The Influenza Genome Sequencing Project

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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)

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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
• Todays 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)

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1918 Spanish flu

• Deaths were reported to occur within 24 hours
• 28 of U.S. population infected
• Mortality rate estimated at 2.5 (versus lt0.1
  for most flu strains)
• Killed 30-40 million worldwide in 4 years

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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

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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?

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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)
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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 (gt 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 dont know whats out there

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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 RD (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 gt3000 samples/year
• PHASE III RD for avian flu sequencing (Aug
  2005-Mar 2006)

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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
lt 500 per genome
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Influenza Project progress
Jan 4 2006 681 genomes
http//www.tigr.org/flu
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http//www.ncbi.nlm.nih.gov/genomes/FLU/FLU.html
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Phylogenetic analysis of the first 156 genomes
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197 neuraminidatse (NA) segments from NYS and
other sites worldwide, 1999-2005
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Re-assortment events inhemagglutinin (HA)
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Antigenic shift event, 2002-2003
H3N2 Clade B (1999-)
H3N2 Clade A(2001-3)
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)
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Genome-wide mutational analysis across five
seasons
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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