Analysis of the 25 ITS sequences affiliated with PnecC obtained from the established clone library for potential PCR artifactsa

Analyzed ITS sequences (source)Affiliation of sequencesNo. of sequencesNo. of analyzed sequence positionsPotential PCR artifacts (no. of sequence positions)No. of potential artifacts in positions:
Conserved in PnecC and PnecDConserved in PnecCVariable within PnecC and PnecD
PnecC reference sequencesbPnecC3417,511No variationdNo variationeVariablef
PnecD reference sequencescPnecD189,279No variationdNo variationeVariablef
Clones (this study)PnecC2512,879131111
  • a Sequence positions, which differed in the cloned sequences from sequences of isolates belonging to the same ITS genotype, were considered potential PCR artifacts. In total 13 positions, i.e., 0.1% of the total number of cloned ITS sequence positions with potential PCR artifacts, were identified. These positions were compared with the homologous positions in a set of 52 reference sequences affiliated with subclusters C and D of the Polynucleobacter cluster. All reference sequences were obtained by direct sequencing. Only one potential PCR artifact was found to be located in a variable sequence position. The other sequence differences were located in sequence positions absolutely invariable in both subclusters or at least within PnecC.

  • b Sequences from directly sequenced isolates and endosymbionts belonging to Polynucleobacter subcluster C (PnecC), including isolates obtained from the dystrophic pond.

  • c Sequences from directtly sequenced isolates belonging to Polynucleobacter subcluster D (PnecD).

  • d No variation within PnecC and PnecD, and identical in both subclusters.

  • e No variation within PnecC and PnecD, but not identical in both subclusters.

  • f Variable within PnecC and PnecD.