Package 'GRaNIE'

Object of class GRN

Character(1). Either auto or a manually defined formula to be used for the model fitting. Default auto. Must include only terms that are part of the metadata as specified with the metadata parameter. If set to auto, the formula will be build automatically based on all metadata variables as specified with the metadata parameter. By default, numerical variables will be modeled as fixed effects, while variables that are defined as factors or can be converted to factors (characters and logical variables) are modeled as random effects as recommended by the variancePartition package.

Character vector. Default all. Vector of column names from the metadata data frame that was provided when using the function addData. Must either contain the special keyword all to denote that all (!) metadata columns from GRN@data$metadata are taken or if not, a subset of the column names from GRN@data$metadatato include in the model fitting for fitExtractVarPartModel..

Character(1). Either all_filtered or all. Default all_filtered. Should variancePartition only be run for the features (TFs, peaks and genes) from the filtered set of connections (the result of filterGRNAndConnectGenes) or for all genes that are defined in the object? If set to all, the running time is greatly increased.

Integer >0. Default 1. Number of cores to use. A value >1 requires the BiocParallel package (as it is listed under Suggests, it may not be installed yet).

TRUE or FALSE. Default FALSE. Force execution, even if the GRN object already contains the result. Overwrites the old results.

Additional parameters passed on to variancePartition::fitExtractVarPartModel beyond exprObj, formula and data. See the function help for more information

Object of class GRN

Character. One of pearson, spearman or bicor. Default pearson. Method for calculating the correlation coefficient. For pearson and spearman , see cor for details. bicor denotes the *biweight midcorrelation*, a correlation measure based on medians as calculated by WGCNA::bicorAndPvalue. Both spearman and bicor are considered more robust measures that are less prone to be affected by outliers.

Integer >0. Default 1. Number of cores to use. A value >1 requires the BiocParallel package (as it is listed under Suggests, it may not be installed yet).

TRUE or FALSE. Default FALSE. Force execution, even if the GRN object already contains the result. Overwrites the old results.

Object of class GRN

Character. "TSS" or "full". Default "TSS". If set to "TSS", only the TSS of the gene is used as reference for finding genes in the neighborhood of a peak. If set to "full", the whole annotated gene (including all exons and introns) is used instead.

Character. One of pearson, spearman or bicor. Default pearson. Method for calculating the correlation coefficient. For pearson and spearman , see cor for details. bicor denotes the *biweight midcorrelation*, a correlation measure based on medians as calculated by WGCNA::bicorAndPvalue. Both spearman and bicor are considered more robust measures that are less prone to be affected by outliers.

Integer >=0. Default 250000. The size of the neighborhood in bp to correlate peaks and genes in vicinity. Only peak-gene pairs will be correlated if they are within the specified range. Increasing this value leads to higher running times and more peak-gene pairs to be associated, while decreasing results in the opposite.

Data frame with TAD domains. Default NULL. If provided, the neighborhood of a peak is defined by the TAD domain the peak is in rather than a fixed-sized neighborhood. The expected format is a BED-like data frame with at least 3 columns in this particular order: chromosome, start, end, the 4th column is optional and will be taken as ID column. All additional columns as well as column names are ignored. For the first 3 columns, the type is checked as part of a data integrity check.

TRUE or FALSE. Default FALSE. Should overlapping TADs be merged? Only relevant if TADs are provided.

NULL or a data frame with exactly two columns. Both columns must be of type character and they must both contain genomic coordinates in the usual format: chr:start-end, while the 2 separators between the three elements can be chosen by the user. The first column denotes the **bait**, the promoter coordinates that are overlapped with the genes (usually their TSS, unless specified otherwise via the parameter overlapTypeGene, while the second column denotes the **other end (OE)** coordinates, which is overlapped with the peaks/enhancers from the GRN object. **NOTE: The provided column names are ignored and column 1 is interpreted as bait column and column 2 as OE column unless column names are exactly 'bait' and 'OE'.**. For more details, see the Workflow vignette.)

Character vector of length 1 or 2. Default c(":", "-"). Separator(s) for the character columns that specify the genomic locations. The first entry splits the chromosome from the position, while the second entry splits the start and end coordinates. If only one separator is given, the same will be used for both.

TRUE or FALSE. Default FALSE. If kept at FALSE (the default), specified knownLinks will be used in addition to the regular peak-gene links that are identified via the default method. If set to TRUE, only the knownLinks will be used.

TRUE or FALSE. Default TRUE. Should the RNA sample labels be shuffled in addition to testing random peak-gene pairs for the background? When set to FALSE, only peak-gene pairs are shuffled, but for each pair, the counts from peak and RNA that are correlated are matched (i.e., sample 1 counts from peak data are compared to sample 1 counts from RNA). If set to TRUE, however, the RNA sample labels are in addition shuffled so that sample 1 counts from peak data are compared to sample 4 data from RNA, for example. Shuffling truly randomizes the resulting background eGRN. Note that this parameter and its influence is still being investigated. Until version 1.0.7, this parameter (although not existent explicitly) was implicitly set to TRUE.

Integer >0. Default 1. Number of cores to use. A value >1 requires the BiocParallel package (as it is listed under Suggests, it may not be installed yet).

TRUE or FALSE. Default TRUE. Run and plot various diagnostic plots? If set to TRUE, PDF files will be produced and saved in the output directory (in a subfolder called plots).

List of character vectors. Default list(c("all"), c("protein_coding")). Each list element may consist of one or multiple gene types that are plotted collectively in one PDF. The special keyword "all" denotes all gene types that are found (be aware: this typically contains 20+ gene types, see https://www.gencodegenes.org/pages/biotypes.html for details).

Character or NULL. Default NULL. If set to NULL, the default output folder as specified when initiating the object in initializeGRN will be used. Otherwise, all output from this function will be put into the specified folder. If a folder is provided, while we recommend specifying an absolute path, a relative one also works.

TRUE or FALSE. Default FALSE. Force execution, even if the GRN object already contains the result. Overwrites the old results.

Object of class GRN

TRUE or FALSE. Default TRUE. Run and plot various diagnostic plots? If set to TRUE, PDF files will be produced and saved in the output directory (in a subfolder called plots).

TRUE or FALSE. Default FALSE. Print additional plots that may help for debugging and QC purposes? Note that these plots are currently less documented or not at all.

Character or NULL. Default NULL. If set to NULL, the default output folder as specified when initiating the object in initializeGRN will be used. Otherwise, all output from this function will be put into the specified folder. If a folder is provided, while we recommend specifying an absolute path, a relative one also works.

Character. One of pearson, spearman or bicor. Default pearson. Method for calculating the correlation coefficient. For pearson and spearman , see cor for details. bicor denotes the *biweight midcorrelation*, a correlation measure based on medians as calculated by WGCNA::bicorAndPvalue. Both spearman and bicor are considered more robust measures that are less prone to be affected by outliers.

Character vector. Default expression. Vector of connection types to include for the TF-peak connections. If an additional connection type is specified here, it has to be available already within the object (EXPERIMENTAL). See the function addData_TFActivity for details.

Vector of TRUE or FALSE. Default FALSE. EXPERIMENTAL. Must be a logical vector of the same length as the parameter connectionType. Should negatively correlated TF-peak connections be removed for the specific connection type? For connection type expression, the default is FALSE, while for any TF Activity related connection type, we recommend setting this to TRUE.

Numeric[0,1]. Default 0.3. Maximum TF-peak FDR value to permanently store a particular TF-peak connection in the object? This parameter has a large influence on the overall memory size of the object, and we recommend not storing connections with a high FDR due to their sheer number.

TRUE or FALSE. Default TRUE. Add connections also for background data. Leave at TRUE unless you know what you are doing.

TRUE or FALSE. Default FALSE. EXPERIMENTAL. Should a GC-matched background be used when calculating FDRs? For more details, see the Package Details vignette.

Numeric[0,100]. Default 75. EXPERIMENTAL. Percentage of the background to use as basis for sampling. If set to 0, an automatic iterative procedure will identify the maximum percentage so that all relevant GC bins with a rel. frequency above 5% from the foreground can be matched. For more details, see the Package Details vignette. Only relevant if useGCCorrection is set to TRUE, ignored otherwise.

TRUE or FALSE. Default TRUE. EXPERIMENTAL. Should resampling be enabled for those GC bins for which not enough background peaks are available?. For more details, see the Package Details vignette. Only relevant if useGCCorrection is set to TRUE, ignored otherwise.

TRUE or FALSE. Default FALSE. Force execution, even if the GRN object already contains the result. Overwrites the old results.

Object of class GRN

Data frame. No default. Counts for the peaks, with raw or normalized counts for each peak (rows) across all samples (columns). In addition to the count data, it must also contain one ID column with a particular format, see the argument idColumn_peaks below. Row names are ignored, column names must be set to the sample names and must match those from the RNA counts and the sample metadata table.

