E P I D E M I C A L E R T A N D R E S P O N S E Laboratory Training for Field Epidemiologists

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May 2007

Sequencing and Phylogeny

E P I D E M I C A L E R T A N D R E S P O N S E Laboratory Training for Field Epidemiologists

Learning objectivesLearning objectives

At the end of the presentation, participants should:

• Understand sequencing

• Interpret phylogenetic trees

E P I D E M I C A L E R T A N D R E S P O N S E Laboratory Training for Field Epidemiologists

SequencingSequencing

Process to determine the exact order of nucleotides in DNA

Parts of the genome are compared with other sequenced samples

• From a databank

• By direct comparison

E P I D E M I C A L E R T A N D R E S P O N S E Laboratory Training for Field Epidemiologists

Review of molecular biologyReview of molecular biology

…AGCUAAAGCUA… Nucleotides code for amino acids (codons)

Genes – DNA and RNA are made of nucleotides

…ala-lys-ala… Amino acids make proteins

Proteins 'R' us; enzymes, cell membranes, etc.

Genetic variation (mutation) breeds trial and error – evolution

RNA based organisms mutate (evolve) much faster than DNA based ones e.g. influenza, HIV

E P I D E M I C A L E R T A N D R E S P O N S E Laboratory Training for Field Epidemiologists

Evaluating organism similarity Evaluating organism similarity

…AGCUAAAGCUA… Gene sequence #1

…AACUAAACCUG… Gene sequence #2

…AACUCCACCUA… Gene sequence #3

…GGUUGCACCGG… Gene sequence #4

E P I D E M I C A L E R T A N D R E S P O N S E Laboratory Training for Field Epidemiologists

Algorithms & programs to compare genesAlgorithms & programs to compare genes

Several available: http://research.amnh.org/~siddall/methods/

Different approaches and assumptions but all startcwith computeralignment e.g. Phylip, PAUP

Parsimony is "a principle that states that the simplest explanation thatexplains the greatest number of observations is preferred to morecomplex explanations".

#1…AGCUAAAGCUA…#2…AACUAAACCUG…#2…AACUAAACCUG…

#1…AGCUAAAGCUA…#2…AACUAAACCUG…

#1…AGCUAAAGCUA…#1…AGCUAAAGCUA…#2…AACUAAACCUG…

#1…AGCUAAAGCUA…#2…AACUAAACCUG…

73% identity

E P I D E M I C A L E R T A N D R E S P O N S E Laboratory Training for Field Epidemiologists

Comparing genes to evaluate Comparing genes to evaluate similarity among organismssimilarity among organisms

…AGCUAAAGCUA… Gene sequence #1

…AACUAAACCUG… Gene sequence #2

…AACUCCACCUA… Gene sequence #3

…GGUUGCACCGG… Gene sequence #4

Trees can be built with nucleic acid or protein sequences

Sequence #1

Sequence #2

Sequence #3

Sequence #4

Unrooted phylogenetic tree by unweighted pair group method

Sequence #4

Sequence #3

Sequence #2Sequence #1

Distance in characters (nt/aa)

E P I D E M I C A L E R T A N D R E S P O N S E Laboratory Training for Field Epidemiologists

Comparing genes to evaluate Comparing genes to evaluate similarity among organismssimilarity among organisms

…AGCUAAAGCUA… Gene sequence #1

…AACUAAACCUG… Gene sequence #2

…AACUCCACCUA… Gene sequence #3

…GGUUGCACCGG… Gene sequence #4

GGLysineGlutamate

Molecular Phylogeny of H5 Avian Influenza viruses (HA and M genes)

Source: Suarez et. al. 2000: J. Virology 74: 6592-6599

E P I D E M I C A L E R T A N D R E S P O N S E Laboratory Training for Field Epidemiologists

Evolution of the 'Z genotype'

Letters to NatureNature 430, 209-213 (8 July 2004)

Genesis of a highly pathogenic and potentially pandemic H5N1 influenza virus in eastern Asia

K. S. Li1, Y. Guan1,2, J. Wang1,2, G. J. D. Smith1,2, K. M. Xu1,2, L. Duan1,2, A. P. Rahardjo3, P. Puthavathana4, C. Buranathai5, T. D. Nguyen6, A. T. S. Estoepangestie3, A. Chaisingh5, P. Auewarakul4, H. T. Long7, N. T. H. Hanh7, R. J. Webby8, L. L. M. Poon2, H. Chen1,2, K. F. Shortridge1,2, K. Y. Yuen2, R. G. Webster2,8 and J. S. M. Peiris1,2

E P I D E M I C A L E R T A N D R E S P O N S E Laboratory Training for Field Epidemiologists

ConclusionsConclusions

A phylogenetic tree is a tool to be used under defined circumstances – it is not absolute

Phylogenetic relationships offer useful information

Phylogenetic relationships often match antigenic relationships, particularly with influenza viruses

Phylogenetic relationships can not predict evolutionary pathways , specific mutations or likelihood of animal viruses becoming human viruses

E P I D E M I C A L E R T A N D R E S P O N S E Laboratory Training for Field Epidemiologists

Developed by the Department of Epidemic and Pandemic Alert and Response of the World Health Organization with assistance from:

European Program for Intervention Epidemiology Training

Canadian Field Epidemiology Program

Thailand Ministry of Health

Institut Pasteur

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