Absolute bacterial abundance
Sequencing solutions provide information on the relative abundance of microorganisms in a sample, but sometimes insight into the total abundance of organisms in a sample is necessary to obtain answers to the presented questions. Such a situation may arise if a treatment does not change the relative abundance of the organisms, but changes the total bacterial load.
Similarly, if the abundance of one bacterium increases it will decrease the relative abundance of other bacteria and based on relative abundance data it is difficult to detect which bacteria actually changed its abundance. We simple see that their relative abundance changed. Statistical solutions have been developed to facilitate the identification of such changes from relative abundance data, however these methods have limitations such as small sample sizes.
Spike-in of bacteria allows absolute quantification
A leading solution to address the problem of relative abundance is to add a spike-in of bacteria of known concentration to the sample. When the bacteria are rare and not found in the environment being investigated, this allow for statistical re-calculation of the data to obtain both relative and absolute abundance tables. The method is still at an early stage and has its limitations. It is for one only available for bacterial profiling, as the spike-in organisms are bacteria. However, we have found that including this step can provide valuable information, especially if a shift in absolute abundance is expected e.g. in evacuations of antibiotic treatment effects.
While “reference frames”-based statistical solutions can compensate for potential shortcommings of relative abduance data for some questions, in some studies, in others such as studies with small samples sizes or in projects profilin less diverse microbiome communities; spike-in solutions are necessary.