We provide a series of tutorials, and analysis walkthroughs to help users get started with eccDNAscope. You can also check out our Reference page which contains a full list of functions available to users.

creat eccDNA object

After the user processes the scATAC data into BAM files using Cell Ranger, eccDNAscope can convert the Cell Ranger results into a eccDNA(Seurat) object and perform dimensionality reduction, clustering, and cell annotation with the Signac R package for subsequent analysis.
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eccDNA feature analysis

eccDNAscope R package provides several powerful functionalities for the analysis of eccDNA features. First, it allows the calculation of the number of fragments with copy number amplification in each cell, and the assignment of CNV_copy information to the eccDNA object. This information can then be visualized to explore the distribution of CNV_copy across all cells and within different cell types.

Next, the package enables the calculation of the number of breakpoints (adapters) of eccDNA in each cell, with the adapter_num information being added to the eccDNA object. The distribution of adapter_num can be visualized both globally and by cell type, providing insights into the structural characteristics of eccDNA.

In addition, eccDNAscope facilitates the calculation of the number of eccDNA genes in each cell. The eccDNA_gene_num information is assigned to the eccDNA object, and its distribution can be visualized across cells and within specific cell types, offering a detailed overview of gene amplification related to eccDNA.

For more in-depth analysis, eccDNAscope allows users to plot the structure of eccDNA. This includes selecting cells from a specific sample, visualizing eccDNA in these cells, and filtering out eccDNA-related genes for analysis. The package also enables the plotting of amplification structures for individual eccDNA-related genes within a specific cell.

The package further supports the visualization of genomic locations of eccDNA, integrating eccDNA-related gene amplifications. Users can explore the relationship between breakpoints and copy numbers of eccDNA-amplified genes across samples. Lastly, it offers the ability to plot the distribution of eccDNA genomic regions and examine the positioning of transcription factor-binding loci relative to the TSS, providing a comprehensive view of the genomic context of eccDNA.
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eccDNA clone analysis

The eccDNAscope R package provides a functionality for eccDNA clone trajectory analysis. First, it merges the copy number amplification information from all samples. Then, it calculates the number of fragments with copy number amplification in each cell and creates a data frame to record the copy number information of each eccDNA-related gene in each cell across all samples.Next, a Seurat object is generated for eccDNA clone analysis. EccDNA clone trajectory analysis is then performed, and all sub-paths from the eccDNA clone trajectory plot are extracted.Finally, a heatmap is plotted to show genes with significant changes along the clone branches.
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shared eccDNA analysis

The eccDNAscope R package also provides a functionality for shared eccDNA analysis, enabling a deeper understanding of how eccDNA-related genes are shared between cells. The analysis begins by clustering cells based on shared specific eccDNA genes, allowing for the identification of groups of cells with similar eccDNA profiles.

Next, hierarchical clustering is performed on cells with amplification of a single eccDNA-related gene, providing insights into the relationships between cells that share amplifications of specific genes. The number of shared eccDNA genes between cells is then calculated, offering a quantitative measure of how eccDNA gene amplification overlaps across different cells.

To validate the similarity between clone clusters, the package assesses chromatin accessibility similarities, ensuring that the clusters are biologically relevant. Finally, the similarity between and within groups of cells is compared based on the transcription start site (TSS) enrichment scores of genes in the single_eccDNA_clone_cluster, further refining the understanding of how gene regulation and eccDNA features are interconnected.
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