Automatic, fast, hierarchical, and non-overlapping gating of flow cytometric data with flowEMMi v2.

Flow cytometry has become a powerful technology for studying microbial community dynamics and ecology. These dynamics are tracked over long periods of time based on two-parameter community fingerprints consisting of subsets of cell distributions with similar cell properties. These subsets are highlighted by cytometric gates which are assembled into a gate template. Gate templates then are used to compare samples over time or between sites. The template is usually created manually by the operator which is time consuming, prone to human error and dependent on human expertise. Manual gating thus lacks reproducibility, which in turn might impact ecological downstream analyses such as various diversity parameters, turnover and nestedness or stability measures. We present a new version of our flowEMMi algorithm - originally designed for an automated construction of a gate template, which now (i) generates non-overlapping elliptical gates within a few minutes. Gate templates (ii) can be created for both single measurements and time-series measurements, allowing immediate downstream data analyses and on-line evaluation. Furthermore, it is possible to (iii) adjust gate sizes to Gaussian distribution confidence levels. This automatic approach (iv) makes the gate template creation objective and reproducible. Moreover, it can (v) generate hierarchies of gates. flowEMMi v2 is essential not only for exploratory studies, but also for routine monitoring and control of biotechnological processes. Therefore, flowEMMi v2 bridges a crucial bottleneck between automated cell sample collection and processing, and automated flow cytometric measurement on the one hand as well as automated downstream statistical analysis on the other hand.