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The Flu Genome Sequencing Venture

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  1. 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)

  2. 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)

  3. 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

  4. 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

  5. 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?

  6. 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)

  7. 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

  8. 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)

  9. 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

  10. Influenza Project progress Jan 4 2006: 681 genomes


  12. Phylogenetic analysis of the first 156 genomes

  13. 197 neuraminidatse (NA) segments from NYS and other sites worldwide, 1999-2005

  14. Re-assortment events inhemagglutinin (HA)

  15. 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)

  16. Genome-wide mutational analysis across five seasons

  17. 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