Character. Default DESeq2_sizeFactors. Normalization procedure for peak data. Must be one of limma_cyclicloess, limma_quantile, limma_scale, csaw_cyclicLoess_orig, csaw_TMM, EDASeq_GC_peaks, gcqn_peaks, DESeq2_sizeFactors, none.

Character. Default peakID. Name of the column in the counts_peaks data frame that contains peak IDs. The required format must be chr:start-end, with chr denoting the abbreviated chromosome name, and start and end the begin and end of the peak coordinates, respectively. End must be bigger than start. Examples for valid peak IDs are chr1:400-800 or chrX:20-25.

Data frame. No default. Counts for the RNA-seq data, with raw or normalized counts for each gene (rows) across all samples (columns). In addition to the count data, it must also contain one ID column with a particular format, see the argument idColumn_rna below. Row names are ignored, column names must be set to the sample names and must match those from the RNA counts and the sample metadata table.

Character. Default limma_quantile. Normalization procedure for peak data. Must be one of limma_cyclicloess, limma_quantile, limma_scale, csaw_cyclicLoess_orig, csaw_TMM, DESeq2_sizeFactors, none.

Character. Default ENSEMBL. Name of the column in the counts_rna data frame that contains Ensembl IDs.

Data frame. Default NULL. Optional, additional metadata for the samples, such as age, sex, gender etc. If provided, the @seealso [plotPCA_all()] function can then incorporate and plot it. Sample names must match with those from both peak and RNA-Seq data. The first column is expected to contain the sample IDs, the actual column name is irrelevant.

Named list. Default list(). Additional parameters for the chosen normalization method. Currently, only the GC-aware normalization methods EDASeq_GC_peaks and gcqn_peaks are supported here. Both support the parameters roundResults (logical flag, TRUE or FALSE) and nBins (Integer > 0), and EDASeq_GC_peaks supports three additional parameters: withinLane_method (one of: "loess","median","upper","full") and betweenLane_method (one of: "median","upper","full"). For more information, see the EDASeq vignette.

TRUE or FALSE. Default FALSE. Should overlapping peaks be allowed (then only a warning is issued when overlapping peaks are found) or (the default) should an error be raised?

TRUE or FALSE. Default FALSE. Should the original read counts as provided to the function be kept in addition to storing the rad counts after a (if any) normalization? This increases the memory footprint of the object because 2 additional count matrices have to be stored.

NULL or Character(1). Default NULL. The Ensembl version to use for genome annotation retrieval via biomaRt, which is only used to populate the gene annotation metadata that is stored in GRN@annotation$genes. By default (NULL), the newest version is selected for the most recent genome assembly versions is used (see biomaRt::listEnsemblArchives() for supported versions). This parameter can override this to use a custom (older) version instead.

AnnotationHub or biomaRt. Default AnnotationHub. Annotation source to retrieve genome annotation data from.

TRUE or FALSE. Default FALSE. Force execution, even if the GRN object already contains the result. Overwrites the old results.

Object of class GRN

Character. Default cyclicLoess. One of cyclicLoess, sizeFactors, quantile, or none. Normalization procedure. When set to cyclicLoess, the csaw package is required (as it is listed under Suggests, it may not be installed).

Name in object under which it should be stored. This corresponds to the connectionType afterwards that some functions iterate over.

TRUE or FALSE. Default FALSE. Force execution, even if the GRN object already contains the result. Overwrites the old results.

Object of class GRN

Character vector. No default. Vector of SNP IDs (rsID) that should be integrated and overlapped with the peaks.

NULL or Character(1). Default NULL. Only relevant if source is not set to custom, ignored otherwise. The Ensembl version to use for the retrieval of gene IDs from their provided database names (e.g., JASPAR) via biomaRt. By default (NULL), the newest version is selected for the most recent genome assembly versions is used (see biomaRt::listEnsemblArchives() for supported versions). This parameter can override this to use a custom (older) version instead.

TRUE or FALSE. Default FALSE. Should SNPs in LD with any of the user-provided SNPs that overlap a peak be identified and added to the peak? If set to TRUE, LDlinkR::LDproxy_batch will be used to identify SNPs in LD based on the user-provided SNP_IDs argument, a specific (set of) populations (argument population) and a value for r2d. The full table (stored in GRN@annotation$SNPs) is then subsequently filtered, see also the filter argument.

TRUE or FALSE. Default FALSE. Only applicable if add_SNPs_LD = TRUE and ignored otherwise. Should LDlinkR::LDproxy_batch be re-executed if already present? As this is very time-consuming, querying the database is only performed if this parameter is set to TRUE or if GRN@annotation$SNPs is missing.

Character vector. Default CEU. Only applicable if add_SNPs_LD = TRUE and ignored otherwise. Population code(s) from the 1000 Genomes project to be used for LDlinkR::LDproxy_batch. Multiple codes are allowed. For all valid codes, see https://ftp.1000genomes.ebi.ac.uk/vol1/ftp/README_populations.md

r2 or d. Default r2. Only applicable if add_SNPs_LD = TRUE and ignored otherwise. See the help of the function LDlinkR::LDproxy_batch for more details.

Character or NULL. Default NULL. Only applicable if add_SNPs_LD = TRUE and ignored otherwise. LDlink provided user token. Register for token at https://ldlink.nih.gov/?tab=apiaccess. Has to be done only once and is very simple and straight forward but unfortunately necessary. An example, non-functional token is 2c49a3b54g04.

Character. Default R2 > 0.8. Only applicable if add_SNPs_LD = TRUE and ignored otherwise. Filter criteria for the output table as generated by LDlinkR::LDproxy_batch. dplyr::filter style is used to specify filters, and multiple filtering criteria can be used (e.g., R2 > 0.8 & MAF > 0.01). The filtered table is stored in GRN@annotation$SNPs_filtered. Note that re-filtering is quick without the need to re-query the database unless requeryLD = TRUE.

TRUE or FALSE. Default FALSE. Force execution, even if the GRN object already contains the result. Overwrites the old results.

Object of class GRN

Character. One of custom, JASPAR2022 or JASPAR2024. Default custom. If a custom source is being used, further details about the motif folder and files will be provided (see the other function arguments). If set to JASPAR2022, the JASPAR2022 database is used. If set to JASPAR2024, the JASPAR2024 database is used.

Character. No default. Only relevant if source = "custom". Path to the folder that contains the TFBS predictions. The files must be in BED format, 6 columns, one file per TF. See the other parameters for more details. The folder must also contain a so-called translation table, see the argument translationTable for details.

Character vector. Default all. Only relevant if source = "custom". Vector of TF names to include. The special keyword all can be used to include all TF found in the folder as specified by motifFolder. If all is specified anywhere, all TFs will be included. TF names must otherwise match the file names that are found in the folder, without the file suffix.

Character. Default translationTable.csv. Only relevant if source = "custom". Name of the translation table file that is also located in the folder along with the TFBS files. This file must have the following structure: at least 2 columns, called ENSEMBL and ID. ID denotes the ID for the TF that is used throughout the pipeline (e.g., AHR) and the prefix of how the corresponding file is called (e.g., AHR.0.B if the file for AHR is called AHR.0.B_TFBS.bed.gz), while ENSEMBL denotes the ENSEMBL ID (dot suffix; e.g., ENSG00000106546, are removed automatically if present).

Character. Default " " (white space character). Only relevant if source = "custom". The column separator for the translationTable file.

Character. Default "_TFBS". Only relevant if source = "custom". Suffix for the file names in the TFBS folder that is not part of the TF name. Can be empty. For example, for the TF CTCF, if the file is called CTCF.all.TFBS.bed, set this parameter to ".all.TFBS".

Character. Default ".bed". Only relevant if source = "custom". File ending for the files from the motif folder.

NULL or Integer[1,]. Default NULL. Maximal number of TFs to import. Can be used for testing purposes, e.g., setting to 5 only imports 5 TFs even though the whole motifFolder has many more TFs defined.

NULL or Character(1). Default NULL. Only relevant if source is not set to custom, ignored otherwise. The Ensembl version to use for the retrieval of gene IDs from their provided database names (e.g., JASPAR) via biomaRt. By default (NULL), the newest version is selected for the most recent genome assembly versions is used (see biomaRt::listEnsemblArchives() for supported versions). This parameter can override this to use a custom (older) version instead.

NULL or Character(1). Default NULL. Should a tax group instead of th specific genome assembly be used for retrieving the TF list? This is useful for genomes that are not human or mouse for which JASPAR otherwise returns too few TFs otherwise. If set to NULL, the specific genome version as provided in the object is used within TFBSTools::getMatrixSet in the opts list for species, while tax_group will be used instead if this argument is not set to NULL. For example, it can be set to vertebrates to use the vertebrates TF collection. For more details, see ?TFBSTools::getMatrixSet.

