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“The Genome Project:On Zoos and Curing Cancer”

Richard K. Wilson, Ph.D.Genome Sequencing Center

Washington University Schoolof Medicine

• “Completed” April 2003• ~3 billion base pairs (bp)• 24 chromosomes: 1-22, X, Y• ~25,000 - 30,000 genes• Only 2% of the genome encodes a gene• The sequence represents ~99% of the

genome. Approximately 99% of thesequence is in a highly accurate “finished”state. The other ~1% is somewhat lowerquality but is ordered & oriented.

The Human Genome

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http://genome.ucsc.edu

http://www.ncbi.nlm.nih.gov

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What’s next?

“applied genomics”

more genomes…

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ChordataAmphibians

Vertebrate genomes:Completed or in progress

Fishes

ReptilesBirds

Marsupials

Monotrem

esRodents

Primates

CarnivoresH. sapiens

WU-GSC: work in progress…• Human: chr 2/4: manuscript in preparation…• Mouse: finishing in progress… (~30 Mb/month; target; Apr 2005)• Chimp:

- 4X draft: assembly released, analysis in progress…- Improved genome sequence: additional WGS, BACs in progress…- Finishing: chr 7 & Y (and ENCODE) in progress…

• Chicken:- 6.6X draft: assembly released, analysis in progress…- ENCODE regions in finishing…- Submitted proposal to improve draft…

• S. mediterranea: 6-8X draft: sequencing in progress…• Drosophila:

- D. yakuba: 8X WGS: assembly complete, analysis & pre-finishing in progress…- D. simulans: 8X WGS: sequencing (multiple strains) in progress…

• Macaque: 7-8X WGS: 7.4M reads scheduled…• Caenorhabditis:

- C. remanei: 7X WGS: sequencing complete, assembly in progress…- C. japonica: 7X WGS: heterozygosity analysis in progress…- CB5161: 7X WGS: heterozygosity analysis in progress…

• Others: Platypus & Lamprey BACs: investigatory sequencing in progress…

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ComparativeGenomics

Developmental biologyAgriculture

Why sequence the chicken genome?

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The evolution of birds,flight and feathers

Gallus gallus• Red jungle fowl• Ancestor of moderndomestic breeds

• Genome size = ~1.1 Gb• Autosomes ~38• Chromosome subtypes -macrochromosomes andmicrochromosomes

• Sex chromosomes: ZW(female is heterogamete)

Why sequence the chicken genome?

MammalsMammalsFishFish BirdsBirdsInvertebratesInvertebratesAmphibiansAmphibians

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Fugu

The comparative genomics playing field

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whole genome

BACs

fosmids

G. gallus genome: Strategy…

plasmids

Genome assembly• Ultracontigs: 82• Avg. ucontig length: 11.4 Mb

Physical map• Total clones for FPC: 142,718• Total contigs: 260• Total contigs anchored to

chicken chromosomes: 202

• Avg. scontig length: 32 kb• N50 scontig length: 8.2 Mb

WGS sequence assembly• Genome size: 1.06 Gb• Total Q20 bp: 7.0 Gb• Sequence coverage: 6.6X• Total repeat bp: 7.6 Mb (7%)• Avg. contig length: 10 kb

• Anchored ucontigs: 60• Anchored ucontig length: 0.77 Gb

G. gallus genome: Strategy…

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Minilda output: Martin Kryswinski

Clone set currently exists as 9,270 clones with 77 kb avg. overlap

BAC minimal tiling path

ensembl Chicken Gene Index• Gene predictions generated using

genewise and exonerate based onprotein, cDNA and EST evidence

• 17,784 gene structure predictions(including 75 pseudogenes)

• 28,491 transcripts• 6.5 exons/transcript

• 95% of chicken RefSeq CDS basescovered

• 87% of available chicken cDNAsaligned

• 18,995 EST genes produced from440,815 ESTs

• http://pre.ensembl.org/chicken

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Genome sequence

Questions to Ponder

Why is the chick genome size constrained?How did sex chromosomes evolve?How did microchromosomes evolve?

What genes are lost/retained in bird to mammal lineage?Why are certain repeat classes absent?

Why is segmental duplication 2x that of human?How do we ascertain causative mutations from QTLs?

What do chicken to mammal alignments tell us about evolution?Do non-genic conserved sequences exist in chicken?

Improving the G. gallus genome1. Sequence selected BACs• Use the physical map• Sequence/local assembly of

difficult/repetitive regions• Sequence underrepresented

regions (e.g., Z & W chr)

2. Pre-finishing• Primer-directed reads to close

gaps & improve low qualityregions

3. Finishing• If necessary, depending on

genome-specific standard…

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“applied genomics”

Variation and mutation

GCG AGG GAT AAT TGT …genome 2

…CysGlyAspArgAla

GCA AGC GAT AAT TGT …genome 4

…CysGlyAspSerAlaGCA AAA GAT AAT TGA …genome 5

…STOPGlyAspLysAla

GCA AAA GAT AAT TGT …genome 3

Cys …GlyAspLysAla

GCA AGA GAT AAT TGT …genome 1

Cys …GlyAspArgAla

correlationto disease?

