Effects of polymerase, template dilution and cycle number on PCR based 16 S rRNA diversity analysis using the deep sequencing method

BMC Microbiol. 2010 Oct 12;10:255. doi: 10.1186/1471-2180-10-255.

Abstract

Background: The primer and amplicon length have been found to affect PCR based estimates of microbial diversity by pyrosequencing, while other PCR conditions have not been addressed using any deep sequencing method. The present study determined the effects of polymerase, template dilution and PCR cycle number using the Solexa platform.

Results: The PfuUltra II Fusion HS DNA Polymerase (Stratagene) with higher fidelity showed lower amount of PCR artifacts and determined lower taxa richness than the Ex Taq (Takara). More importantly, the two polymerases showed different efficiencies for amplifying some of very abundant sequences, and determined significantly different community structures. As expected, the dilution of the DNA template resulted in a reduced estimation of taxa richness, particularly at the 200 fold dilution level, but the community structures were similar for all dilution levels. The 30 cycle group increased the PCR artifacts while comparing to the 25 cycle group, but the determined taxa richness was lower than that of the 25 cycle group. The PCR cycle number did not changed the microbial community structure significantly.

Conclusions: These results highlight the PCR conditions, particularly the polymerase, have significant effect on the analysis of microbial diversity with next generation sequencing methods.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Artifacts
  • Bacteria / classification*
  • Bacteria / genetics
  • Bacteria / isolation & purification*
  • Base Sequence
  • Biodiversity*
  • DNA Primers
  • DNA, Bacterial / genetics
  • DNA-Directed DNA Polymerase
  • Genes, rRNA
  • Geologic Sediments / microbiology*
  • Oligonucleotide Array Sequence Analysis
  • Polymerase Chain Reaction / methods*
  • RNA, Ribosomal, 16S / genetics*
  • Sequence Analysis, DNA / methods*
  • Sequence Homology, Nucleic Acid
  • Soil Microbiology*
  • Species Specificity
  • Taq Polymerase / metabolism
  • Templates, Genetic

Substances

  • DNA Primers
  • DNA, Bacterial
  • RNA, Ribosomal, 16S
  • Taq Polymerase
  • DNA-Directed DNA Polymerase