TRUE or FALSE. Default TRUE. Remove TFs for which the name as provided b JASPAR cannot be mapped uniquely to one and only Ensembl ID? By default (NULL), the newest version is selected (see biomaRt::listEnsemblArchives() for supported versions). This parameter can override this to use a custom (older) version instead.

TRUE or FALSE. Default FALSE. Force execution, even if the GRN object already contains the result. Overwrites the old results.

Additional named elements for the opts function argument from ?TFBSTools::getMatrixSet that is used to query the JASPAR database.

Object of class GRN

Numeric[0,1]. Default 0.05. Significance threshold for Wilcoxon test that is run in the end for the final classification. See the Vignette and *diffTF* paper for details.

Integer[1,]. Default 100. Minimum number of TFBS for a TF to be included in the heatmap that is part of the output of this function.

TRUE or FALSE. Default TRUE. Should intermediate data be deleted before returning the object after a successful run? Due to the size of the produced intermediate data, we recommend setting this to TRUE, but if memory or object size are not an issue, the information can also be kept.

TRUE or FALSE. Default TRUE. Run and plot various diagnostic plots? If set to TRUE, PDF files will be produced and saved in the output directory (in a subfolder called plots).

Character or NULL. Default NULL. If set to NULL, the default output folder as specified when initiating the object in initializeGRN will be used. Otherwise, all output from this function will be put into the specified folder. If a folder is provided, while we recommend specifying an absolute path, a relative one also works.

Character. One of pearson, spearman or bicor. Default pearson. Method for calculating the correlation coefficient. For pearson and spearman , see cor for details. bicor denotes the *biweight midcorrelation*, a correlation measure based on medians as calculated by WGCNA::bicorAndPvalue. Both spearman and bicor are considered more robust measures that are less prone to be affected by outliers.

TRUE or FALSE. Default FALSE. Force execution, even if the GRN object already contains the result. Overwrites the old results.

Object of class GRN

TRUE or FALSE. Default FALSE. Should TF and gene nodes be modeled separately? If set to TRUE,this may lead to unwanted effects in case of TF-TF connections (i.e., a TF regulating another TF)

TRUE or FALSE. Default FALSE. Allow loops in the network (i.e., a TF that regulates itself)

TRUE or FALSE. Default FALSE. Remove loops with the same start and end point? This can happen if multiple TF originate from the same gene, for example.

TRUE or FALSE. Default FALSE. Should the network be directed?

TRUE or FALSE. Default FALSE. Force execution, even if the GRN object already contains the result. Overwrites the old results.

Object of class GRN

Character vector of ontologies. Default c("GO_BP", "GO_MF"). Valid values are "GO_BP", "GO_MF", "GO_CC", "KEGG", "DO", and "Reactome", referring to GO Biological Process, GO Molecular Function, GO Cellular Component, KEGG, Disease Ontology, and Reactome Pathways, respectively. GO ontologies require the topGO, "KEGG" the clusterProfiler, "DO" the DOSE, and "Reactome" the ReactomePA packages, respectively. As they are listed under Suggests, they may not yet be installed, and the function will throw an error if they are missing.

Character. Default "weight01". One of: "classic", "elim", "weight", "weight01", "lea", "parentchild". Only relevant if ontology is GO related (GO_BP, GO_MF, GO_CC), ignored otherwise. Name of the algorithm that handles the GO graph structures. Valid inputs are those supported by the topGO library. For general information about the algorithms, see https://academic.oup.com/bioinformatics/article/22/13/1600/193669. weight01 is a mixture between the elim and the weight algorithms.

Character. Default "fisher". One of: "fisher", "ks", "t". Statistical test to be used. Only relevant if ontology is GO related (GO_BP, GO_MF, GO_CC), and valid inputs are a subset of those supported by the topGO library (we had to remove some as they do not seem to work properly in topGO either), ignored otherwise. For the other ontologies the test statistic is always Fisher.

Character. Default "neighborhood". One of: "all_annotated", "all_RNA", "all_RNA_filtered", "neighborhood". Set of genes to be used to construct the background for the enrichment analysis. This can either be all annotated genes in the reference genome (all_annotated), all genes from the provided RNA data (all_RNA), all genes from the provided RNA data excluding those marked as filtered after executing filterData (all_RNA_filtered), or all the genes that are within the neighborhood of any peak (before applying any filters except for the user-defined promoterRange value in addConnections_peak_gene) (neighborhood).

Character vector of gene types that should be considered for the background. Default "all". Only gene types as defined in the GRN object, slot GRN@annotation$genes$gene.type are allowed. The special keyword "all" means no filter on gene type.

Character. Default "byRank". One of: "byRank", "byLabel". Specify whether the communities enrichment will by calculated based on their rank, where the largest community (with most vertices) would have a rank of 1, or by their label. Note that the label is independent of the rank.

NULL or numeric vector or character vector. Default NULL. If set to NULL, all community enrichments that have been calculated before are plotted. If a numeric vector is specified (when selection = "byRank"), the rank of the communities is specified. For example, communities = c(1,4) then denotes the first and fourth largest community. If a character vector is specified (when selection = "byLabel"), the name of the communities is specified instead. For example, communities = c("1","4") then denotes the communities with the names "1" and "4", which may or may not be the largest and fourth largest communities among all.

Character. Default "BH". One of: "holm", "hochberg", "hommel", "bonferroni", "BH", "BY", "fdr". This parameter is only relevant for the following ontologies: KEGG, DO, Reactome. For the other ontologies, the algorithm serves as an adjustment.

TRUE or FALSE. Default FALSE. Force execution, even if the GRN object already contains the result. Overwrites the old results.

Object of class GRN

Character. Default louvain. One of: louvain, leiden, leading_eigen, fast_greedy, optimal, walktrap. The community detection algorithm to be used. Please bear in mind the robustness and time consumption of the algorithms when opting for an alternative to the default.

TRUE or FALSE. Default FALSE. Force execution, even if the GRN object already contains the result. Overwrites the old results.

Additional parameters for the used clustering method, see the igraph::cluster_* methods for details on the specific parameters and what they do. For leiden clustering, for example, you may add a resolution_parameter to control the granularity of the community detection or n_iterations to modify the number of iterations.

Object of class GRN

Character vector of ontologies. Default c("GO_BP", "GO_MF"). Valid values are "GO_BP", "GO_MF", "GO_CC", "KEGG", "DO", and "Reactome", referring to GO Biological Process, GO Molecular Function, GO Cellular Component, KEGG, Disease Ontology, and Reactome Pathways, respectively. GO ontologies require the topGO, "KEGG" the clusterProfiler, "DO" the DOSE, and "Reactome" the ReactomePA packages, respectively. As they are listed under Suggests, they may not yet be installed, and the function will throw an error if they are missing.

Character. Default "weight01". One of: "classic", "elim", "weight", "weight01", "lea", "parentchild". Only relevant if ontology is GO related (GO_BP, GO_MF, GO_CC), ignored otherwise. Name of the algorithm that handles the GO graph structures. Valid inputs are those supported by the topGO library. For general information about the algorithms, see https://academic.oup.com/bioinformatics/article/22/13/1600/193669. weight01 is a mixture between the elim and the weight algorithms.

Character. Default "fisher". One of: "fisher", "ks", "t". Statistical test to be used. Only relevant if ontology is GO related (GO_BP, GO_MF, GO_CC), and valid inputs are a subset of those supported by the topGO library (we had to remove some as they do not seem to work properly in topGO either), ignored otherwise. For the other ontologies the test statistic is always Fisher.

Character. Default "neighborhood". One of: "all_annotated", "all_RNA", "all_RNA_filtered", "neighborhood". Set of genes to be used to construct the background for the enrichment analysis. This can either be all annotated genes in the reference genome (all_annotated), all genes from the provided RNA data (all_RNA), all genes from the provided RNA data excluding those marked as filtered after executing filterData (all_RNA_filtered), or all the genes that are within the neighborhood of any peak (before applying any filters except for the user-defined promoterRange value in addConnections_peak_gene) (neighborhood).

Character vector of gene types that should be considered for the background. Default "all". Only gene types as defined in the GRN object, slot GRN@annotation$genes$gene.type are allowed. The special keyword "all" means no filter on gene type.

Character. Default "BH". One of: "holm", "hochberg", "hommel", "bonferroni", "BH", "BY", "fdr". This parameter is only relevant for the following ontologies: KEGG, DO, Reactome. For the other ontologies, the algorithm serves as an adjustment.

TRUE or FALSE. Default FALSE. Force execution, even if the GRN object already contains the result. Overwrites the old results.

