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 FORCHANGES 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