MCB 317Genetics and Genomics

Topic 11, part 2Genomics

Need to Add to part 2 or 3

A. Chip-seqB. Deep sequencing for expression profilingC. Illumina? movie

Genomics Summary

A. Microarrays: expression profiling and other usesB. Global Gene Knockouts C. Global protein localization in yeast D. Global complex identification in yeast E. Global two-hybrid analysis in yeast and other

organismsF. RNAiG. Transgenics, gene “knock-outs” (genetics not

genomics)H. Human Genome Project, Next Generation

Sequencing, and Comparative Genomics

Yeast “Knockout” Library

Delete YFG

Delete all genes (individually)

Disruption of “All” Yeast Genes

• Approx 6000 genes• Make 6000 sets of disruption primers• Disrupt each gene in a diploid• Dissect all 6000 diploids

– Identify set of essential genes– Identify set of non-essential genes

Yeast “Knockout” Library

• Delete one copy of each gene in diploid– 5,916 “genes” deleted– 5,916 diploid strains constructed

• Dissect to determine if gene is essential– 1,105 genes = essential– 18.7% of genes = essential

• Construct an ordered library of haploids for non-essential genes– 4,811 mutant strains in library

Genomics Summary

A. Microarrays: expression profiling and other usesB. Global Gene Knockouts C. Global protein localization in yeast D. Global complex identification in yeast E. Global two-hybrid analysis in yeast and other organismsF. RNAiG. Transgenics, gene “knock-outs” (genetics not genomics)H. Human Genome Project, Next Generation Sequencing,

and Comparative Genomics

Ab

Protein

TxnProfile

Gene

Orthologs and Paralogs

Mutant Gene

Biochemistry

Genetics

Mutant Organism

A

C

F

Subunits of Protein Complex

B, G

D E

ProteinProfile/Localization

Genomics:

High-throughput genetics

Genomics

B, G

H

Genomics Summary

A. Microarrays: expression profiling (and other uses)B. Global Gene Knockouts C. Global protein localization in yeast D. Global complex identification in yeast E. Global two-hybrid analysis in yeast and other organismsF. RNAiG. Transgenics, gene “knock-outs” (genetics not genomics)H. Human Genome Project, Next Generation Sequencing,

and Comparative Genomics

Ab

Protein

TxnProfile

Gene

Orthologs and Paralogs

Mutant Gene

Biochemistry

Genetics

Mutant Organism

A

C

F

Subunits of Protein Complex

B, G

D E

ProteinProfile/Localization

Genomics:

High-throughput genetics

Genomics

B, G

H

Genomics Summary

A. Microarrays: expression profiling and other usesB. Global Gene Knockouts C. Global protein localization in yeast D. Global complex identification in yeast E. Global two-hybrid analysis in yeast and other organismsF. RNAiG. Transgenics, gene “knock-outs” (genetics not genomics)H. Human Genome Project, Next Generation Sequencing,

and Comparative Genomics

Ab

Protein

TxnProfile

Gene

Orthologs and Paralogs

Mutant Gene

Biochemistry

Genetics

Mutant Organism

A

C

F

Subunits of Protein Complex

B, G

D E

ProteinProfile/Localization

Genomics:High-throughput genetics

Genomics

B, G

H

8100 Human DBD-ORFs x 8100 Human AD-ORFs

Genomics Summary

A. Microarrays: expression profiling and other usesB. Global Gene Knockouts C. Global protein localization in yeast D. Global complex identification in yeast E. Global two-hybrid analysis in yeast and other organismsF. RNAiG. Transgenics, gene “knock-outs” (genetics not genomics)H. Human Genome Project, Next Generation Sequencing,

and Comparative Genomics

Evolution of RNAi (current model)

1. a. Viruses are bad (so are transposons). b. Many viruses have dsRNA genomes c. euks originally lacked dsRNAs d. invent mechanism to kill dsRNA

2. Evolve mechanism to regulate endogenous genes a. RNA degradation b. inhibit translation c. form heterochromatin

3. Use as experimental technique

Genomics Summary

A. Microarrays: expression profiling and other usesB. Global Gene Knockouts C. Global protein localization in yeast D. Global complex identification in yeast E. Global two-hybrid analysis in yeast and other organismsF. RNAiG. Transgenics, gene “knock-outs” (genetics not genomics)H. Human Genome Project, Next Generation Sequencing,

and Comparative Genomics

Knockout Mouse: The Goal

YFG

Replace the coding region of YFG with a selectable marker gene

Marker Gene

Knockout Mouse

Transfected DNA can integrate at random sites (standard transgenic organism). This is a relatively common event.