Object of class GRN

Character. Default "degree". One of: "degree", "EV", "custom". This parameter will determine the criterion to be used to identify the "top" TFs. If set to "degree", the function will select top TFs based on the number of connections to genes they have, i.e. based on their degree-centrality. If set to "EV" it will select the top TFs based on their eigenvector-centrality score in the network. If set to custom, a set of TF IDs will have to be passed to the "TF.IDs" parameter.

Numeric. Default 3. If this parameter is passed as a value between 0 and 1, it is treated as a percentage of top nodes. If the value is passed as an integer it will be treated as the number of top nodes. This parameter is not relevant if rankType = "custom".

Character vector. Default NULL. If the rank type is set to "custom", a vector of TF IDs for which the GO enrichment should be calculated should be passed to this parameter.

Character vector of ontologies. Default c("GO_BP", "GO_MF"). Valid values are "GO_BP", "GO_MF", "GO_CC", "KEGG", "DO", and "Reactome", referring to GO Biological Process, GO Molecular Function, GO Cellular Component, KEGG, Disease Ontology, and Reactome Pathways, respectively. GO ontologies require the topGO, "KEGG" the clusterProfiler, "DO" the DOSE, and "Reactome" the ReactomePA packages, respectively. As they are listed under Suggests, they may not yet be installed, and the function will throw an error if they are missing.

Character. Default "weight01". One of: "classic", "elim", "weight", "weight01", "lea", "parentchild". Only relevant if ontology is GO related (GO_BP, GO_MF, GO_CC), ignored otherwise. Name of the algorithm that handles the GO graph structures. Valid inputs are those supported by the topGO library. For general information about the algorithms, see https://academic.oup.com/bioinformatics/article/22/13/1600/193669. weight01 is a mixture between the elim and the weight algorithms.

Character. Default "fisher". One of: "fisher", "ks", "t". Statistical test to be used. Only relevant if ontology is GO related (GO_BP, GO_MF, GO_CC), and valid inputs are a subset of those supported by the topGO library (we had to remove some as they do not seem to work properly in topGO either), ignored otherwise. For the other ontologies the test statistic is always Fisher.

Character. Default "neighborhood". One of: "all_annotated", "all_RNA", "all_RNA_filtered", "neighborhood". Set of genes to be used to construct the background for the enrichment analysis. This can either be all annotated genes in the reference genome (all_annotated), all genes from the provided RNA data (all_RNA), all genes from the provided RNA data excluding those marked as filtered after executing filterData (all_RNA_filtered), or all the genes that are within the neighborhood of any peak (before applying any filters except for the user-defined promoterRange value in addConnections_peak_gene) (neighborhood).

Character vector of gene types that should be considered for the background. Default "all". Only gene types as defined in the GRN object, slot GRN@annotation$genes$gene.type are allowed. The special keyword "all" means no filter on gene type.

Character. Default "BH". One of: "holm", "hochberg", "hommel", "bonferroni", "BH", "BY", "fdr". This parameter is only relevant for the following ontologies: KEGG, DO, Reactome. For the other ontologies, the algorithm serves as an adjustment.

TRUE or FALSE. Default FALSE. Force execution, even if the GRN object already contains the result. Overwrites the old results.

Object of class GRN

Character. Default .. New output directory for all output files unless overwritten via the parameter outputFolder.

Object of class GRN

Object of class GRN

TRUE or FALSE. Default TRUE. Should all connections be included for plotting? If set to TRUE, all connections are marked for plotting and everything else is ignored. This resets any previous setting. If set FALSE, the filter expressions (if any) are used to determine which connection to plot

An arbitrary set of arguments that is used directly, without modification, as input for dplyr::filter and therefore has to be valid expression that dplyr::filter understands. The filtering is based on the all.filtered table as stored in GRN@connections$all.filtered$`0`. Thus, the specific filters can be completely arbitrary for ultimate flexibility and must only adhere to the column names and types as defined in GRN@connections$all.filtered$`0`. See the examples also for what you can do.

TRUE or FALSE. Default FALSE. Force execution, even if the GRN object already contains the result. Overwrites the old results.

Object of class GRN

Numeric[0,] or NULL. Default 5. Minimum mean across all samples for a peak to be retained for the normalized counts table. Set to NULL for not applying the filter. Be aware that depending on the chosen normalization, this filter may not make sense and should NOT be applied. See the notes for this function.

Numeric[0,] or NULL. Default NULL. Maximum mean across all samples for a peak to be retained for the normalized counts table. Set to NULL for not applying the filter. Be aware that depending on the chosen normalization, this filter may not make sense and should NOT be applied. See the notes for this function.

Numeric[0,] or NULL. Default 5. Minimum mean across all samples for a gene to be retained for the normalized counts table. Set to NULL for not applying the filter. Be aware that depending on the chosen normalization, this filter may not make sense and should NOT be applied. See the notes for this function.

Numeric[0,] or NULL. Default NULL. Maximum mean across all samples for a gene to be retained for the normalized counts table. Set to NULL for not applying the filter. Be aware that depending on the chosen normalization, this filter may not make sense and should NOT be applied. See the notes for this function.

Character vector or NULL. Default NULL. Vector of chromosomes that peaks are allowed to come from. This filter can be used to filter sex chromosomes from the peaks, for example (e.g, c(paste0("chr", 1:22), "chrX", "chrY"))

Integer[1,] or NULL. Default 20. Minimum peak size (width, end - start) for a peak to be retained. Set to NULL for not applying the filter.

Integer[1,] or NULL. Default 10000. Maximum peak size (width, end - start) for a peak to be retained. Set to NULL for not applying the filter.

Numeric[0,] or NULL. Default NULL. Minimum CV (coefficient of variation, a unitless measure of variation) for a peak to be retained. Set to NULL for not applying the filter. Be aware that depending on the chosen normalization, this filter may not make sense and should NOT be applied. See the notes for this function.

Numeric[0,] or NULL. Default NULL. Maximum CV (coefficient of variation, a unitless measure of variation) for a peak to be retained. Set to NULL for not applying the filter. Be aware that depending on the chosen normalization, this filter may not make sense and should NOT be applied. See the notes for this function.

Numeric[0,] or NULL. Default NULL. Minimum CV (coefficient of variation, a unitless measure of variation) for a gene to be retained. Set to NULL for not applying the filter. Be aware that depending on the chosen normalization, this filter may not make sense and should NOT be applied. See the notes for this function.

Numeric[0,] or NULL. Default NULL. Maximum CV (coefficient of variation, a unitless measure of variation) for a gene to be retained. Set to NULL for not applying the filter. Be aware that depending on the chosen normalization, this filter may not make sense and should NOT be applied. See the notes for this function.

TRUE or FALSE. Default FALSE. Force execution, even if the GRN object already contains the result. Overwrites the old results.

Object of class GRN

Numeric[0,1]. Default 0.2. Maximum FDR for the TF-peak links. Set to 1 or NULL to disable this filter.

Character vector. Default all. TF-peak connection types to consider. The special keyword all denotes all connection types (e.g., expression and TFActivity) that are found in the GRN object. By default, only expression is present in the object, so all and expression are usually equivalent unless calculation of TF-peak links based on TF activity has also been enabled.

Named list. Default list(min_nSNPs = 0, filterType = "orthogonal"). Filters related to SNP data if they have been added with the function addSNPData, ignored otherwise. The named list must contain at least two elements: First, min_nSNPs, an integer >= 0 that denotes how many SNPs a peak has to overlap with at least to pass the filter or be considered for inclusion. Second, filterType, a character that must either be orthogonal or extra and denotes whether the SNP filter is orthogonal to the other filters (i.e, an alternative way of when a peak is considered for being kept) or whether the SNP filter is in addition to all other filters. For more help, see the Vignettes.

Numeric[0,1]. Default NULL. Threshold for the peak-gene connections, based on the raw p-value. All peak-gene connections with a larger raw p-value will be filtered out.

Numeric[0,1]. Default 0.2. Threshold for the peak-gene connections, based on the FDR. All peak-gene connections with a larger FDR will be filtered out.

Character. Default "BH". One of: "holm", "hochberg", "hommel", "bonferroni", "BH", "BY", "fdr", "none", "IHW". Method for adjusting p-values for multiple testing. If set to "IHW", the package IHW is required (as it is listed under Suggests, it may not be installed), and independent hypothesis weighting will be performed, and a suitable covariate has to be specified for the parameter peak_gene.IHW.covariate.

Character. Default NULL. Name of the covariate to use for IHW (column name from the table that is returned with the function getGRNConnections. Only relevant if peak_gene.fdr.method is set to "IHW". You have to make sure the specified covariate is suitable for IHW, see the diagnostic plots that are generated in this function for this. For many datasets, the peak-gene distance (called peak_gene.distance in the object) seems suitable.

