| Abstract [eng] |
Single-cell sequencing is a powerful tool in microbiological research, as it enables the exploration of cellular heterogeneity, the identification of rare populations, and the analysis of gene expression at the individual cell level. The technology of semi-permeable capsules allows for high-throughput, multi-step reactions necessary for single-cell nucleic acid analysis. Ideally, information about biomolecular composition and distribution would be obtained from live-cell measurements in real time; however, few methods currently exist to achieve this. Although considerable scientific effort has been devoted to developing fixation protocols, data on the effects of fixatives and storage conditions on microbial communities remain limited. The accessibility of nucleic acids is a key criterion for obtaining high-quality sequencing data. For this reason, various cell lysis conditions were tested in this study. Fixation with paraformaldehyde leads to the formation of cross-links between proteins and other biomolecules, resulting in a stiffer and less permeable cell envelope that hampers lysis reagent efficiency. Following optimization of the lysis conditions for fixed bacteria using potassium hydroxide, two sequencing approaches were pursued: whole-genome sequencing and targeted sequencing of AmpR and 16S rRNA genes. Analysis of the sequencing data revealed that the quality of genetic information retrieved from fixed samples at the single-cell level was reduced. Significant differences in the number of reads between fixed and unfixed bacterial samples were observed, and reference genome coverage differed by orders of magnitude. To enable sensitive analysis of fixed single cells in future applications, further optimization of lysis protocols, DNA repair strategies, and cross-link reversal techniques is required. |
