Tourlousse Dieter M, Yoshiike Satowa, Ohashi Akiko, Matsukura Satoko, Noda Naohiro, Sekiguchi Yuji
Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305-8566, Japan.
Nucleic Acids Res. 2017 Feb 28;45(4):e23. doi: 10.1093/nar/gkw984.
High-throughput sequencing of 16S rRNA gene amplicons (16S-seq) has become a widely deployed method for profiling complex microbial communities but technical pitfalls related to data reliability and quantification remain to be fully addressed. In this work, we have developed and implemented a set of synthetic 16S rRNA genes to serve as universal spike-in standards for 16S-seq experiments. The spike-ins represent full-length 16S rRNA genes containing artificial variable regions with negligible identity to known nucleotide sequences, permitting unambiguous identification of spike-in sequences in 16S-seq read data from any microbiome sample. Using defined mock communities and environmental microbiota, we characterized the performance of the spike-in standards and demonstrated their utility for evaluating data quality on a per-sample basis. Further, we showed that staggered spike-in mixtures added at the point of DNA extraction enable concurrent estimation of absolute microbial abundances suitable for comparative analysis. Results also underscored that template-specific Illumina sequencing artifacts may lead to biases in the perceived abundance of certain taxa. Taken together, the spike-in standards represent a novel bioanalytical tool that can substantially improve 16S-seq-based microbiome studies by enabling comprehensive quality control along with absolute quantification.
16S rRNA基因扩增子的高通量测序（16S测序）已成为一种广泛应用于复杂微生物群落分析的方法，但与数据可靠性和定量相关的技术缺陷仍有待充分解决。在这项工作中，我们开发并应用了一组合成16S rRNA基因作为16S测序实验的通用内参标准。这些内参代表全长16S rRNA基因，包含与已知核苷酸序列相似度可忽略不计的人工可变区，从而能够在来自任何微生物组样本的16S测序读取数据中明确鉴定内参序列。使用定义的模拟群落和环境微生物群，我们表征了内参标准的性能，并证明了它们在逐个样本基础上评估数据质量的效用。此外，我们表明在DNA提取时添加的交错内参混合物能够同时估计适用于比较分析的绝对微生物丰度。结果还强调，特定模板的Illumina测序假象可能导致某些分类群的感知丰度出现偏差。综上所述，这些内参标准代表了一种新型生物分析工具，通过实现全面的质量控制和绝对定量，可大幅改进基于16S测序的微生物组研究。
