Measuring the absolute abundance of the microbiome by adding yeast containing 16S rRNA gene from a hyperthermophile

We aimed to develop a standardized method for quantifying the absolute abundance of bacteria in microbiome studies. The 16S rRNA gene of a hyperthermophile,Thermus aquaticus, was cloned intoPichia pastoris (yeast) genome, and an equivalent amount of the yeast was added to the stool and cecal samples of mice before high-throughput sequencing and 16S rRNA gene amplicon analysis. The absolute abundances of bacteria were calculated usingT.  aquaticus reads. AbstractHigh-throughput sequencing (HTS) of 16S rRNA gene amplicons provides compositional information regarding the microbial community, but not the absolute abundance 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 intoPichia 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 usingT.  aquaticus reads. The average relative abundances ofT.  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 s...
Source: MicrobiologyOpen - Category: Microbiology Authors: Tags: ORIGINAL ARTICLE Source Type: research