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
E.coli:
4.6 Mbp
Yeast:
13.1 Mbp
Drosophila:
122 Mbp
Zebrafish:
1.2 Gbp
Human:
3.3 Gbp

Basic Yeast Statistics

16 chromosomes

13.1 Mbp of sequence

6,183 open reading frames

73% of the genome codes for genes
E.coli:
4,377
Yeast:
6,183
Drosophila:
17,000
Zebrafish:
15,800
Human:
23,000

Basic Yeast Statistics

16 chromosomes

13.1 Mbp of sequence
Left arm Crick Strand
Y A L 014 C


6,183 open reading frames
Chromosome I 14 th gene from
73% of the genome codes for genes the centromere

Genes are named by position.

Where to learn more:

Saccharomyces Genome Database

Where to learn more: Browser

Saccharomyces Genome Database

Yeast as a Model System
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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 plasmid cosmid fosmid
BAC
YAC
Host
E.Coli
E.Coli / phage
E.Coli – F’ element
E.coli
Yeast
Amount of DNA
1-20 kb
37-52 kb
40 kb 1/cell
150-350 kb
100 – 3,000 kb

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

So… How do you sequence a Genome?

Walking

Shotgunning

Mixed Approach

Prescaffolding 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
Copy Number Variation
… and more

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
GFP Library
STRESS
Cntl
 -factor HU MMS
FIX and STAIN
IMAGE
Quantify changes in intensity and location
Protein: RNR4
Control

-factor HU
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”
ALL STRAINS

Experiments can be done by looking at the variations in frequency of the pool after changing the environment of the library.
RICH MEDIA MINIMAL MINIMAL + 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