Next, chromosomes are traced in each cell and the image data is passed through various image analysis pipelines that provide measurements and statistics on structural genome features at the single-cell level as well as for sub- and whole cell populations.
WORKFLOW OVERVIEW
jebFISH Workflow Steps
jebFISH involves genome painting by hybridizing a cocktail of special LociPaint oligonucleotide probes to simultaneously paint multiple genomic regions—either along one chromosome, across multiple chromosomes, or genome-wide across all chromosomes.
Once the LociPaint probes are hybridized to the genomic regions of interest, a set of fluorescently labeled secondary readout probes (called jebReader probes) are then hybridized to the LociPaints.
Each LociPaint oligo is thus “lit up”, imaged, and identified using multiplex fluorescence imaging. The innovative jebSmart™ multi-color barcoding scheme ensures high detection efficiency and accurate readout. During the imaging phase, fluorescent signals from the jebReaders appear as puncta that are then located in 3D space and identified by decoding the associated barcodes.
The concomitant image of genomic loci targets in each nucleus is pulled through various imaging data analysis pipelines to calculate measurements such as distances between the targeted genomic regions and to generate chromosome traces.
The chromosome traces are passed through further data analyses revealing insights such as copy number variation, ecDNA formation, translocations, chromatin compaction, and multi-loci proximities. Importantly, data is calculated not only for each single cell but also pooled to cell populations and sub-populations to identify biological trends among them.
