Although methods are used to normalize amplicon data after sequencing,
the composititonal nature of the data (i.e. relative abundance) does limit the ability to perform comparative analyses of the quantity of individual taxa or groups of interest.
Thus, researchers have developed experimental approaches that allow amplicon sequencing data to become more quantitative. Due to their nature, amplicon profiling data cannot inform about absolute abundances of the target taxa, unless internal DNA standards are co-amplified during amplicon generation (Jones and Kustka 2017; Piwosz et al. 2018). Efforts have previously been invested into absolute quantification of the target taxa, through combining the NGS analyses with microbial quantification by phospholipid fatty acid (PLFA) profiling, direct cell counts or qPCR (Barlow et al. 2020; Galazzo et al. 2020; Jian et al. 2020; Nkongolo and Narendrula-Kotha 2020).
The use of PLFA profiling provides a broad-scale, yet quantitative approach to assessing microbial community diversity. It can be used to determine ratios of fungi to bacteria, as well as gram-positive to gram-negative bacteria based on specific phospholipid fatty acid signatures in a given sample. Recently, studies have compared the insights gained through the use of PLFAs and those from amplicon-based sequencing studies. These analyses suggest that PLFAs are as sensitive or even more sensitive than metabarcoding approaches in detecting changes in microbial community structure \cite{Orwin_2018,Ramsey_2006}. When taken together, the use of PLFAs and amplicon sequencing improve resolution for detecting quantitative differences in community composition over sequence-based approaches alone. However, differences in the fatty acid signature produced by a microbial guild as a result of their biomass must also be considered (Duncan et al. 2016; Galazzo et al. 2020)
Another, more time intensive approach toward absolute abundance data from soil communities is the use of cell counts. Direct cell counts can be achieved by staining cells with nucleic acid stains (e.g. DAPI, SYBR Green) and counting using fluorescence microscopy (REFs). However, this approach is relatively lower throughput and less accurate than flow cytometric enumeration (45). In this approach, cells are separated from large soil particles and incubated with nucleic acid stain prior to flow cytometric enumeration. Counting of cells using flow cytometry may circumvent overestimation of microbial diversity related to extracellular DNA by counting only intact cells (48, 49).
An additional approach to improve the quantitative nature of amplicon sequencing is Catalyzed Reporter Deposition coupled to Fluorescence in situ hybridization (CARD-FISH). Recently, Piwosz and colleagues combined CARD-FISH with sequencing (50). Although the use of CARD-FISH is more labor-intensive than amplicon sequencing alone, it allows sequencing to become more quantitative through direct cell counts and phylogenetic staining of microorganisms of interest. This approach is restricted to the analysis of bacteria and archaea, as high-throughput sequencing data and CARD-FISH analysis of eukaryotic organisms correlate poorly with one another (50). The correlation between datasets was higher for bacteria and taxa that were highly underrepresented in the sequencing data could be captured using CARD-FISH.
One
relatively accessible experimental approach is the use of qPCR to
improve the quantitative nature of sequencing data. Numerous studies
have applied qPCR in soil and other sample types in order to reduce the
effects of compositional sequencing data on interpretation of microbial
community studies (e.g.
(41, 43–46), and
others). Using this highly sensitive and reproducible approach,
amplicons are copied using polymerase chain reactions, however at each
cycle the fluorescence of amplified material is measured
(47). In this way,
qPCR in combination with sequencing can be used for detection,
quantification and identification of bacteria, archaea and eukaryotes in
complex communities. Zhang and colleagues applied 16S amplicon
sequencing, as well as qPCR and other cell-counting approaches, and
found that qPCR as a cell enumeration method correlates with other forms
of cell quantification
(45). This and
other studies support the use of multiple approaches to improve the
inferences that can be made from microbial community studies,
particularly when these studies are conducted in heterogeneous
environments or compare across different sampling sites or time periods. A limitation of this approach is the need for standards...ddPCR as an alternative/next step
Some of these approaches remain limited at present due to the
sheer biological diversity of soil ecosystems. However, with an
expanding view of the diversity of microorganisms and growing number of
published reference genomes, more accurate quantitative approaches are
within reach. In combining sequencing with quantitative measurements,
one can obtain absolute abundances of organisms in a given sample,
making investigations of complex microbial communities more robust.