Toyomane Kochi, Yokota Ryo, Watanabe Ken, Akutsu Tomoko, Asahi Ai, Kubota Satoshi
National Research Institute of Police Science, Kashiwa, Chiba, Japan
National Research Institute of Police Science, Kashiwa, Chiba, Japan.
mSystems. 2021 Feb 2;6(1):e01255-20. doi: 10.1128/mSystems.01255-20.
The highly personalized human skin microbiome may serve as a viable marker in personal identification. Amplicon sequencing resolution using 16S rRNA cannot identify bacterial communities sufficiently to discriminate between individuals. Thus, novel higher-resolution genetic markers are required for forensic purposes. The clustered regularly interspaced short palindromic repeats (CRISPRs) are prokaryotic genetic elements that can provide a history of infections encountered by the bacteria. The sequencing of CRISPR spacers may provide phylogenetic information with higher resolution than other markers. However, using spacer sequencing for discrimination of personal skin microbiome is difficult due to limited information on CRISPRs in human skin microbiomes. It remains unclear whether personal microbiome discrimination can be achieved using spacer diversity or which CRISPRs will be forensically relevant. We identified common CRISPRs in the human skin microbiome via metagenomic reconstruction and used amplicon sequencing for deep sequencing of spacers. We successfully reconstructed 24 putative CRISPR arrays using metagenomic data sets. A total of 1,223,462 reads from three CRISPR arrays revealed that spacers in the skin microbiome were highly personalized, and conserved repeats were commonly shared between individuals. These individual specificities observed using CRISPR typing were confirmed by comparing the CRISPR diversity to microbiome diversity assessed using 16S rRNA amplicon sequencing. CRISPR typing achieved 95.2% accuracy in personal classification, whereas 16S rRNA sequencing only achieved 52.6%. These results suggest that sequencing CRISPRs in the skin microbiome may be a more powerful approach for personal identification and ecological studies compared to conventional 16S rRNA sequencing. Microbial community diversity analysis can be utilized to characterize the personal microbiome that varies between individuals. CRISPR sequences, which reflect virome structure, in the human skin environment may be highly personalized similar to the structures of individual viromes. In this study, we identified 24 putative CRISPR arrays using a shotgun metagenome data set of the human skin microbiome. The findings of this study expand our understanding of the nature of CRISPRs by identifying novel CRISPR candidates. We developed a method to efficiently determine the diversity of three CRISPR arrays. Our analysis revealed that the CRISPR spacer diversity in the human skin microbiome is highly personalized compared with the microbiome diversity assessed by 16S rRNA sequencing, providing a new perspective on the study of the skin microbiome.
高度个性化的人类皮肤微生物群可能成为个人识别的可行标记。使用16S rRNA的扩增子测序分辨率不足以充分识别细菌群落以区分个体。因此，法医用途需要新的高分辨率遗传标记。成簇规律间隔短回文重复序列（CRISPRs）是原核生物遗传元件，可提供细菌遇到的感染历史。CRISPR间隔序列的测序可能提供比其他标记更高分辨率的系统发育信息。然而，由于人类皮肤微生物群中关于CRISPRs的信息有限，使用间隔序列测序来区分个人皮肤微生物群很困难。目前尚不清楚是否可以通过间隔序列多样性实现个人微生物群的区分，或者哪些CRISPRs在法医上具有相关性。我们通过宏基因组重建在人类皮肤微生物群中鉴定了常见的CRISPRs，并使用扩增子测序对间隔序列进行深度测序。我们使用宏基因组数据集成功重建了24个假定的CRISPR阵列。来自三个CRISPR阵列的总共1,223,462条读数显示，皮肤微生物群中的间隔序列高度个性化，保守重复序列在个体之间普遍共享。通过将CRISPR多样性与使用16S rRNA扩增子测序评估的微生物群多样性进行比较，证实了使用CRISPR分型观察到的这些个体特异性。CRISPR分型在个人分类中达到了95.2%的准确率，而16S rRNA测序仅达到了52.6%。这些结果表明，与传统的16S rRNA测序相比，对皮肤微生物群中的CRISPRs进行测序可能是一种更强大个人识别和生态研究方法。微生物群落多样性分析可用于表征个体间不同的个人微生物群。人类皮肤环境中反映病毒组结构的CRISPR序列可能与个体病毒组结构一样高度个性化。在本研究中，我们使用人类皮肤微生物群的鸟枪法宏基因组数据集鉴定了24个假定的CRISPR阵列。本研究的结果通过鉴定新的CRISPR候选物扩展了我们对CRISPRs性质的理解。我们开发了一种有效确定三个CRISPR阵列多样性的方法。我们的分析表明，与通过16S rRNA测序评估的微生物群多样性相比，人类皮肤微生物群中的CRISPR间隔序列多样性高度个性化,为皮肤微生物群的研究提供了新的视角。
