The effects of PCR cycle number and DNA polymerase type on 16S rRNA gene pyrosequencing analysis were investigated using an artificially prepared bacterial community (mock community). The bacterial richness was overestimated at increased PCR cycle number mostly due to the occurence of chimeric sequences, and this was more serious with a DNA polymerase having proofreading activity than with Taq DNA polymerase. These results suggest that PCR cycle number must be kept as low as possible for accurate estimation of bacterial richness and that particular care must be taken when a DNA polymerase having proofreading activity is used.