Integer or "auto". Default "auto". Number of bins for IHW. Only relevant if peak_gene.fdr.method is set to "IHW".

Character or NULL. Default NULL. If set to NULL, the default output folder as specified when initiating the object in initializeGRN will be used. Otherwise, all output from this function will be put into the specified folder. If a folder is provided, while we recommend specifying an absolute path, a relative one also works.

Character vector of supported gene types. Default "all". Filter for gene types to retain, genes with gene types not listed here are filtered. The special keyword "all" indicates no filter and retains all gene types. To filter for only protein-coding genes, use "protein_coding". The specified names must match the names as stored in the GRN object (see GRN@annotation$genes$gene.type) and correspond 1:1 to the gene type names as provided by biomaRt, with the exception of lncRNAs, which is internally renamed to lincRNAs when first fetching all gene types. This is done due to a recent change in biomaRt and aims at keeping backwards compatibility with GRN objects.

TRUE or FALSE. Default FALSE. Should connections be returned for which the TF is NA (i.e., connections consisting only of peak-gene links?). If set to TRUE, this generally greatly increases the number of connections but it may not be what you aim for.

TRUE or FALSE. Default TRUE. Should connections be returned for which the gene is NA (i.e., connections consisting only of TF-peak links?). If set to TRUE, this generally increases the number of connections.

Numeric(2). Default c(0,1). Filter for lower and upper limit for the peak-gene links. Only links will be retained if the correlation coefficient is within the specified interval. This filter is usually used to filter out negatively correlated peak-gene links.

"all" or "closest". Default "all". Filter for the selection of genes for each peak. If set to "all", all previously identified peak-gene are used, while "closest" only retains the closest gene for each peak that is retained until the point the filter is applied.

Integer >0. Default NULL. Maximum peak-gene distance to retain a peak-gene connection.

Character vector. Default NULL. Vector of TFs (as named in the GRN object) to retain. All TFs not listed will be filtered out.

Character vector. Default NULL. Vector of gene IDs (as named in the GRN object) to retain. All genes not listed will be filtered out.

Character vector. Default NULL. Vector of peak IDs (as named in the GRN object) to retain. All peaks not listed will be filtered out.

TRUE or FALSE. Default FALSE. Use a modified procedure for selecting TF-peak links. Instead of selecting solely based on the user-specified FDR, this procedure first identifies the correlation bin closest to 0 that contains at least one significant TF-peak link according to the chosen TF_peak.fdr.threshold. This is done spearately for both FDR directions. It then retains all TF-peak links that have a correlation bin at least as extreme as the identified pair. For example, if the correlation bin [0.35,0.40] contains a significant TF-peak link while [0,0.05], [0.05,0.10], ..., [0.30,0.35] do not, all TF-peak links with a correlation of at least 0.35 or above are selected (i.e, bins [0.35,0.40], [0.40,0.45], ..., [0.95,1.00]). Thus, for the final selection, also links with a higher FDR but a more extreme correlation may be selected.

TRUE or FALSE. Default TRUE. If a TF regulates itself (i.e., the TF and the gene are the same entity), should such loops be filtered from the GRN?

TRUE or FALSE. Default TRUE. If set to TRUE, the stored eGRN graph (slot graph) is reset due to the potentially changed connections that would otherwise cause conflicts in the information stored in the object. Also, a GRN object is returned. If set to FALSE, only the new filtered connections are returned and the object is not altered.

TRUE or FALSE. Default FALSE. Print progress messages and filter statistics.

TRUE or FALSE. Default FALSE. Force execution, even if the GRN object already contains the result. Overwrites the old results.

Object of class GRN

Numeric vector[0,1]. Default c(0.001, 0.01, 0.05, 0.1, 0.2). TF-peak FDR values to iterate over.

Character vector. Default all. TF-peak connection types to consider. The special keyword all denotes all connection types (e.g., expression and TFActivity) that are found in the GRN object. By default, only expression is present in the object, so all and expression are usually equivalent unless calculation of TF-peak links based on TF activity has also been enabled.

Numeric vector[0,1]. Default c(0.001, 0.01, 0.05, 0.1, 0.2). Peak-gene FDR values to iterate over.

Numeric vector of length 2[-1,1]. Default c(0,1). The correlation range of peak-gene connections to keep.

Character vector of supported gene types. Default "all". Filter for gene types to retain, genes with gene types not listed here are filtered. The special keyword "all" indicates no filter and retains all gene types. To filter for only protein-coding genes, use "protein_coding". The specified names must match the names as stored in the GRN object (see GRN@annotation$genes$gene.type) and correspond 1:1 to the gene type names as provided by biomaRt, with the exception of lncRNAs, which is internally renamed to lincRNAs when first fetching all gene types. This is done due to a recent change in biomaRt and aims at keeping backwards compatibility with GRN objects.

Logical vector of length 1 or 2. Default c(FALSE, TRUE). Allow genes to be missing for peak-gene connections? If both FALSE and TRUE are given, the code loops over both

Logical vector of length 1 or 2. Default c(FALSE). Allow TFs to be missing for TF-peak connections? If both FALSE and TRUE are given, the code loops over both

TRUE or FALSE. Default FALSE. Force execution, even if the GRN object already contains the result. Overwrites the old results.

Object of class GRN

Character. Either peaks or rna. peaks corresponds to the counts for the open chromatin data, while rna refers to th RNA-seq counts. If set to rna, both real (foreground) and background data can be retrieved, while for peaks, only the real (i.e., the one with index 0) can be retrieved.

TRUE or FALSE. Default FALSE. Should the permuted data be taken (TRUE) or the non-permuted, original one (FALSE)?

Logical. TRUE or FALSE. Default FALSE. If set to FALSE, counts are returned as a data frame with or without an ID column (see includeIDColumn). If set to TRUE, counts are returned as a matrix with the ID column as row names.

Logical. TRUE or FALSE. Default TRUE. Only relevant if asMatrix = FALSE. If set to TRUE, an explicit ID column is returned (no row names). If set to FALSE, the IDs are in the row names instead.

Logical. TRUE or FALSE. Default FALSE. If set to FALSE, genes or peaks marked as filtered (after running the function filterData) will not be returned. If set to TRUE, all elements are returned regardless of the currently active filter status.

Object of class GRN

Character. One of TF_peaks, peak_genes, TF_genes or all.filtered. Default all.filtered. The type of connections to retrieve.

Integer (0 or 1). Default 0. Either 0 or 1). Here, 0 refers to the real (foreground) while 1 to the background.

Logical. TRUE or FALSE. Default FALSE. Should TFs and gene correlations be returned as well? If set to TRUE, they must have been computed beforehand with add_TF_gene_correlation.

Logical. TRUE or FALSE. Default FALSE. Should TF metadata be returned as well?

Logical. TRUE or FALSE. Default FALSE. Should peak metadata be returned as well?

Logical. TRUE or FALSE. Default FALSE. Should gene metadata be returned as well?

Logical. TRUE or FALSE. Default FALSE. Should the results from the function add_featureVariation be included? If set to TRUE, they must have been computed beforehand with add_featureVariation; otherwise, an error is thrown.

Object of class GRN

TRUE or FALSE. Default FALSE. Should the function be silent and print nothing?

Object of class GRN

Character. Either function or parameter. Default parameter. When set to function, a valid GRaNIE function name must be given that has been run before. When set to parameter, in combination with name, returns a specific parameter (as specified in GRN@config)).

Character. Default all. Name of parameter or function name to retrieve. Set to the special keyword all to retrieve all parameters.

Object of class GRN

Character. One of: "gene" or "TF". Node type.

Character. One of: "degree", "EV". This parameter will determine the criterion to be used to identify the "top" nodes. If set to "degree", the function will select top nodes based on the number of connections they have, i.e. based on their degree-centrality. If set to "EV" it will select the top nodes based on their eigenvector-centrality score in the network.

Numeric. Default 0.1. If this parameter is passed as a value between [0,1], it is treated as a percentage of top nodes. If the value is passed as an integer >=1 it will be treated as the number of top nodes.

TRUE or FALSE. Default TRUE. Should the TF-gene network be used (TRUE) or the TF-peak-gene network (FALSE)?

Object of class GRN

Data frame. No default. Data with TF data.

Name in object under which it should be stored. This corresponds to the connectionType afterwards that some functions iterate over.

Character. Default ENSEMBL. Name of the ID column. Must not be unique as some TFs may correspond to the same ID.

Character. Default TF.name. Must be unique for each TF / row.

Character. Default cyclicLoess. One of cyclicLoess, sizeFactors, quantile, or none. Normalization procedure. When set to cyclicLoess, the csaw package is required (as it is listed under Suggests, it may not be installed).

