MiSeq-derived artificial sequences appeared to be of good quality, thus bioinformatics tools failed to remove MiSeq artefacts. Even after removing singleton sequences or operational taxonomic units (OTUs), it is not clear how many sequence artefacts remained. Here, 16S rRNA genes were amplified from soil, human feces, pig feces, and groundwater. These were sequenced with five separate runs of MiSeq. Subsequently, each run of MiSeq was compared through alpha and beta-diversity analyses. We found more than half the OTUs were not in consensus through the multiple MiSeq runs, resulting in varying group-specific biomarker OTUs in each MiSeq run. Thus, differential abundance test should be interpreted with caution, and we suggest that results also should be verified further with other quantification methods such as qPCR.

中文翻译：

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MiSeq可重复性在生物标志物鉴定中的研究

MiSeq衍生的人工序列看起来质量很高，因此生物信息学工具无法消除MiSeq的伪像。即使删除了单例序列或操作分类单位（OTU），也不清楚还剩下多少序列伪像。在这里，从土壤，人类粪便，猪粪和地下水中扩增了16S rRNA基因。用MiSeq的五次独立运行对它们进行测序。随后，通过alpha和beta多样性分析比较了MiSeq的每次运行。我们发现，通过多次MiSeq运行，一半以上的OTU不一致，从而导致每次MiSeq运行中特定于组的生物标志物OTU不同。因此，应谨慎解释差异丰度测试，我们建议还应使用其他定量方法（例如qPCR）进一步验证结果。