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

Fluorescence-Activated Cell Sorting (FACS) followed by 16S rRNA gene profiling is commonly used to probe the composition of targeted microbial cell subpopulations. 16S rRNA gene profiling of samples with low microbial biomass after sorting is, however, prone to contamination and systematic biases introduced during sorting and DNA extraction. To improve the 16S rRNA gene profiling accuracy and precision, we first maximized the DNA yield obtained by the DNeasy® PowerSoil® Pro DNA extraction kit. Using this optimized DNA extraction method, we found that FACS distorted the composition of the sorted microbiota by introducing sheath fluid contaminants that we termed the FACSome. Proper controls allowed for characterization of the FACSome and subsequent in silico decontamination of PacBio and Illumina 16S rRNA gene sequencing data. The optimized extraction and decontamination were validated in soil, water, feces, saliva, gut reactor and mock communities. The 16S rRNA gene profiles of non-selectively sorted and unsorted samples derived from these ecosystems accurately and precisely matched, except for the mock community. A follow-up relic DNA spike-in showed that enzyme- and magnetic bead-based purification of sorted samples shifted the sorted microbiota and proved unnecessary to remove relic DNA. Since sorting depletes 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. Addition of this cryoprotectants induced larger compositional shifts in the viable sorted microbiota compared to fresh samples.