Using PEBBLE for the Evolutionary Analysis of Serially Sampled Molecular Sequences

Matthew Goode1, Allen G. Rodrigo1

1 Bioinformatics Institute and The Allan Wilson Centre for Molecular Ecology and Evolution, University of Auckland, Auckland
Publication Name:  Current Protocols in Bioinformatics
Unit Number:  Unit 6.8
DOI:  10.1002/0471250953.bi0608s05
Online Posting Date:  May, 2004
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The PEBBLE (Phylogenetics, Evolutionary Biology, and Bioinformatics in a moduLar Environment) application is a relative newcomer to the field of phylogenetic applications. Although designed as a customizable generalist application, PEBBLE was initially developed to implement procedures for the analysis of sequences associated with different sampling times, e.g., rapidly evolving viral genes sampled over the course of infection, or ancient DNA sequences. The basic protocol describes the use of PEBBLE to infer a phylogenetic tree using the sUPGMA algorithm, and the inference of substitution rate parameters using maximum likelihood. The alternate and support protocols describe the simulation capabilities of PEBBLE, and general use of the PEBBLE application, respectively.

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Table of Contents

  • Basic Protocol 1: Using PEBBLE for Analysis
  • Support Protocol 1: General Use of PEBBLE
  • Alternate Protocol 1: Using PEBBLE for Simulation
  • Guidelines for Understanding Results
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Using PEBBLE for Analysis

  Necessary Resources
  • Hardware
  • Any computer capable of running Java 1.1. This includes Apple Macintosh computers (running MacOS 8 or higher) and personal computers running Microsoft Windows or Linux.
  • Software
  • PEBBLE main application, and the Java Runtime Environment (JRE) version 1.1 or higher. The PEBBLE application can be downloaded from (by following the Software link). Notification of updates and milestone releases of the PEBBLE application can be obtained via E‐mail by sending an E‐mail to . Bug reports regarding the PEBBLE application may be sent to .
  • Files
  • PEBBLE accepts sequence alignment files in Nexus (unit 6.4), PHYLIP (unit 6.3), Clustal (unit 2.3), or FASTA ( appendix 1B) formats. PEBBLE accepts tree files in Nexus (unit 6.4) and Newick (unit 6.2) formats.
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Literature Cited

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   Drummond, A. and Rodrigo, A.G. 2000. Reconstructing genealogies of serial samples under the assumption of a molecular clock using serial‐sample UPGMA (sUPGMA). Mol. Biol. Evol. 17:1807‐1815.
   Drummond, A. and Strimmer, K. 2001. PAL: An object‐oriented programming library for molecular evolution and phylogenetics. Bioinformatics 17:662‐663.
   Drummond, A., Forsberg, R., and Rodrigo, A.G. 2001. Estimating stepwise changes in substitution rates using serial samples. Mol. Biol. Evol. 18:1365‐1371.
   Drummond, A., Nicholls, G.K., Rodrigo, A.G., and Solomon, W. 2002. Estimating mutation parameters, population history and genealogy simultaneously from temporally spaced sequence data. Genetics 161:1307‐1320.
   Drummond, A.J., Pybus, O.G., Rambaut, A., Forsberg, R., and Rodrigo, A.G. 2003. Measurably evolving populations. Trends Ecol. Evol. 18:481‐488.
   Felsenstein, J. 1981. Evolutionary trees from DNA sequences: A maximum likelihood approach. J. Mol. Evol. 17:368‐376.
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   Fu, Y.X. 2001. Estimating mutation rate and generation time from longitudinal samples of DNA sequences. Mol. Biol. Evol. 18:620‐626.
   Goldman, N. 1990. Maximum likelihood inferences of phylogenetic trees, with special reference to the Poisson process model of DNA substitutions and to parsimony analysis. Syst. Zool. 39:345‐361.
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   Lambert, D.M., Ritchie, P.A., Millar, C.D., Holland, B., Drummond, A.J., and Baroni, C. 2002. Rates of evolution in ancient DNA from Adelie penguins. Science 295:2270‐2273.
   Leitner, T. and Albert, J. 1999. The molecular clock of HIV‐1 unveiled through analysis of a known transmission history. Proc. Natl. Acad. Sci. U.S.A. 96:10752‐10757.
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   Rodrigo, A.G. and Felsenstein, J. 1999. Coalescent approaches to HIV population genetics. In The Evolution of HIV (K.A. Crandall, ed.) pp. 223‐271. Johns Hopkins University Press, Baltimore.
   Rodrigo, A.G., Shpaer, E.G., Delwart, E.L., Iverson, A.K.N., Gallo, M.V., Brojatsch, J., Hirsch, M.S., Walker, B.D., and Mullins, J.I. 1999. Coalescent estimates of HIV‐1 generation time in vivo. Proc. Natl. Acad. Sci. U.S.A. 96:2187‐2191.
   Rodriguez, F., Oliver, J.F., Marin, A., and Medina, J.R. 1990. The general stochastic model of nucleotide substitution. J. Theor. Biol. 142:485‐501.
   Shankarappa, R., Margolick, J.B., Gange, S.J., Rodrigo, A.G., Upchurch, D., Farzadegan, H., Gupta, P., Rinaldo, C.R., Learn, G.H., He, X., Huang, X.L., and Mullins, J.I. 1999. Consistent viral evolutionary dynamics associated with the progression of HIV‐1 infection. J. Virol. 73:10489‐10502.
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Internet Resources
  The PEBBLE application can be downloaded from the above URL by following the Software link. Notification of updates and milestone releases of the PEBBLE application can be obtained via E‐mail by sending an E‐mail to Bug reports regarding the PEBBLE application may be sent to‐project
  The home page of the PAL project can be found at the above URL.
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