I am working on the effect of pH on functional genes in the nitrogen cycle (amoA, nosZ..). Some of the papers i have read present results based on the ratio between functional gene (like nosZ) and 16S. Why do they compare the ratio between functional genes and 16S genes? Any assistance will be highly appreciated.
In general, the gene number copies are expressed in relation to the suspension volume or sample mass (i.e. number of 16S gene copies per mL or number of 16S gene copies per gram of soil). The number of gene copies can also be expressed in Log10. While functional genes are expressed in relation to the total abundance of the bacterial community (16S rRNA). For example, a number of nifH gene copies/number of 16S rRNA gene copies.
Nitrous oxide reductase (nosZ) gene is largely unique to denitrifying bacteria found in environment, soil and marine samples (David and Lee, 1998). The amoA gene encoding AMO subunit A is functional and phylogenetic marker gene for bacterial and archaeal ammonia oxidizers (Rotthauwe et al., 1997; Purkhold et al., 2000; Junier et al., 2008; Pester et al., 2012). The 16S gene is also the phylogenetic marker for bacteria. That’s why researchers search and/or compare these two genes against 16 S gene using public database, mining for sequences related to the unique nosZ and amoA putatively present in various environments including soils (paddy rice soils, other agricultural soils, forest soils, grassland soils), freshwater habitats (wetlands, rivers, lakes, groundwater basins), groundwater wells (GGWs), full-scale wastewater treatment plants (WWTPs), and drinking water treatment plants (DWTPs) (Daims et al., 2015; van Kessel et al., 2015).
In general, the gene number copies are expressed in relation to the suspension volume or sample mass (i.e. number of 16S gene copies per mL or number of 16S gene copies per gram of soil). The number of gene copies can also be expressed in Log10. While functional genes are expressed in relation to the total abundance of the bacterial community (16S rRNA). For example, a number of nifH gene copies/number of 16S rRNA gene copies.
Functional genes give us an idea of what is happening, 16S genes an idea of the bacteria in a sample. Comparing them allows us to infer something about who is doing what, assuming we ignore all of the vagaries of PCR.
Thank you very much Andrew Macrae , Adijailton Jose de Souza and Md. Nazmul Hoque for your detailed and insightful answers. I have a lot more clarify in the matter now.
To complete the previous answers, when you run a qPCR you add genomic DNA which contain not only bacterial DNA. Expressing the functional genes, such as the ones involved in the N cycles (nosZ, amoA, nifH, nirS/K) as a proportion of the total bacterial community (assessed using the 16S rDNA gene) can help to take into account the variation of the bacterial DNA. This can be important if you are not adding a fix amount of DNA for each sample.
This is not the only way to express you gene copies number for functional genes. I copy below an extract from a book chapter I wrote about qPCR for N functional genes in soil:
The gene copy number can either be expressed as g-1 of dry soil, ng-1 DNA, or as a proportion of the bacterial 16S rRNA gene in the case of a functional gene such as bacterial amoA gene. The gene copy number expressed as the total amount of
DNA g-1 of dry soil will give the total number of genes in the soil. The gene copy number expressed ng-1 DNA can be chosen to reduce the potential bias due to DNA extraction efficiency between samples [7]. However, similar samples (e.g.,
soil with similar texture or organic matter content) may not be affected significantly by DNA extraction efficiency, or the effect of extraction efficiency may not be higher than biological variation. Furthermore, DNA samples also vary significantly in their amount (e.g., arable vs. grassland soil) and composition
(i.e., plant, bacteria, fungi etc.). Thus, the gene copy number can be expressed as a proportion of the bacterial 16S rRNA gene to estimate the relative abundance of a functional gene. Overall, these three ways to calculate gene copy number give
different information and one is not better than the other but their use depends on the research question and the experiment design, and can all be used in the same study.
You can find the reference of the chapter here.
Chapter Quantification of Ammonia Oxidizing Bacterial Abundances in ...
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