The University of Arizona Cancer Center, University of Arizona, Tucson, AZ, USA.
Department of Pharmacology, University of Arizona, Tucson, AZ, USA.
J Appl Microbiol. 2017 Dec;123(6):1584-1596. doi: 10.1111/jam.13590. Epub 2017 Nov 7.
Test the choice of 16S rRNA gene amplicon and data analysis method on the accuracy of identification of clinically important bacteria utilizing a benchtop sequencer.
Nine 16S rRNA amplicons were tested on an Ion Torrent PGM to identify 41 strains of clinical importance. The V1-V2 region identified 40 of 41 isolates to the species level. Three data analysis methods were tested, finding that the Ribosomal Database Project's SequenceMatch outperformed BLAST and the Ion Reporter Metagenomics analysis pipeline. Lastly, 16S rRNA gene sequencing mixtures of four species through a six log range of dilution showed species were identifiable even when present as 0·1% of the mixture.
Sequencing the V1-V2 16S rRNA gene region, made possible by the increased read length Ion Torrent PGM sequencer's 400 base pair chemistry, may be a better choice over other commonly used regions for identifying clinically important bacteria. In addition, the SequenceMatch algorithm, freely available from the Ribosomal Database Project, is a good choice for matching filtered reads to organisms. Lastly, 16S rRNA gene sequencing's sensitivity to the presence of a bacterial species at 0·1% of a mixture suggests it has sufficient sensitivity for samples in which important bacteria may be rare.
We have validated 16S rRNA gene sequencing on a benchtop sequencer including simple mixtures of organisms; however, our results highlight deficits for clinical application in place of current identification methods.
利用台式测序仪测试 16S rRNA 基因扩增子和数据分析方法对临床重要细菌鉴定准确性的选择。
在 Ion Torrent PGM 上测试了九个 16S rRNA 扩增子，以鉴定 41 株临床重要菌株。V1-V2 区将 41 株分离株中的 40 株鉴定到种水平。测试了三种数据分析方法，发现核糖体数据库项目的 SequenceMatch 优于 BLAST 和 Ion Reporter Metagenomics 分析管道。最后，通过六个对数稀释范围的四个物种的 16S rRNA 基因测序混合物表明，即使以混合物的 0.1%存在，也可以鉴定出物种。
通过 Ion Torrent PGM 测序仪的 400 碱基化学物质增加的读长，可以对 V1-V2 16S rRNA 基因区域进行测序，这可能是比其他常用区域更好的选择，用于鉴定临床重要细菌。此外，Ribosomal Database Project 免费提供的 SequenceMatch 算法是将过滤后的读数与生物体匹配的不错选择。最后，16S rRNA 基因测序对混合物中 0.1%细菌物种存在的敏感性表明，它对重要细菌可能很少的样本具有足够的敏感性。
我们已经在台式测序仪上验证了 16S rRNA 基因测序，包括简单的生物体混合物；然而，我们的结果突出了临床应用的缺陷，而不是当前的鉴定方法。
