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Quantitatively evaluating mistaken clone assignments by RFLP analysis of 16S rRNA genes: a case study

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Abstract:

We quantitatively evaluated the errors of clone assignment based on the restriction fragment length polymorphism (RFLP) pattern of 16S rRNA genes. Eighty clones were randomly selected from a 16S rRNA gene library and were categorized into 35 operational taxonomic units (OTU) based on their indistinguishable enzyme restriction patterns of 3 tetrameric restriction enzymes RsaI, BsuRI, and HinfI. All of these clones were then sequenced and were reassigned into 36-53 OTUs using the DOTUR program when sequence similarities of 95%-100% were used. The number of the identically assigned clones ranged from 53 to 61 and the percentage varied from 66.3% to 76.3%. The Shannon-Weaver index for the bacterial community observed by RFLP analysis was 2.75, equal to that estimated by DOTUR at a 97% sequence similarity. Compared with clones assigned with the DOTUR program at a 97% sequence similarity, only 61 clones (76.3%) were correctly assigned by RFLP analysis. Six clones (7.5%) were assigned mistakenly at the phylum level, and the positions of 13 clones (16.2%) were phylogenetically different at a lower taxonomic rank.

Nous avons évalué quantitativement les erreurs d’identification de clones basée sur le polymorphisme de longueur des fragments de restriction (RFLP) du gène de l’ARNr 16S. Quatre-vingt clones ont été choisis au hasard à partir d’une banque de gènes d’ARNr 16S, et ont été catégorisés en 35 unités taxonomiques opérationnelles (UTO) à partir de leurs patrons de restriction enzymatique jugés identiques, obtenus avec les 3 enzymes de restriction tétramères RsaI, BsuRI et HinfI. Tous ces clones ont alors été séquencés et ont été réassignés en 36 à 53 UTO par le programme DOTUR, lorsqu’un seuil de 95 % à 100 % de similarité de séquence était utilisé. Le nombre de clones assignés de façon identique allait de 53 à 61 et le pourcentage variait de 66,3 % à 76,3 %. L’index de Shannon-Weaver de la communauté bactérienne observé par l’analyse en RFLP était de 2,75, égal à celui estimé par DOTUR à un seuil de 97 % de similarité de séquence. En comparant l’affectation des clones par DOTUR à un seuil de 97 % de similarité de séquence, 61 clones seulement (76,3 %) étaient correctement assignés par l’analyse en RFLP. Six clones (7,5 %) ont été assignés de façon erronée à l’échelle du phylum et les positions de 13 clones (16,2 %) étaient différentes d’un point de vue phylogénique à des niveaux taxonomiques inférieurs.

Document Type: Research Article

Publication date: June 1, 2008

More about this publication?
  • Published since 1954, this monthly journal contains new research in the field of microbiology including applied microbiology and biotechnology; microbial structure and function; fungi and other eucaryotic protists; infection and immunity; microbial ecology; physiology, metabolism and enzymology; and virology, genetics, and molecular biology. It also publishes review articles and notes on an occasional basis, contributed by recognized scientists worldwide.
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