Abstract: A method that harnesses the cell membrane as a natural reaction chamber for DNA amplification, enabling optional downstream sequencing and optional downstream cell sorting is described herein. Unlike traditional single-cell techniques, methods herein utilize a strand-displacing, isothermal polymerase to amplify DNA within fixed, permeabilized cells while maintaining the cell's structural integrity. This approach overcomes challenges associated with both typical single-cell DNA sequencing and hindrances encountered when previously attempting to sequence genetic material from fixed microbial cells, allowing amplification of genomic regions up to 100 kb and optional downstream sequencing of whole genomes from diverse cell types, including S. cerevisiae, B. subtilis, and E. coli. Additionally, methods herein (e.g., FISA) can optionally be adapted for targeted DNA enrichment using biotinylated primers and for fluorescence-based cell sorting.

Abstract: Rare genotypes in bacterial or eukaryotic populations are essential for understanding adaptation, ecology, and disease, but assembling the full genome of rare cell types remains challenging. Bulk sequencing of mixed populations obscures rare genotypes and haplotype information, while random single-cell isolation is labor-intensive, particularly for rare cell types. Described here is a novel method that enriches cells with a known genetic marker, enabling efficient sequencing of their partial or full genome. This approach integrates intracellular genomic DNA amplification, Fluorescent In Situ Amplification (FISA), Fluorescence-Activated Cell Sorting (FACS), and whole-genome sequencing. It allows robust amplification and sequencing of single genes and entire genomes from formaldehyde-fixed cells, preserving cellular integrity and minimizing false positives.

Abstract: Disclosed herein is an in situ, high throughput, single-cell region(s) of interest (ROI) or whole-genome sequencing technology developed for sequencing gene(s) or genomes in large heterogeneous cell populations combined with single-cell RNA sequencing. More specifically, the technology disclosed herein does not require cell sorting or isolation because it uses the cell membrane to separate each genome whereupon single genomes and transcriptomes are concurrently sequenced. While other in situ single-cell sequencing technologies are only able to work with RNA, and must first convert DNA to RNA, the method of this disclosure operates directly on DNA as well as on RNA. Thus, it simultaneously sequences each single cell's genome and transcriptome.

Abstract: Methods and kits for uniquely labeling nucleic acid molecules within a plurality of microbial cells are described. In an embodiment, the method comprises fixing and permeabilizing the plurality of microbial cells; dissociating microbial cell aggregates within a suspension comprising the plurality of microbial cells; reverse transcribing mRNA within the plurality of microbial cells to provide cDNA; and combinatorially labelling the cDNA to provide labelled cDNA.

Abstract: Disclosed herein is an in situ, high throughput, single-cell whole-genome sequencing technology developed for sequencing genomes in large heterogeneous cell populations. More specifically, the invention disclosed herein does not require cell sorting or isolation because it uses the cell membrane to separate each genome.

Abstract: Methods and kits for uniquely labeling nucleic acid molecules within a plurality of microbial cells are described. In an embodiment, the method comprises fixing and permeabilizing the plurality of microbial cells; dissociating microbial cell aggregates within a suspension comprising the plurality of microbial cells; reverse transcribing mRNA within the plurality of microbial cells to provide cDNA; and combinatorially labelling the cDNA to provide labelled cDNA.

Abstract: Methods and kits for uniquely labeling nucleic acid molecules within a plurality of microbial cells are described. In an embodiment, the method comprises fixing and permeabilizing the plurality of microbial cells; dissociating microbial cell aggregates within a suspension comprising the plurality of microbial cells; reverse transcribing mRNA within the plurality of microbial cells to provide cDNA; and combinatorially labelling the cDNA to provide labelled cDNA.

Abstract: Methods and kits for uniquely labeling nucleic acid molecules within a plurality of microbial cells are described. In an embodiment, the method comprises fixing and permeabilizing the plurality of microbial cells; dissociating microbial cell aggregates within a suspension comprising the plurality of microbial cells; reverse transcribing mRNA within the plurality of microbial cells to provide cDNA; and combinatorially labelling the cDNA to provide labelled cDNA.