[ODP1B] b. 微生物の検出・同定の技術

[ODP-006] 古細菌ゲノムのSpike-inを利用した定量的16Sメタゲノム解析法の評価

○大野 歩¹，髙橋 麻乃¹，羽原 拓哉¹，Kirill Kryukov²，中川 草¹，今西 規¹ (¹東海大・医・分子生命科学，²国立遺伝研・ゲノム・進化研究系)

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.