TRUE or FALSE. Default FALSE. Force execution, even if the GRN object already contains the result. Overwrites the old results.

List. Default list(). Optional (named) list with an arbitrary number of elements, all of which capture metadata for the object. Only atomic data types are allowed for each list element (see ?is.atomic for more help: logical, integer, numeric, complex, character, raw, and NULL), and this slot is not supposed to store real data. This is mainly used to distinguish GRN objects from one another by storing object-specific metadata along with the data.

Output folder, either absolute or relative to the current working directory. Default ".". Default output folder where all pipeline output will be put unless specified otherwise. We recommend specifying an absolute path. Note that for Windows-based systems, the path must be correctly specified with "/" as path separator.

Character. No default. The genome assembly of all data that to be used within this object. Currently, supported genomes are: hg19, hg38, mm9, mm10, mm39, rn6, rn7, dm6. If you need additional genomes, let us know. See function description for further information and notes.

TRUE or FALSE. Default FALSE. Should the download be enforced even if the local cached file is already present?

Character. Default https://git.embl.de/grp-zaugg/GRaNIE/-/raw/master/data/GRN.rds. URL to the GRN example object in rds format.

Object of class GRN

TRUE or FALSE. Default TRUE. Should genes marked as filtered be included in the count?

Object of class GRN

TRUE or FALSE. Default TRUE. Should peaks marked as filtered be included in the count?

Object of class GRN

Object of class GRN

Integer >0. Default 1. Number of cores to use. A value >1 requires the BiocParallel package (as it is listed under Suggests, it may not be installed yet).

TRUE or FALSE. Default FALSE. Force execution, even if the GRN object already contains the result. Overwrites the old results.

No default. Only relevant if source = "JASPAR" has been selected in addTFBS, ignored otherwise. Additional arguments for motifmatchr::matchMotifs such as custom background nucleotide frequencies or p-value cutoffs. For more information, type ?motifmatchr::matchMotifs

Object of class GRN

Character vector of ontologies. Default c("GO_BP", "GO_MF"). Valid values are "GO_BP", "GO_MF", "GO_CC", "KEGG", "DO", and "Reactome", referring to GO Biological Process, GO Molecular Function, GO Cellular Component, KEGG, Disease Ontology, and Reactome Pathways, respectively. GO ontologies require the topGO, "KEGG" the clusterProfiler, "DO" the DOSE, and "Reactome" the ReactomePA packages, respectively. As they are listed under Suggests, they may not yet be installed, and the function will throw an error if they are missing.

Character. Default "weight01". One of: "classic", "elim", "weight", "weight01", "lea", "parentchild". Only relevant if ontology is GO related (GO_BP, GO_MF, GO_CC), ignored otherwise. Name of the algorithm that handles the GO graph structures. Valid inputs are those supported by the topGO library. For general information about the algorithms, see https://academic.oup.com/bioinformatics/article/22/13/1600/193669. weight01 is a mixture between the elim and the weight algorithms.

Character. Default "fisher". One of: "fisher", "ks", "t". Statistical test to be used. Only relevant if ontology is GO related (GO_BP, GO_MF, GO_CC), and valid inputs are a subset of those supported by the topGO library (we had to remove some as they do not seem to work properly in topGO either), ignored otherwise. For the other ontologies the test statistic is always Fisher.

Character. Default "neighborhood". One of: "all_annotated", "all_RNA", "all_RNA_filtered", "neighborhood". Set of genes to be used to construct the background for the enrichment analysis. This can either be all annotated genes in the reference genome (all_annotated), all genes from the provided RNA data (all_RNA), all genes from the provided RNA data excluding those marked as filtered after executing filterData (all_RNA_filtered), or all the genes that are within the neighborhood of any peak (before applying any filters except for the user-defined promoterRange value in addConnections_peak_gene) (neighborhood).

Character. Default louvain. One of: louvain, leiden, leading_eigen, fast_greedy, optimal, walktrap. The community detection algorithm to be used. Please bear in mind the robustness and time consumption of the algorithms when opting for an alternative to the default.

NULL or numeric vector or character vector. Default NULL. If set to NULL, all community enrichments that have been calculated before are plotted. If a numeric vector is specified (when selection = "byRank"), the rank of the communities is specified. For example, communities = c(1,4) then denotes the first and fourth largest community. If a character vector is specified (when selection = "byLabel"), the name of the communities is specified instead. For example, communities = c("1","4") then denotes the communities with the names "1" and "4", which may or may not be the largest and fourth largest communities among all.

Character. Default "byRank". One of: "byRank", "byLabel". Specify whether the communities will be selected based on their rank or explicitly by their label. Note that the label is independent of the rank. When set to "byRank", the largest community (with most vertices) always has a rank of 1.

Integer > 0. Default 20. Number of genes to plot, sorted by their rank or label.

Integer > 0. Default 20. Number of TFs to plot, sorted by their rank or label.

Integer (>=10). Default 50. Maximum number of characters for a term before it is truncated.

TRUE or FALSE. Default FALSE. Is the p-value being displayed in the plots the adjusted p-value? This parameter is relevant for KEGG, Disease Ontology, and Reactome enrichments, and does not affect GO enrichments.

Character or NULL. Default NULL. If set to NULL, the default output folder as specified when initiating the object in initializeGRN will be used. Otherwise, all output from this function will be put into the specified folder. If a folder is provided, while we recommend specifying an absolute path, a relative one also works.

TRUE or FALSE. Default FALSE. Force execution, even if the GRN object already contains the result. Overwrites the old results.

Object of class GRN

Character. Either "heatmap" or "boxplot". Default "heatmap". Which plot type to produce?

Character or NULL. Default NULL. If set to NULL, the default output folder as specified when initiating the object in initializeGRN will be used. Otherwise, all output from this function will be put into the specified folder. If a folder is provided, while we recommend specifying an absolute path, a relative one also works.

NULL or character. Default NULL. Basename of the output files that are produced. If set to NULL, a default basename is chosen. If a custom basename is specified, all output files will have the chosen basename as file prefix, be careful with not overwriting already existing files (if forceRerun is set to TRUE)

TRUE or FALSE. Default TRUE.Should the plots be printed to a PDF file? If set to TRUE, a PDF file is generated, the name of which depends on the value of basenameOutput. If set to FALSE, all plots are printed to the currently active device. Note that most functions print more than one plot, which means you may only see the last plot depending on your active graphics device.

Number>0. Default 12. Width of the PDF, in cm.

Number >0. Default 12. Height of the PDF, in cm.

Integer vector or NULL. Default NULL. Page number(s) to plot. Can be used to plot only specific pages to a PDF or the currently active graphics device.

TRUE or FALSE. Default FALSE. Force execution, even if the GRN object already contains the result. Overwrites the old results.

Object of class GRN

Character or NULL. Default NULL. If set to NULL, the default output folder as specified when initiating the object in initializeGRN will be used. Otherwise, all output from this function will be put into the specified folder. If a folder is provided, while we recommend specifying an absolute path, a relative one also works.

NULL or character. Default NULL. Basename of the output files that are produced. If set to NULL, a default basename is chosen. If a custom basename is specified, all output files will have the chosen basename as file prefix, be careful with not overwriting already existing files (if forceRerun is set to TRUE)

Character. Default "byRank". One of: "byRank", "byLabel". Specify whether the communities will be selected based on their rank or explicitly by their label. Note that the label is independent of the rank. When set to "byRank", the largest community (with most vertices) always has a rank of 1.

NULL or numeric vector or character vector. Default NULL. If set to NULL, all community enrichments that have been calculated before are plotted. If a numeric vector is specified (when selection = "byRank"), the rank of the communities is specified. For example, communities = c(1,4) then denotes the first and fourth largest community. If a character vector is specified (when selection = "byLabel"), the name of the communities is specified instead. For example, communities = c("1","4") then denotes the communities with the names "1" and "4", which may or may not be the largest and fourth largest communities among all.

Numeric. Default 30. Maximum number of ontology terms that meet the p-value significance threshold to display in the enrichment dot plot

Numeric. Default 0.05. p-value threshold to determine significance.

Numeric > 0. Default 3. Threshold to filter out an ontology term with less than nSignificant overlapping genes.

Numeric > 0. Default 10. For the reduced summary heatmap, number of top terms to select per community / for the general enrichment.

Integer (>=10). Default 50. Maximum number of characters for a term before it is truncated.

TRUE or FALSE. Default FALSE. Is the p-value being displayed in the plots the adjusted p-value? This parameter is relevant for KEGG, Disease Ontology, and Reactome enrichments, and does not affect GO enrichments.

