Phase determination from direct sequencing of length-variable DNA regions

Authors

  • JEAN-FRANÇOIS FLOT,

    1. UMR UPMC-CNRS-MNHN-IRD 7138, Département Systématique et Évolution, Muséum National d’Histoire Naturelle, Case Postale 26, 57 rue Cuvier, 75231 Paris Cedex 05, France,
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  • ANNIE TILLIER,

    1. Service de Systématique Moléculaire, Département Systématique et Évolution, Muséum National d’Histoire Naturelle, Case Postale 26, 57 rue Cuvier, 75231 Paris Cedex 05, France
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  • SARAH SAMADI,

    1. UMR UPMC-CNRS-MNHN-IRD 7138, Département Systématique et Évolution, Muséum National d’Histoire Naturelle, Case Postale 26, 57 rue Cuvier, 75231 Paris Cedex 05, France,
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  • SIMON TILLIER

    1. UMR UPMC-CNRS-MNHN-IRD 7138, Département Systématique et Évolution, Muséum National d’Histoire Naturelle, Case Postale 26, 57 rue Cuvier, 75231 Paris Cedex 05, France,
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Jean-François Flot, Fax: +33 1 40 79 38 44; E-mail: jfflot@mnhn.fr

Abstract

For diploid organisms, haplotype determination usually requires sequencing cloned polymerase chain reaction (PCR) products or comparing the genotypes of several individuals. We found out that phase could be reconstructed from direct sequencing of mixed PCR products by combining for each individual the complementary information contained in its forward and reverse chromatograms, provided these products had different lengths. When applied to the internal transcribed spacer 2 (ITS2) from corals of the genus Pocillopora, this new method allowed us to identify two dominant sequence types in some specimens; however, sequencing cloned PCR products from the same specimens yielded more variants, including possible PCR-generated recombination artifacts.

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