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“Mutational Profiling”

gene of interest

multiple individuals

Mutational Profiling

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PCR amplification

Mutational Profiling

DNA sequencing

High-throughput M.P.

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High-throughput M.P.

High-throughput M.P.

a new pipeline

• High quality data• Perfect sample tracking• Low cost

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Mutational Profiling

• Pulmonary surfactant protein B(SPB) deficiency

• Prostate cancer• Non-small cell lung cancer• Acute myelogenous leukemia

Acute myelogenous leukemia• A group of diseases caused by a variety of inherited

and acquired genetic and epigenetic changes• Most frequently reported form of leukemia among

adults. Approximately 10,000 new cases per year inthe U.S.

• Generic therapeutics exist, but most patients stilldie from this disease.

• Risk stratification:• Age• De novo vs. secondary• Cytogenetic and genetic alterations• Response to initial therapy, relapse

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Acute myelogenous leukemia

A variety of genetic and epigenetic events areprobably responsible for the heterogeneity of AML.

Acute myelogenous leukemia• ~450 target genes• “Discovery Set”:

Matched DNAsamples from 47 AMLpatients.

• “Validation Set”: Anadditional 94matched patientsamples.

• Expression profiling,array CGH, functionalgenomics/mousemodels.

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Acute myelogenous leukemia

Target genesReceptor tyrosine kinasesCytoplasmic tyrosine kinasesHOX genesAbundant ProteasesTranscription factorsTumor suppressorsDNA repairRAS pathway genes

Signaling modifiersPhosphatasesCytokine receptorsCell cycle genesImmune surveillanceApoptosis relatedDrug resistanceMiscellaneous

Acute myelogenous leukemiaGene Mutations

CBF-! R151C

c-KIT M541L

c-MYC N11S Y32H V170I

FLT3 D835Y exon 11 internal tandem duplication (ITD)

NRAS G13R

PML R307C

RAR-" T43I

nonsynonomous

synonomous

20% of AML patients10% of AML patients

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Acute myelogenous leukemia

TM TK TKFLT3

expansion

FLT3 *

20-25%

~10%D835Y

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Non-small cell lung cancer• Lung cancer is the leading cause of cancer deaths in

men and women (US: 164,100 new cases and 156,900deaths in 2000).

• Non-small cell lung cancer is the most common typeof lung cancer. It typically metastasizes more slowlythan small cell lung cancer.

• Cigarette smoking is the most common cause of lungcancer: 87% of all cases are associated with smoking.

• Treatment is typically a combination of surgery,chemotherapy and radiation therapy.

NSC lungcancer

• Mutational profilingis focused on kinasegenes.

• Samples includetissue biopsies frompatients who haveresponded well tothe kinase inhibitorsIressa and Tarceva.

• Just underway…

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Non-small cell lung cancer

05.22.04 EGFR Paraffin Extracted Samples and Germline Controls, tested over exons 18-24

IR18 E19 IR19 IR19 E20 E21 E23

155432

156146

156286

162603

162740

173192

180094

H_AO-0005038 GG ++ AA TT AG TT CT

H_AO-0005346 GG -- AA TT GG TT TT E19 156146del TTAAGAGAAGCAACATCT

H_AO-0005039 AG ++ AA TT AA TT CC

H_AO-0005344 AG ++ AA TT AA GT CC

H_AO-0005040 GG ++ AG CT AG TT CC

H_AO-0005342 GG ++ AA TT AG GT CC

H_AO-cntr101 GG ++ NN CT AG TT CC

H_AO-cntr102 GG ++ NN CT AG TT CC

egfrcon.c1 GG ++ AA TT GG TT TT

Exons examined: 18, 19, 20, 21, 22, 23, 24

Indels

Mutational profiling data for three Iressa responders

Revolutionizing medicine!

• A better understanding of thegenetics and the molecularevents underlying human disease.

• Diagnostic DNA sequencing• “Designer therapeutics”

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Gene-based designer drugs

bcr-abl bcr-ablTargetprotein

Targetprotein

Challenges!

• DNA samples- Collection & records- Sample type & purity- Informed consent/HIPPA

• Technology & throughput improvements• Software tools & data management• Data visualization & statistical analysis• Correlation of complex datasets

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Acknowledgements• WU Genome Sequencing Center

Lucinda Fulton, Bob Fulton, Pat Minx, Tina Graves, TracyMiner, Kim Delahaunty, Bill Nash, Kym Pepin, Ginger Fewell,Jim Eldred, Dave Dooling, Ph.D., Asif Chinwalla, LaDeanaHillier, Doris Kupfer, Ph.D., John Spieth, Ph.D., Wes Warren,Ph.D., Sandy Clifton, Ph.D., Elaine Mardis, Ph.D., many others…

• Acute myelogenous leukemiaTimothy J. Ley, M.D. - WUSM

• Non-small cell lung cancerHarold E. Varmus, M.D. & William Pao, M.D., Ph.D. - MSKCC

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