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The Influenza Genome Sequencing Project Steven Salzberg Center for Bioinformatics and Computational Biology Institute for Advanced Computer Studies University of Maryland and The Institute for Genomic Research (TIGR) Dominant influenza strains

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the influenza genome sequencing project

The Influenza Genome Sequencing Project

Steven Salzberg

Center for Bioinformatics and Computational Biology

Institute for Advanced Computer Studies

University of Maryland

and

The Institute for Genomic Research (TIGR)

dominant influenza strains
Dominant influenza strains
  • Current human influenza A: strains H3N2 and H1N1
    • 30,000 people die from flu each year in the U.S.
  • Avian influenza A: strain H5N1 (and others)
  • 1918 “Spanish flu”: H1N1
    • Today’s H1N1 is a descendant of this strain
    • Originally an avian flu
  • 1889 and 1957 pandemics: H2N2
  • 1968 “Hong Kong” pandemic: H3N2
  • 1977 “Russian” outbreak: H1N1 (no pandemic)
1918 spanish flu
1918 Spanish flu
  • Deaths were reported to occur within 24 hours
  • 28% of U.S. population infected
  • Mortality rate estimated at 2.5% (versus <0.1% for most flu strains)
  • Killed 30-40 million worldwide in 4 years
why do a flu genome project
Why do a flu genome project?
  • Predict the dominant strain for vaccine formulation
    • it takes 6 months to ramp up vaccine production
  • Use the flu sequence to get a clear picture of positive selection in each gene
  • Track the temporal and geographic distribution of strains
research questions
Research questions
  • Study genotypic correlates of virulence
  • Study frequency of genetic re-assortment
    • good background data non-existent
  • Estimate evolutionary rate in different hosts
    • Where do pandemic strains adapt to human host?
  • Avian flu genome comparisons
    • How does genetic material move around among wild and domestic birds?
    • What changes permit the flu to infect humans?
flu genome
Flu genome
  • negative strand RNA virus
  • Viral particles contain 8 “segments”
  • Genome length: 13,627 nucleotides

Photo credits: G.R. Whitaker (above), L. Stannards (left)

paucity of public sequence data
Paucity of Public Sequence Data
  • Until 2005, only 7 complete H3N2 genomes
  • As of 2004, 50 complete HA segments (H3N2)
    • For HA1 in 2003, 19 H3N2 (> half from S. Africa)
    • 2002, 11 H3N2, 12 H1N2, 37 H1N1 (mostly from Japan & Korea)
    • For NA in 2003, none!
    • Other segments have only a handful/year
  • GenBank had 54 avian flu has genomes but 41/54 from Hong Kong markets
  • Basically, we don’t know what’s out there
influenza genome sequencing project
Influenza genome sequencing project
  • White paper proposal (Dec 2003)
    • submitted by David Lipman, Steven Salzberg, et al
    • approved by NIAID January 2004
  • Preliminary Testing (Mar – Aug 2004)
    • Protocol testing (micro-libraries, random priming, directed walking)
    • Laboratory set-up
  • PHASE I: R&D (Sept 2004 – Aug 2005)
    • Optimize methodologies
    • Develop high-throughput pipeline
    • Process first 500 Wadsworth Center (NY) samples
  • PHASE II: High-throughput(Mar 2005 – Feb 2008)
    • Build capacity to 400 samples/month
    • Process >3000 samples/year
  • PHASE III: R&D for avian flu sequencing (Aug 2005-Mar 2006)
slide9

TIGR High-throughput pipeline

Seg1 (PB2)

Seg2 (PB1)

Seg3 (PA)

Seg4 (HA)

Seg5 (NP)

Seg6 (NA)

Seg7 (M)

Seg8 (NS)

circularize

Sample

(All in one tube)

RNA

ligation

pp1

  • RT/PCR
  • Clean-up

Sequencing reactions

5’ 3’

degenerate Primers

with M13 tags

Rearray into

384-well plates

pp2

pp4

1 sample/plate

pp3

Sequencing

< $500 per genome

slide10

Influenza Project progress

Jan 4 2006:

681 genomes

http://www.tigr.org/flu

antigenic shift event 2002 2003
Antigenic shift event, 2002-2003

H3N2 Clade A(2001-3)

H3N2 Clade B (1999-)

Seg1 (PB2)

Seg2 (PB1)

Seg3 (PA)

Seg4 (HA)

Seg5 (NP)

Seg6 (NA)

Seg7 (M)

Seg8 (NS)

Seg1 (PB2)

Seg2 (PB1)

Seg3 (PA)

Seg4 (HA)

Seg5 (NP)

Seg6 (NA)

Seg7 (M)

Seg8 (NS)

H3N2 dominant strain (2003-)

Seg1 (PB2)

Seg2 (PB1)

Seg3 (PA)

Seg4 (HA)

Seg5 (NP)

Seg6 (NA)

Seg7 (M)

Seg8 (NS)

project team and collaborators
Project team and collaborators
  • TIGR/UMD project mgmt: Elodie Ghedin, Steven Salzberg, David Spiro
  • TIGR sequencing mgmt: Tamara Feldblyum, Claire Fraser
  • TIGR bioinformatics mgmt: Martin Shumway
  • TIGR lab and bioinformatics: Naomi Sengamalay, Jennie Zaborsky, Vik Subbu, Jeff Sitz, ...
  • NCBI/NIH – David Lipman, Yiming Bao, ...
  • Wadsworth Center – Jill Taylor, Kirsten St. George
  • Armed Forces Inst. of Pathology – Jeff Taubenberger
  • Mt. Sinai School of Medicine – Peter Palese
  • NIAID/NIH – Maria Giovanni, Linda Lambert, Karen Lacourciere, Lone Simonsen, Valentina Di Francesco

http://www.tigr.org/flu