BUDDING TECHNOLOGIES AND BUDDING YEAST
2012 HHMI Summer Workshop for High School Science Teachers
The Genomics of S.cerevisiae
GOALSIntroduction 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 CChromosome 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 SystemYeast 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.
GOALSIntroduction 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 kbcosmid E.Coli / phage 37-52 kbfosmid E.Coli – F’
element40 kb 1/cell
BAC E.coli 150-350 kbYAC 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: GeneticsMutation in alk in 224A/+
R>H D>N homozygous
GOALSIntroduction 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
STRESSCntl-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 EndIntroduction to the
Genomics of Yeast
Sequencing Technologies and how they are evolving
Introduction to Systems Biology and modern Yeast Genetics