High-throughput sequencing (HTS) of 16S rRNA gene amplicons provides compo sitional information regarding the microbial community, but not the absolute abun dance of the bacteria. We aimed to develop a standardized method for quantifying
the absolute abundance of bacteria in microbiome studies. To demonstrate the utility
of our approach, we quantified the number of bacteria from the compositional data of
the fecal and cecal microbiomes. The 16S rRNA gene of a hyperthermophile, Thermus
aquaticus, was cloned into Pichia pastoris (yeast) genome, and an equivalent amount of
the yeast was added to the stool and cecal samples of mice before DNA extraction.
16S rRNA gene library construction and HTS were performed after DNA extraction.
The absolute abundances of bacteria were calculated using T. aquaticus reads. The
average relative abundances of T. aquaticus in the five stool and five cecal samples
were 0.95% and 0.33%, respectively, indicating that the number of bacteria in a cecum
sample is 2.9 times higher than that in a stool sample. The method proposed for quan tifying the absolute abundance of the bacterial population in this study is expected
to overcome the limitation of showing only compositional data in most microbiome
studies.