Assessing soil bacterial community and dynamics by integrated high-throughput absolute abundance quantification

Abstract

Microbial ecological studies have been remarkably promoted by the high-throughput sequencing approach with explosive information of taxonomy and relative abundance. However, relative abundance does not reflect the quantity of the microbial community and the inter-sample differences among taxa. In this study, we refined and applied an integrated high-throughput absolute abundance quantification (iHAAQ) method to better characterize soil quantitative bacterial community through combining the relative abundance (by high-throughput sequencing) and total bacterial quantities (by quantitative PCR). The proposed iHAAQ method was validated by an internal reference strain EDL933 and a laboratory strain WG5. Application of the iHAAQ method to a soil phenanthrene biodegradation study showed that for some bacterial taxa, the changes of relative and absolute abundances were coincident, while for others the changes were opposite. With the addition of a microbial activity inhibitor (NaN3), the absolute abundances of soil bacterial taxa, including several dominant genera of Bacillus, Flavobacterium, and Paenibacillus, decreased significantly, but their relative abundances increased after 28 days of incubation. We conclude that the iHAAQ method can offer more comprehensive information to reflect the dynamics of soil bacterial community with both relative and absolute abundances than the relative abundance from high-throughput sequencing alone.

A quantitative sequencing framework for absolute abundance measurements of mucosal and lumenal microbial communities

“…This method will be useful in other areas that benefit from quantitative analysis, such as monitoring microbial communities during manufacturing of complex probiotic mixtures 56 and monitoring changes of host-associated microbial communities over time (e.g., in health, aging and development, disease progression, and during probiotic or other treatments). Applying absolute quantification [19][20][21][23][24][25][26][27][28]32,33 of microbial taxa to biogeographically relevant GI locations will provide researchers with new insights in how microbial communities affect host phenotypes.…”

“…These methods require a purified DNA sequence of known concentration from an organism not present in the sample and an estimate of the initial sample concentration to determine the amount of exogenous DNA to spike-in. Another group of anchoring methods, such as those that use flow cytometry 24 , total DNA 25 , or quantitative PCR (qPCR) [26][27][28] , measure the total concentration of cells, DNA, or amplicons to transform the relative abundances to absolute numbers. These methods have already demonstrated the value of quantitative microbiome analysis, yet microbiome researchers have not yet uniformly adopted these methods.…”

Changes in soil microbial community structure during long‐term secondary succession

“…All gene copy numbers were calculated from the standard curves of 16S rRNA and the ITS genes by using the ΔCt (cycle threshold) method. All qPCRs were conducted in triplicate (Fierer et al, ; Guinane et al, ; Lou, Li, Wang, Wu, & Xu, ; Reddy et al, ). Amplicons were extracted from 2% agarose gels and purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA) according to the manufacturer's instructions and quantified with QuantiFluor‐ST (Promega).…”

Rhizosphere bacterial and fungal communities during the growth of Angelica sinensis seedlings cultivated in an Alpine uncultivated meadow soil

“…Finally, all of results in our study are based on the taxonomic relative abundance, but the reported studies have demonstrated that the observed differences with relative abundances can cover those with the actual taxonomic abundances Tkacz, Hortala & Poole, 2018). The method of combining the quantification and relative abundance of the microbial communities should be used in the further studies (Lou et al, 2018;Guo et al, 2019).…”