Genome Sequencing Technology and The Cancer Genome Atlas TCGA

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Genome Sequencing Technology and The Cancer
Genome Atlas (TCGA)

• Brad Ozenberger, PhD
• Program Director, The Cancer Genome Atlas
• Program Director, Technology Development
• National Human Genome Research Institute
• National Institutes of Health
• September 29, 2009

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Collins et al., Nature 4/24/03
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Human Genome Project Sequencing Centers
Slide credit Eric Green, NHGRI
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Emulsion Based Clonal Amplification
PCR Reagents
Emulsion Oil
Micro-reactors
Create Water-in-oil emulsion
Mix DNA Library capture beads (limited
dilution)
Prepare adapter-carrying library DNA
Break micro-reactors Isolate DNA containing
beads
Perform emulsion PCR

• Generation of millions of clonally amplified
  sequencing templates on each bead
• No cloning and colony picking

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Sequencing by cyclic synthesis (SBS)
Cycle 1 Add sequencing reagents
First base incorporated
Remove unincorporated bases
Detect signal
Cycle 2-n Add sequencing reagents and repeat
Do this on a microarray with hundreds of
thousands or millions of different molecules on a
surface!
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Applied Biosystems SOLiD 2007
Polonator 2009
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NHGRI Centers - Installed base and experience
3 Large-scale sequencing centers Washington
University The Broad Institute Baylor
College of Medicine
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University of North Carolina Chapel Hill J.
Michael Ramsey
Nanopore Sequencing
For the sensor configuration shown at left, with
electrodes in the walls of a nanopore, modeling
shows that the distributions of current values
for each nucleotide will be sufficiently
different to allow for rapid sequencing.
M Di Ventra 2006 Nano Lett. 6,779-782
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Cancer in the U.S.

• gt10,000,000 in the US have cancer (1 in 30
  people)
• 1,400,000 people were diagnosed in 2008
• 700,000 will die from cancer this year (a death
  every 45 seconds)

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Cancer A Disease of the Genome

• Goals of NHGRI Cancer Genomics Research
• Catalog genomic changes in tumors
• Identify new targets for therapy
• Enable individualized therapy based on the
  genomic signature of a tumor
• Develop technologies to investigate
  heterogeneous and small tumor samples

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NCI-NHGRI Partnership
Cancer Genomics Ultimate Goal Create
comprehensive public catalog of all genomic
alterations present at significant frequency for
all major cancer types National Cancer Advisory
Board Report - Feb, 2005
Integrate
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TCGA Project Pipeline
Supplementary Figure 1
Tissue Sample
Analysis
Pathology QC
Sequencing
Data and Results Storage QC
Integrative Analysis
DNA RNAIsolation, QC
Expression,CNA LOH,Epigenetics
Comprehensive Knowledge of a Cancer
Analysis
Process Data Results
BCR
GSCs
CGCCs
DCC
Collaborators
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TCGA Pilot Components
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TCGA Connecting multiple sources, experiments,
and data types
Three Cancers - TCGA Pilot glioblastoma
multiforme(brain) squamous carcinoma(lung) s
erouscystadenocarcinoma(ovarian)
Biospecimen CoreResource with more than 13
Tissue Source Sites 7 Cancer GenomicCharacterizat
ion Centers 3 GenomeSequencingCenters Data
Coordinating Center
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GBM Findings

• September 2008, TCGA published study of
  glioblastoma (GBM), reported discovery of new
  mutations confirmed many maybes (Nature)
• Data types integrated across labs and across the
  genome, transcriptome, epigenome clinical data
  and outcomes
• Performed in-depth, integrated characterization
  of the tumor genomes of 206 GBM patients
• Identified three genes and three core biological
  pathways commonly altered in GBM tumors
• Discovered possible mechanism by which GBM tumors
  become resistant to TMZ

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TCGA By the numbers

• GBM Project
• 262 cases complete for gene exp., SNP array,
  methylation
• Sequencing 144 cases / 1,300 genes, 56 cases /
  6,000 genes, 12 whole genomes
• Ovarian Project
• 379 cases undergoing comprehensive analysis
• Sequencing 238 cases / 6,000 genes, 12 whole
  genomes
• gt8 Terabases of sequence data generated in 4
  months (equivalent to 1,000 Human Genome Projects)

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OVARIAN
Lost BRCA1 germline indel
NF1-EFCAB5 fusion gene probably
inactivatingvalidated by RNA-seq
Courtesy of Gad Getz Broad Institute
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Status TCGA Pilot Program

• Set up and functionalized all part of TCGA
  network (10 centers, over 150 scientists) and
  developed pipeline from samples to data
  availability
• Built an unprecedented team of scientists,
  oncologists, pathologists, bioethicists,
  technologists and bioinformaticists and a working
  pipeline from sample to data release
• Set a high bar for sample quality and percentage
  of tumor nuclei drove data quality
• Implemented 2nd generation sequencing methods -
  with intensive effort on computational methods
• WE CAN DO IT

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TCGA expansion

• Project will scale production level pipeline
  for 20-25 tumors
• Increased emphasis on an analysis pipeline
• Implementation of 2nd generation genome
  sequencing technologies
• Specific goals
• Standards for biospecimen acquisition - high
  quality of all aspects of samples, clinical
  information and data
• Complete genome characterization each cancer case
• Two levels of data integration and analysis
  advanced approaches and tools for visualization
  and management of data

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Potential Tumors for TCGA
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Game Changing Research

• NIH to accelerate TCGA program with funds
  provided by the American Recovery and
  Reinvestment Act total commitment of 275
  million over next 2 years
• During two years of ARRA funding TCGA will
  complete comprehensive genome characterization of
  10 tumor types and initiate 10 more
• The Cancer Genome Atlas project will forever
  change genomic and cancer research technology
  and analysis, tumor biomarkers, therapeutic
  targets, individualized approaches to therapy,
  and new hope for cancer patients

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Many to acknowledge

• The cancer patients who contribute specimens
• My staff and TCGA teammates at NIH
• The 100 researchers who work daily on The Cancer
  Genome Atlas project
• cancergenome.nih.gov