create ArchRProject

create ArchRProject is a latch.bio workflow for generating R objects and data for downstream analysis of epigenomic DBiT-seq experiments. Provided fragments from a single-cell ATAC-seq preprocessing and alignment workflow and spatial information, create ArchRProjectr performs the heavy computational steps of ArchR and Seurat and returns files that can be easily input into custom scripts for more neuanced analysis without the need to perform intensive computation.

The workflow utilizes ArchR to perform epigenomic single-cell analysis and Seurat to spatially align the data. The workflow can take data from either a single tissue-sample analyzed via DBiT-seq or multiple tissue-samples; in ATX parlance, tissue-samples analyzed via DBIT-seq are termed 'Runs'. All Runs input to create ArchRProject are merged into a single ArchRProject for analysis.

Inputs

All input files for create ArchRProject must be on the latch.bio file system. Each run in the workflow takes the following parameters,

• fragments.tsv.gz file: A BED-like, tab-delimited file in which each row contains an ATAC-seq fragment

• tissue_positions_list.csv: A comma-separated file in which each row contains a unique barcode, an indicator for whether the tixel is 'on-tissue' (1, 0), and a row/column index

• Spatial folder: A directory containing tissue images and experiment metadata

• Run ID: An identifier for the run

• Condition (optional): An experimental Condition descriptor (ie. 'control', 'diseased')

Individual runs are batched in a Project with the following global parameters,

• Project Name: A name for the output folder

• Genome: A reference genome to be used for alignment

• Upload to SLIMS (ATX-internal only): A T/F toggle for whether to push QC results to LIMS

• LSI resolution: A decimal value used as input to the clusterParams parameter of the addIterativeLSI function in ArchR;

• LSI varFeatures: An integer used as input to the varFeatures parameter of the addIterativeLSI function in ArchR;

• clustering resolution: A decimal value used as input to the resolution parameter of the addClusters function in ArchR.

The Project also takes a series of single-value parameters that can be found under the 'Hidden Parameters' dropdown; these parameters are less commonly varied inputs to ArchR functions.

Running the workflow (in progress)

The create ArchRProject workflow can be found in the Workflows module in your latch.bio workspace. For access to an ATX-collaborator workspace, please contact your AtlasXomics Support Scientist or email [email protected]. See here for general instructions for running workflows in latch.bio.

1. Navigate to the optimize archr workflow in the Workflows module in your latch.bio workspace. Ensure you are on the 'Parameters' tab of the workflow.

2. To add Runs to the Project, select the '+ runs' icon. Add values for the Run parameters described above; repeat for each Run in the Project.

3. Scroll to the bottom of the page and input values for global project parameters.

4. Click the 'Hidden Parameters' button and change the global parameters as needed.

5. Click the 'Launch Workflow' button on the bottom-right of the parameters page. This will automatically navigate you to the Executions tab of the workflow.

6. From the Executions tab, you can view the status of the launched workflow. Once the workflow has completed running, the status will change to 'Succeeded'; if the workflow has the status 'Failed', please contact an AtlasXomics Support Scientist. You can click on the workflow execution to view a more granular workflow status and see output logs.

7. Workflow outputs are loaded into the latch.bio Data module in the ArchRProjects directory.

Outputs

Outputs from create ArchRProject are loaded into latch.bio Data module in the ArchRProjects directory.

• ArchRProject/
  • This folder contains all of arrow/peak files and the ArchR object to load an pre-computed ArchRProject.
• _SeuratObj.rds
  • A Seurat object for each of the samples in ArchRProject with all metadata. The count matrix is the gene accessibility matrix that is stored in the Arrow files as GeneScoreMatrix.
• _SeuratObjMotif.rds.
  • Seurat object for each of the samples in ArchRProject with all metadata. The count matrix is the motif deviation score matrix calculated by getDeviation ArchR script.
• UMAPHarmony.csv
  • Coordinates of the UMAP plot for the merged samples, extracted from the ArchRProject.
• enrichMotifs_clusters.rds
  • SummarizedExperiment object where rows are marker motifs per cluster and columns are the clusters. Pvalues, FDR and other informations per clusters can be found in the assays of this object.
• enrichMotifs_sample.rds
  • Same as enrichMotifs_clusters.rds but per sample.
• enrichMotifs_treatment.rds
  • Same as enrichMotifs_clusters.rds but per condition.
• genes_per_cluster_hm.csv
  • The general matrix data for gene accessibility heatmap of clusters.
• genes_per_sample_hm.csv
  • The general matrix data for gene accessibility heatmap of samples.
• genes_per_treatment_hm.csv
  • The general matrix data for gene accessibility heatmap of conditions.
• inpMarkers.txt
  • A table with columns avg_log2FC, p_val, p_val_adj, gene, cluster, Significance that is used for volcano plots of genes per group.
• inpMarkers_motif.txt
  • A table with columns avg_log2FC, p_val, p_val_adj, gene, cluster, Significance that used for volcano plots of motifs per group.
• markersGS_clusters.rds
  • SummarizedExperiment object where rows are marker genes per cluster and columns are the clusters. Pvalues, FDR and other informations per clusters can be found in the assays of this object.
• markersGS_sample.rds
  • Same as markersGS_clusters.rds but per sample.
• markersGS_treatment.rds
  • Same as markersGS_clusters.rds but per condition.
• motif_per_cluster_hm.csv
  • The general matrix data for motif enrichment heatmap of clusters.
• motif_per_sample_hm.csv
  • The general matrix data for motif enrichment heatmap of samples.
• motif_per_treatment_hm.csv
  • The general matrix data for motif enrichment heatmap of conditions.
• req_genes1.csv
  • List of the genes used as default in heatmap of clusters.
• req_genes2.csv
  • List of the genes used as default in heatmap of conditions.
• req_genes3.csv
  • List of the genes used as default in heatmap of samples.
• req_motifs1.csv
  • List of the motifs used as default in heatmap of clusters.
• req_motifs2.csv
  • List of the motifs used as default in heatmap of conditions.
• req_motifs3.csv
  • List of the motifs used as default in heatmap of samples.
• seqlogo.rds
  • Position weight matrices class for plotting sequence logos using the seqlogo package.

Next Steps

Analysis can be performed locally or in a latch.bio Pod. For access to ATX-specific Pods, please contact your AtlasXomics Support Scientist.

Output from create ArchRProject can be provided to the atlasShiny workflow to create input for a DBiT-seq-specific R Shiny App. For access to this workflow and app, please contact your AtlasXomics Support Scientist.

Support

Questions? Comments? Contact [email protected] or post in AtlasXomics Discord.