Or the Transfected DNA can Replace the Endogenous Copy of the Gene via Homologous Recombination. This is a relatively rare event.

Gene Deletion Deletion by Homologous Recombination

MarkerGene

Knockout Mouse

Knockout Mouse

How to select for the cells in which the Occludin gene is replaced with a mutant allele (a null allele) in the face of the fact that most of the transformed DNA will integrate at random sites?

neor gene makes mammalian cells resistance to the drug G418

tkNSV makes mammalian cells sensitive to the drug ganciclovir

Knockout Mouse

YFG

Red = Mouse DNA including YFG and regions upstream and downstream of YFGBlue = neor gene, Green = tkHSV geneBlack = plasmid DNA (not homologous to any mouse DNA)

neor tkHSV

Knockout Mouse

Lodish 5-40

Knockout Mouse

Different Types of Transgenic Organisms

Genomics Summary

A. Microarrays: expression profiling and other usesB. Global Gene Knockouts C. Global protein localization in yeast D. Global complex identification in yeast E. Global two-hybrid analysis in yeast and other organismsF. RNAiG. Transgenics, gene “knock-outs” (genetics not genomics)H. Human Genome Project, Next Generation Sequencing,

and Comparative Genomics

Goals of Human Genome Project

1. Generate Genetic, Physical and Sequence maps of the human genome

2. Sequence genomes of a variety of model organisms: Comparative Genomics

3. Develop improved technology for mapping and sequencing

4. Develop computational tools for capturing, storing, analyzing, displaying, and distributing map and sequence data

5. Sequence ESTs and cDNAs

6. Consider social, ethical and legal challenges posed by genetic information

Genomicists look at two basic features of genomes: sequence and polymorphism

• Major challenges to determine sequence of each chromosome in genome and identify many polymorphisms– How does one sequence a 500 Mb chromosome 600 bp at a time?– How accurate should a genome sequence be?

• DNA sequencing error rate is about 1 per 600 bp– How does one distinguish sequence errors from polymorphisms?

• Rate of polymorphism in diploid human genome is about 1 in 1000 bp– Repeat sequences may be hard to place– Unclonable DNA cannot be sequenced

• Up to 30% of genome is heterochromatic DNA that can not be cloned

Whole-genome shotgun sequencingPrivate company Celera used to sequence whole human genome

• Whole genome randomly sheared three times– Plasmid library constructed

with ~ 2kb inserts– Plasmid library with ~10 kb

inserts– BAC library with ~ 200 kb

inserts• Computer program assembles

sequences into chromosomes• No physical map construction• Only one BAC library• Overcomes problems of repeat

sequences

Fig. 10.13

Pyrosequencing, pt 1

Rxn2Adenosine phosphosulfate = APS

APS + PPi ATP

Rxn1(DNA)n + dNTP (DNA)n+1 + PPi

ATP sulfurylase

DNAP

Pyrosequencing, pt 2

LuciferinLuciferase

Oxyuciferin + Light

ATP ADP

Apyrase: dNTP -> dNDP + Pi -> dNMP + Pi + Pi

Pyrosequencing, overview

GCTACACTCGATGTGACTGTA

dTTP

PPi

APS

ATPLuciferin

LuciferaseOxyuciferin + Light

Pyrosequencing

Add one nt (A) -> detect light (yes or no)

Apyrase degrades excess nt (A)

Add next nt (C) -> detect light (yes or no)

Apyrase degrades excess nt (C)

Repeat cycle 100’s of times

Pyrosequencing

Pyrosequencing

Emulsion PCR1. Add linkers (primers) to ends of genomic fragments2. Attach frags to 1000’s of beads in a mixture3. Add PCR reagents4. Add oil and make an emulsion so that each bead is in it’s own droplet (it’s own PCR reaction)5. Amplify DNA to make millions of identical copies. Each bead has millions of copies of a single DNA

Pyrosequencing

Pico-titer plate 200,000-400,000 wells per plate1. Add beads to picotiter plate, only one bead fits in each well2. Add a second type of bead, smaller, that holds the DNA bead in the wells and delivers enzymes to the wells3. Flow the nts into the wells one at a time and record the light emitted from each well using a CCD camera

Pyrosequencing

Currently the best machines can sequence 400 - 600 million base pairs in one 10 hour run

Haploid human genome = 3,000,000,000 bp therefore sequence haploid human genome to 1x depth in 6 days with one machine.

The current target goal for sequencing individual human genomes is to get the cost down to $1,000 per genome. At present the cost is around “$5,000-$10,000” per individual (last year)… Illumina claims to have hit the $1,000 cost per genome in January of 2014

Illumina Sequencing Technology

See Movie