TRUE or FALSE. Default TRUE.Should the plots be printed to a PDF file? If set to TRUE, a PDF file is generated, the name of which depends on the value of basenameOutput. If set to FALSE, all plots are printed to the currently active device. Note that most functions print more than one plot, which means you may only see the last plot depending on your active graphics device.

Number>0. Default 12. Width of the PDF, in cm.

Number >0. Default 12. Height of the PDF, in cm.

Integer vector or NULL. Default NULL. Page number(s) to plot. Can be used to plot only specific pages to a PDF or the currently active graphics device.

TRUE or FALSE. Default FALSE. Force execution, even if the GRN object already contains the result. Overwrites the old results.

Object of class GRN

Character or NULL. Default NULL. If set to NULL, the default output folder as specified when initiating the object in initializeGRN will be used. Otherwise, all output from this function will be put into the specified folder. If a folder is provided, while we recommend specifying an absolute path, a relative one also works.

NULL or character. Default NULL. Basename of the output files that are produced. If set to NULL, a default basename is chosen. If a custom basename is specified, all output files will have the chosen basename as file prefix, be careful with not overwriting already existing files (if forceRerun is set to TRUE)

Character. Default "byRank". One of: "byRank", "byLabel". Specify whether the communities will be selected based on their rank or explicitly by their label. Note that the label is independent of the rank. When set to "byRank", the largest community (with most vertices) always has a rank of 1.

NULL or numeric vector or character vector. Default NULL. If set to NULL, all community enrichments that have been calculated before are plotted. If a numeric vector is specified (when selection = "byRank"), the rank of the communities is specified. For example, communities = c(1,4) then denotes the first and fourth largest community. If a character vector is specified (when selection = "byLabel"), the name of the communities is specified instead. For example, communities = c("1","4") then denotes the communities with the names "1" and "4", which may or may not be the largest and fourth largest communities among all.

Integer > 0. Default 20. Number of genes to plot, sorted by their rank or label.

Integer > 0. Default 20. Number of TFs to plot, sorted by their rank or label.

TRUE or FALSE. Default TRUE.Should the plots be printed to a PDF file? If set to TRUE, a PDF file is generated, the name of which depends on the value of basenameOutput. If set to FALSE, all plots are printed to the currently active device. Note that most functions print more than one plot, which means you may only see the last plot depending on your active graphics device.

Number>0. Default 12. Width of the PDF, in cm.

Number >0. Default 12. Height of the PDF, in cm.

Integer vector or NULL. Default NULL. Page number(s) to plot. Can be used to plot only specific pages to a PDF or the currently active graphics device.

TRUE or FALSE. Default FALSE. Force execution, even if the GRN object already contains the result. Overwrites the old results.

Object of class GRN

Character(1). Default "all.filtered". Either "TF-peak", "peak-gene", "TF-gene", or "all.filtered". Default "all.filtered". Which connections to plot? "all.filtered" plots all TF-peak and peak-gene pairs from the filtered eGRN as stored in the object.

Character(). Default NULL. Character vector of TF IDs to include. If set to NULL, this filter will be ignored. Only applicable when type = "TF-peak" or type = "TF-gene".

Character(). Default NULL. Character vector of peak IDs to include. If set to NULL, this filter will be ignored. Only applicable when type = "TF-peak" or type = "peak-gene".

Character(). Default NULL. Character vector of gene IDs (Ensembl) to include. If set to NULL, this filter will be ignored. Only applicable when type = "TF-gene" or type = "peak-gene".

Numeric[0,1]. Default 0. Filter for all types of pairs: Minimum correlation coefficient (absolute value) required to include a particular pair.

Numeric[0,1]. Default 0.2. Filter for TF-peak pairs: Which maximum FDR should a pair to plot have? Only applicable when type = "TF-peak".

Numeric[0,1]. Default 0.2. Filter for peak-gene pairs: Which maximum FDR should a pair to plot have? Only applicable when type = "peak-gene".

Numeric[0,1]. Default 0.2. Filter for TF-gene pairs: Which maximum FDR should a pair to plot have? Only applicable when type = "TF-gene".

Numeric(1). Default 10. Filter for all types of pairs: maximum number of selected pairs that fulfill all other filters that should be plotted. If set to 0, this filter will be disabled and all pairs that fulfill the user-defined criteria will be plotted. If set to a value > 0, different pairs may be selected each time the function is run (if the total number of remaining pairs is large enough)

random or top. Default top. Only applicable if nMax is set to a value > 0. If set to top, only the top features will be plotted while random plots randomly selected features.

Character vector. One of, or both of, real or background. Default real. For which type, real or background data, to produce the diagnostic plots?

Character. Either NULL (the default) or one of pearson, spearman or bicor. Method for calculating the correlation coefficient. If set to NULL, the correlation method that has been used in the GraNIE object is used. For testing nd visualizing data, this can be changed, though. For pearson and spearman , see cor for details. bicor denotes the *biweight midcorrelation*, a correlation measure based on medians as calculated by WGCNA::bicorAndPvalue. Both spearman and bicor are considered more robust measures that are less prone to be affected by outliers.

Character or NULL. Default NULL. If set to NULL, the default output folder as specified when initiating the object in initializeGRN will be used. Otherwise, all output from this function will be put into the specified folder. If a folder is provided, while we recommend specifying an absolute path, a relative one also works.

NULL or character. Default NULL. Basename of the output files that are produced. If set to NULL, a default basename is chosen. If a custom basename is specified, all output files will have the chosen basename as file prefix, be careful with not overwriting already existing files (if forceRerun is set to TRUE)

TRUE or FALSE. Default TRUE.Should the plots be printed to a PDF file? If set to TRUE, a PDF file is generated, the name of which depends on the value of basenameOutput. If set to FALSE, all plots are printed to the currently active device. Note that most functions print more than one plot, which means you may only see the last plot depending on your active graphics device.

Integer vector of length 2. Default c(2,2). How man plots should be put on one page? The first number denotes the number of rows, the second one the number of columns.

Number>0. Default 12. Width of the PDF, in cm.

Number >0. Default 12. Height of the PDF, in cm.

TRUE or FALSE. Default FALSE. Force execution, even if the GRN object already contains the result. Overwrites the old results.

Object of class GRN

Character or NULL. Default NULL. If set to NULL, the default output folder as specified when initiating the object in initializeGRN will be used. Otherwise, all output from this function will be put into the specified folder. If a folder is provided, while we recommend specifying an absolute path, a relative one also works.

NULL or character. Default NULL. Basename of the output files that are produced. If set to NULL, a default basename is chosen. If a custom basename is specified, all output files will have the chosen basename as file prefix, be careful with not overwriting already existing files (if forceRerun is set to TRUE)

List of character vectors. Default list(c("all"), c("protein_coding")). Vectors of gene types to consider for the diagnostic plots. Multiple distinct combinations of gene types can be specified. For example, if set to list(c("protein_coding", "lincRNA"), c("protein_coding"), c("all")), 3 distinct PDFs will be produced, one for each element of the list. The first file would only consider protein-coding and lincRNA genes, while the second plot only considers protein-coding ones. The special keyword "all" denotes all gene types found (usually, there are many gene types present, also more exotic and rare ones).

Logical. TRUE or FALSE. Default FALSE. If set to FALSE, the diagnostic plots will be produced based on all peak-gene connections. This is the default and will usually be best to judge whether the background behaves as expected. If set to TRUE, the diagnostic plots will be produced based on the filtered set of connections. For this, the function filterGRNAndConnectGenes must have been run before.

TRUE or FALSE. Default FALSE. Print additional plots that may help for debugging and QC purposes? Note that these plots are currently less documented or not at all.

Logical. TRUE or FALSE. Default FALSE. If set to FALSE, the diagnostic plots will be done across all TF (the default), while setting it to TRUE will generate the QC plots TF-specifically, including "all" TF, sorted by the number of connections.

TRUE or FALSE. Default TRUE.Should the plots be printed to a PDF file? If set to TRUE, a PDF file is generated, the name of which depends on the value of basenameOutput. If set to FALSE, all plots are printed to the currently active device. Note that most functions print more than one plot, which means you may only see the last plot depending on your active graphics device.

Number>0. Default 12. Width of the PDF, in cm.

Number >0. Default 12. Height of the PDF, in cm.

Integer vector or NULL. Default NULL. Page number(s) to plot. Can be used to plot only specific pages to a PDF or the currently active graphics device.

TRUE or FALSE. Default FALSE. Force execution, even if the GRN object already contains the result. Overwrites the old results.

Object of class GRN

Character or NULL. Default NULL. If set to NULL, the default output folder as specified when initiating the object in initializeGRN will be used. Otherwise, all output from this function will be put into the specified folder. If a folder is provided, while we recommend specifying an absolute path, a relative one also works.

