The researchers have developed artificial nucleic acid reference materials as internal standards for accuracy control of analysis of microbiomes (mixed microbial flora), which consists of various microbial species, by next-generation sequencing.

Developed artificial nucleic acid reference materials (artificial 16S rRNA genes) and a mixture of the reference materials to be used as an internal standard

The 16S rRNA gene is widely used for the phylogenetic classification of microorganisms such as bacteria. The researchers designed artificial nucleic acid reference materials to be amplified by PCR and to allow the amplified base sequences to be differentiated from natural genes during data analysis. They produced twelve types of such artificial nucleic acid reference materials. When a mixture of the reference materials is used as an internal standard and microbiome analysis is performed, the accuracy of the obtained base sequences can be evaluated and the performance of quantitative analysis over a wide range of concentration can be controlled.

Microbiomes are drawing attention as diagnostic markers and drug discovery targets. Determining the type and quantity of microorganisms by next-generation sequencing is the starting point of the microbiome analysis. However, sample pretreatment and other processes have a significant effect on the measurement, which may result in a lack of accuracy in the obtained base sequences and poor reliability and comparability in the determined quantities. Consequently, standardization for accuracy control is being promoted in many different countries. Human factors significantly affect the measurement and accuracy control is thus required for each analysis; however, no effective accuracy control technique had been developed.

To establish the reliability of the accuracy control technique based on the developed reference materials, the researchers will apply the technique widely to microbiome analyses in areas such as medicine, food, and the environment.