High-throughput amplicon sequencing is a critical tool for studying microbiota; however, it results only in relative abundance data. Thus, changes in absolute abundance of microbiota cannot be determined, which hinders further microbiology research. We have therefore established a gradient internal standard absolute quantification (GIS-AQ) method to overcome this issue, which can simultaneously obtain the absolute abundances of bacteria and fungi. Deviations from the quantitative equations of microbes and internal standards were eliminated through calibration. Compared with traditional quantitative real-time PCR and microscopy quantifications, this method is reliable (R²_average = 0.998; P < 0.001) and accurate (P_{internals versus microscopy} > 0.05). The GIS-AQ method can be adapted to any amplicon primer choice (e.g., 336F/806R and ITS3/ITS4), rendering it applicable to ecosystem studies including food, soil, and water samples. Crucially, when using solid-state fermentation samples from various temporal dimensions, the results obtained from the relative and absolute abundance are different. The absolute abundance can be used to study the difference in communities between different samples, and the GIS-AQ method allows this to be done rapidly. Therefore, combining the absolute abundance with relative abundance can accurately reflect the microbiota composition.

中文翻译：

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微生物扩增测序数据绝对定量的梯度内标法

高通量扩增子测序是研究微生物群的关键工具。但是，它仅导致相对丰度数据。因此，无法确定微生物群落绝对丰度的变化，这阻碍了进一步的微生物学研究。因此，我们建立了一种梯度内标绝对定量法（GIS-AQ）来解决此问题，该方法可以同时获取细菌和真菌的绝对丰度。通过校准消除了与微生物和内标物定量方程的偏差。与传统的定量实时PCR和显微镜定量相比，该方法可靠（R ²_均值= 0.998；P < 0.001）且准确（P_{内镜与显微镜}> 0.05）。GIS-AQ方法可适应任何扩增子引物选择（例如336F / 806R和ITS3 / ITS4），使其适用于包括食物，土壤和水样在内的生态系统研究。至关重要的是，当使用不同时间维度的固态发酵样品时，从相对和绝对丰度获得的结果是不同的。绝对丰度可用于研究不同样本之间的群落差异，而GIS-AQ方法可快速完成这项工作。因此，将绝对丰度与相对丰度相结合可以准确反映微生物群组成。