Abstract
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 (R 2 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.IMPORTANCE To solve the problem of amplicon sequencing cannot discern the microbiota absolute abundance, we proposed a gradient internal standard absolute quantification method. We used Chinese liquor fermentation as a model system to demonstrate the reliability and accuracy of the method. By comparing the relative and absolute abundances of microbiota in various temporal dimensions, we found dynamic changes in the absolute abundance of communities under various temporal dimensions from the relative abundance. Based on its design principle, this method can be widely applied to different ecosystems. Therefore, we believe that the GIS-AQ method can play an immeasurably useful role in microbiological research.
Highlights
High-throughput amplicon sequencing is a critical tool for studying microbiota; it results only in relative abundance data
Each internal standard contained a specific pair of primers and recognition sequences (Table 1) that were not present in the microbial genomes based on a search of the National Center for Biotechnology Information (NCBI) database and were flanked by the universal primers of the bacterial 16S rRNA V3-V4 and fungal ITS2 regions (Fig. 1A)
To resolve the shortcomings of traditional amplicon sequencing, various methods have been proposed to assess the absolute abundance of microbiota, such as flow cytometry [11, 16, 17], quantitative real-time PCR (qPCR) [17], fluorescence in situ hybridization coupled with catalyzed reporter deposition (CARD-FISH) [23], and spike-in [12, 14, 15, 22, 24,25,26]
Summary
High-throughput amplicon sequencing is a critical tool for studying microbiota; it results only in relative abundance data. 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. IMPORTANCE To solve the problem of amplicon sequencing cannot discern the microbiota absolute abundance, we proposed a gradient internal standard absolute quantification method. Comparing microbial abundances between different samples in various temporal dimensions is important for expanding the breadth and depth of the research. To compare samples across temporal and spatial dimensions, we must use microbiota absolute quantitative results [13]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
