Borthong Jednipit, Omori Ryosuke, Sugimoto Chihiro, Suthienkul Orasa, Nakao Ryo, Ito Kimihito
Division of Bioinformatics, Research Center for Zoonosis Control, Hokkaido University, Sapporo, Japan.
Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, Kawaguchi, Japan.
Front Microbiol. 2018 Jun 19;9:1272. doi: 10.3389/fmicb.2018.01272. eCollection 2018.
Metagenomic analysis has become a powerful tool to analyze bacterial communities in environmental samples. However, the detection of a specific bacterial species using metagenomic analysis remains difficult due to false positive detections of sequences shared between different bacterial species. In this study, 16S rRNA amplicon and shotgun metagenomic analyses were conducted on samples collected along a stream and ponds in the campus of Hokkaido University. We compared different database search methods for bacterial detection by focusing on . In this study, we used -specific nested PCR as a gold standard to evaluate the results of the metagenomic analysis. Comparison with the results from -specific nested PCR indicated that a blastn search of shotgun reads against the NCBI-NT database led to false positive results and had problems with specificity. We also found that a blastn search of shotgun reads against a database of the catalase-peroxidase () gene detected with the highest area under the receiver operating characteristic curve among the tested search methods; indicating that a blastn search against the gene database had better diagnostic ability than searches against other databases. Our results suggest that sequence searches targeting long genes specifically associated with the bacterial species of interest is a prerequisite to detecting the bacterial species in environmental samples using metagenomic analyses.
宏基因组分析已成为分析环境样本中细菌群落的有力工具。然而，由于不同细菌物种之间共享序列的假阳性检测，使用宏基因组分析检测特定细菌物种仍然很困难。在本研究中，对北海道大学校园溪流和池塘采集的样本进行了16S rRNA扩增子和鸟枪法宏基因组分析。我们通过关注……比较了不同的数据库搜索方法用于细菌检测。在本研究中，我们使用……特异性巢式PCR作为金标准来评估宏基因组分析的结果。与……特异性巢式PCR结果的比较表明，对NCBI-NT数据库进行鸟枪法读段的blastn搜索会导致假阳性结果，并且存在特异性问题。我们还发现，对过氧化氢酶-过氧化物酶（……）基因数据库进行鸟枪法读段的blastn搜索在测试的搜索方法中，以最高的受试者工作特征曲线下面积检测到……；这表明对……基因数据库进行blastn搜索比针对其他数据库的搜索具有更好的诊断能力。我们的结果表明，针对与感兴趣细菌物种特异性相关的长基因进行序列搜索是使用宏基因组分析检测环境样本中细菌物种的先决条件。
