Gradient Internal Standard Method for Absolute Quantification of Microbial Amplicon Sequencing Data

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 (R2average = 0.998; P < 0.001) and accurate (Pinternals 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.

Quantitative 16S rRNA Gene Amplicon Sequencing for Comprehensive Pathogenic Bacterial Tracking in a Municipal Wastewater Treatment Plant

“…Simultaneous quantification and composition analysis of environmental microbes has been studied by incorporating different internal standards (bacterial species/strains and chimeric DNA). , The use of synthetic spike-in internal standards (harboring conserved identical regions) into the 16S rRNA gene for quantitative sequencing (qSeq) has proven for analysis of absolute abundance across the samples. − Moreover, quantification accuracy in qSeq has tested with qPCR and microscopic methods. The quantitative data derived by each method had no significant difference with qSeq quantification. , This ensures qSeq as a powerful approach for quantitative studies. But qSeq has not yet been used to quantify bacterial pathogens in environmental samples.…”

“…The quantitative data derived by each method had no significant difference with qSeq quantification. 26,27 This ensures qSeq as a powerful approach for quantitative studies. But qSeq has not yet been used to quantify bacterial pathogens in environmental samples.…”

Absolute quantification of the microbiota spatial distribution in the murine large intestine

“…16 Real-time polymerase chain reaction (RT-PCR), a classic method for absolute quantification, has been used to count absolute microbial numbers. 17,18 Recently, a new internal standard method has been proposed to determine the absolute number of microorganisms in food and environment using the sequencing. 19,20 Since the efficiency of bacterial DNA extraction is difficult to achieve 100%, heterogeneous among taxa, and the extraction may cause damage to some DNA, using DNA from previous studies as an internal standard may have resulted in some inaccuracies.…”

A quantitative sequencing method using synthetic internal standards including functional and phylogenetic marker genes

“…In recent years, quantitative sequencing methods have been developed to spike microbial DNA (Lin et al, 2018; Smets et al, 2016) or viable bacteria absent in the sample (Stämmler et al, 2016), or to add synthetic 16S rRNA (Tourlousse et al, 2017) or 16S‐18S‐internal transcribed spacer regions (Tkacz et al, 2018; Wang et al, 2021) as standards for high‐throughput quantitative microbial community analysis. These quantitative sequencing methods can be divided into two quantitative patterns: (i) the total number of a target gene in a sample is calculated from the internal standard gene (ISG) read ratio and the total number of ISG molecules added to the sample (Galagoda et al, 2023; Lin et al, 2018; Smets et al, 2016; Stämmler et al, 2016; Tkacz et al, 2018) or (ii) the target gene read numbers are converted to copy numbers using a dose–response curve as an internal standard curve, which is estimated based on the ISG read numbers with known gene copy numbers (Tourlousse et al, 2017; Wang et al, 2021). However, neither method is widespread enough to replace qPCR.…”