[ODP1B] b. Methods in Detection and Identification of Microbes

[ODP-006] Evaluation of quantitative 16S metagenomic analysis using spike-in archaeal genome

○Ayumu Ohno¹, Mano Takahashi¹, Takuya Habara¹, Kirill Kryukov², So Nakagawa¹, Tadashi Imanishi¹ (¹Dept. Molecular Life Science., Sch, Med., Tokai Univ, ²Dept. Genomics and Evolutionary Biology, National Institute of Genetics)

The 16S metagenomic analysis is an effective method to examine bacterial compositions in various samples. However, the number of reads in NGS analyses is not sufficient to make reliable comparisons between different experiments and to judge contaminated bacteria. We need to evaluate the absolute concentrations of bacteria in original samples. To realize this, we can use spike-in genomes of known concentrations as a control. We thus developed a method of quantitative 16S metagenomic analysis by using spike-in of an archaeal genome, which exist only in environments. Here we used an extremely halophilic archaeal species, Haloarcula japonica, that was isolated from a Japanese salten soil. Ten-fold serial dilutions of Haloarcula, genome were prepared and spiked into 1 ng Microbial Community DNA Standard (Zymo Research). Then, the 16S rRNA genes were amplified by PCR and sequenced by the Nanopore DNA sequencer. As a result, it appeared that we could efficiently quantify bacterial genomes when 10³ copies/µl of archaeal genomes were spiked-in. The quantitative 16S metagenomic analysis that we propose here will be useful in various applications.