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TheAnanseSeurat package takes pre-processed clustered single cellobjects of scRNAseq and scATACseq or a multiome combination, andgenerates files for gene regulatory network (GRN) analysis. It is partof the scANANSE pipeline.https://doi.org/10.12688/f1000research.130530.1

AnanseSeurat can be installed from CRAN using

install.packages('AnanseSeurat')

Or to install the developmental branch from github:

library(devtools)# Tools to Make Developing R Packages Easier # Tools to Make Developing R Packages EasierSys.unsetenv("GITHUB_PAT")remotes::install_github("JGASmits/AnanseSeurat@main")

library("AnanseSeurat")rds_file<-'./scANANSE/preprocessed_PDMC.Rds'pbmc<- readRDS(rds_file)

Next you can output the data from your single cell object, the fileformat, config file and sample file are all ready to automate GRNanalysis usinganansnake.https://github.com/vanheeringen-lab/anansnake

export_CPM_scANANSE(pbmc,min_cells=25,output_dir='./scANANSE/analysis',cluster_id='predicted.id',RNA_count_assay='RNA')export_ATAC_scANANSE(pbmc,min_cells=25,output_dir='./scANANSE/analysis',cluster_id='predicted.id',ATAC_peak_assay='peaks')# Specify additional contrasts:contrasts<-  c('B-naive_B-memory','B-memory_B-naive','B-naive_CD14-Mono','CD14-Mono_B-naive')config_scANANSE(pbmc,min_cells=25,output_dir='./scANANSE/analysis',cluster_id='predicted.id',additional_contrasts=contrasts)DEGS_scANANSE(pbmc,min_cells=25,output_dir='./scANANSE/analysis',cluster_id='predicted.id',additional_contrasts=contrasts)

Follow the instructions its respective github page,https://github.com/vanheeringen-lab/anansnake After activating theconda environment, use the generated files to run GRN analysis usingyour single cell cluster data:

anansnake \--configfile scANANSE/analysis/config.yaml \--resources mem_mb=48_000 --cores 12

After running Anansnake, you can import the TF influence scores backinto your single cell object of choice

pbmc<- import_seurat_scANANSE(pbmc,cluster_id='predicted.id',anansnake_inf_dir="./scANANSE/analysis/influence")TF_influence<- per_cluster_df(pbmc,cluster_id='predicted.id',assay='influence')

scANANSE gene regulatory network and motif analysis of single-cell clusters [version 1; peer review: awaiting peer review]Jos G.A. Smits, Julian A. Arts, Siebren Frölich, Rebecca R. Snabel, Branco M.H. Heuts, Joost H.A. Martens, Simon J van Heeringen, Huiqing ZhouF1000Research 2023, 12:243 (https://doi.org/10.12688/f1000research.130530.1)

• Julian A. Arts and his Pycharm equivalent of this packagehttps://github.com/Arts-of-coding/AnanseScanpy
• Siebren Frohlich and his anansnake implementationhttps://github.com/vanheeringen-lab/anansnake
• Rebecca R. Snabel for her implementation of the motif expressioncorrelation analysis
• Branco Heuts for testing

The hex sticker is generated using thehexSticker package.