An optimized flow cytometry sorting-sequencing workflow reduces storage, sorting and extraction bias in sorted microbial communities

Fluorescence-Activated Cell Sorting (FACS) followed by metataxonomics is commonly used to probe the composition of microbial subpopulations with a functionality of interest. Metataxonomic analysis of sorted low microbial biomass samples is prone to contamination and bias introduced during sorting and DNA extraction. To improve metataxonomic accuracy, the DNA yield obtained by the DNeasy® PowerSoil® Pro kit was first maximized. FACS-sequencing using the optimized DNA extraction method distorted the composition of sorted microbiota by introducing sheath fluid contaminants, termed the FACSome. Carefully selected controls allowed for FACSome characterization and subsequent in silico decontamination of PacBio and Illumina metataxonomic sequencing data. The optimized extraction and decontamination were validated in soil, water, feces, saliva, gut reactor and mock microbial communities. Metataxonomic profiles of non-selectively sorted and unsorted samples derived from these ecosystems did not perfectly match due to relic DNA depletion during FACS. A nuclease-based digest enhanced the depletion of spiked and naturally present relic DNA, which can interfere with function-driven FACS. Since FACS depleted relic DNA, the preservation of intact cells prior to sorting is essential for activity– or viability-targeted FACS. Intact cells were best preserved during long-term –80°C storage of fecal samples that were not amended with glycerol-1X Tris-EDTA cryoprotectant.