NULL or character. Default NULL. Basename of the output files that are produced. If set to NULL, a default basename is chosen. If a custom basename is specified, all output files will have the chosen basename as file prefix, be careful with not overwriting already existing files (if forceRerun is set to TRUE)

TRUE or FALSE. Default FALSE. Print additional plots that may help for debugging and QC purposes? Note that these plots are currently less documented or not at all.

Character vector. One of, or both of, "real" or "background". For which type, real or background data, to produce the diagnostic plots?

NULL or Integer > 0. Default NULL. Maximum number of TFs to process. Can be used for testing purposes by setting this to a small number i(.e., 10)

TRUE or FALSE. Default TRUE.Should the plots be printed to a PDF file? If set to TRUE, a PDF file is generated, the name of which depends on the value of basenameOutput. If set to FALSE, all plots are printed to the currently active device. Note that most functions print more than one plot, which means you may only see the last plot depending on your active graphics device.

Number>0. Default 12. Width of the PDF, in cm.

Number. Default 8. Base height of the PDF, in cm, per connection type. The total height is automatically determined based on the number of connection types that are found in the object (e.g., expression or TF activity). For example, when two connection types are found, the base height is multiplied by 2.

Integer vector or NULL. Default NULL. Page number(s) to plot. Can be used to plot only specific pages to a PDF or the currently active graphics device.

TRUE or FALSE. Default FALSE. Force execution, even if the GRN object already contains the result. Overwrites the old results.

Object of class GRN

Character or NULL. Default NULL. If set to NULL, the default output folder as specified when initiating the object in initializeGRN will be used. Otherwise, all output from this function will be put into the specified folder. If a folder is provided, while we recommend specifying an absolute path, a relative one also works.

NULL or character. Default NULL. Basename of the output files that are produced. If set to NULL, a default basename is chosen. If a custom basename is specified, all output files will have the chosen basename as file prefix, be careful with not overwriting already existing files (if forceRerun is set to TRUE)

Character vector. One of, or both of, "real" or "background". For which type, real or background data, to produce the diagnostic plots?

NULL or Integer > 0. Default NULL. Maximum number of TFs to process. Can be used for testing purposes by setting this to a small number i(.e., 10)

TRUE or FALSE. Default TRUE.Should the plots be printed to a PDF file? If set to TRUE, a PDF file is generated, the name of which depends on the value of basenameOutput. If set to FALSE, all plots are printed to the currently active device. Note that most functions print more than one plot, which means you may only see the last plot depending on your active graphics device.

Number>0. Default 12. Width of the PDF, in cm.

Number. Default 8. Base height of the PDF, in cm, per connection type. The total height is automatically determined based on the number of connection types that are found in the object (e.g., expression or TF activity). For example, when two connection types are found, the base height is multiplied by 2.

Integer vector or NULL. Default NULL. Page number(s) to plot. Can be used to plot only specific pages to a PDF or the currently active graphics device.

TRUE or FALSE. Default FALSE. Force execution, even if the GRN object already contains the result. Overwrites the old results.

Object of class GRN

Character or NULL. Default NULL. If set to NULL, the default output folder as specified when initiating the object in initializeGRN will be used. Otherwise, all output from this function will be put into the specified folder. If a folder is provided, while we recommend specifying an absolute path, a relative one also works.

NULL or character. Default NULL. Basename of the output files that are produced. If set to NULL, a default basename is chosen. If a custom basename is specified, all output files will have the chosen basename as file prefix, be careful with not overwriting already existing files (if forceRerun is set to TRUE)

Character. NULL or vector of ontology names. Default NULL. Vector of ontologies to plot. The results must have been previously calculated otherwise an error is thrown.

Numeric. Default 30. Maximum number of ontology terms that meet the p-value significance threshold to display in the enrichment dot plot

Numeric. Default 0.05. p-value threshold to determine significance.

TRUE or FALSE. Default FALSE. Is the p-value being displayed in the plots the adjusted p-value? This parameter is relevant for KEGG, Disease Ontology, and Reactome enrichments, and does not affect GO enrichments.

Integer (>=10). Default 50. Maximum number of characters for a term before it is truncated.

TRUE or FALSE. Default TRUE.Should the plots be printed to a PDF file? If set to TRUE, a PDF file is generated, the name of which depends on the value of basenameOutput. If set to FALSE, all plots are printed to the currently active device. Note that most functions print more than one plot, which means you may only see the last plot depending on your active graphics device.

Number>0. Default 12. Width of the PDF, in cm.

Number >0. Default 12. Height of the PDF, in cm.

Integer vector or NULL. Default NULL. Page number(s) to plot. Can be used to plot only specific pages to a PDF or the currently active graphics device.

TRUE or FALSE. Default FALSE. Force execution, even if the GRN object already contains the result. Overwrites the old results.

Object of class GRN

Character or NULL. Default NULL. If set to NULL, the default output folder as specified when initiating the object in initializeGRN will be used. Otherwise, all output from this function will be put into the specified folder. If a folder is provided, while we recommend specifying an absolute path, a relative one also works.

NULL or character. Default NULL. Basename of the output files that are produced. If set to NULL, a default basename is chosen. If a custom basename is specified, all output files will have the chosen basename as file prefix, be careful with not overwriting already existing files (if forceRerun is set to TRUE)

TRUE or FALSE. Default TRUE.Should the plots be printed to a PDF file? If set to TRUE, a PDF file is generated, the name of which depends on the value of basenameOutput. If set to FALSE, all plots are printed to the currently active device. Note that most functions print more than one plot, which means you may only see the last plot depending on your active graphics device.

Number>0. Default 12. Width of the PDF, in cm.

Number >0. Default 12. Height of the PDF, in cm.

Integer vector or NULL. Default NULL. Page number(s) to plot. Can be used to plot only specific pages to a PDF or the currently active graphics device.

TRUE or FALSE. Default FALSE. Force execution, even if the GRN object already contains the result. Overwrites the old results.

Object of class GRN

Character or NULL. Default NULL. If set to NULL, the default output folder as specified when initiating the object in initializeGRN will be used. Otherwise, all output from this function will be put into the specified folder. If a folder is provided, while we recommend specifying an absolute path, a relative one also works.

NULL or character. Default NULL. Basename of the output files that are produced. If set to NULL, a default basename is chosen. If a custom basename is specified, all output files will have the chosen basename as file prefix, be careful with not overwriting already existing files (if forceRerun is set to TRUE)

Character. Either "peaks" or "rna" or "all". Default c("rna", "peaks"). Type of data to plot a PCA for. "peaks" corresponds to the the open chromatin data, while "rna" refers to the RNA-seq counts. If set to "all", PCA will be done for both data modalities. In any case, PCA will be based on the original provided data before any additional normalization has been run (i.e., usually the raw data).

Integer vector. Default c(500,1000,5000). Number of top variable features to do PCA for. Can be a vector of different numbers (see default).

Character. Must be "normalized". On which data type (raw or normalized) should the PCA plots be done? We removed support for raw and currently only support normalized.

Logical. TRUE or FALSE. Default TRUE. Should features marked as filtered as determined by filterData be removed?

TRUE or FALSE. Default TRUE.Should the plots be printed to a PDF file? If set to TRUE, a PDF file is generated, the name of which depends on the value of basenameOutput. If set to FALSE, all plots are printed to the currently active device. Note that most functions print more than one plot, which means you may only see the last plot depending on your active graphics device.

Number>0. Default 12. Width of the PDF, in cm.

Number >0. Default 12. Height of the PDF, in cm.

Integer vector or NULL. Default NULL. Page number(s) to plot. Can be used to plot only specific pages to a PDF or the currently active graphics device.

TRUE or FALSE. Default FALSE. Force execution, even if the GRN object already contains the result. Overwrites the old results.

Object of class GRN

Character. One of: "degree", "EV", "custom". This parameter will determine the criterion to be used to identify the "top" nodes. If set to "degree", the function will select top nodes based on the number of connections they have, i.e. based on their degree-centrality. If set to "EV" it will select the top nodes based on their eigenvector-centrality score in the network.

NULL or numeric. Default NULL. If set to NULL, all previously calculated TF enrichments will be plotted. If set to a value between (0,1), it is treated as a percentage of top nodes. If the value is passed as an integer it will be treated as the number of top nodes. This parameter is not relevant if rankType = "custom".

NULL or character vector. Default NULL. For rankType="custom" the IDs of the TFs to plot. Ignored otherwise.

Numeric. Default 30. Maximum number of ontology terms that meet the p-value significance threshold to display in the enrichment dot plot

Numeric. Default 0.05. p-value threshold to determine significance.

Numeric > 0. Default 3. Threshold to filter out an ontology term with less than nSignificant overlapping genes.

Numeric > 0. Default 10. For the reduced summary heatmap, number of top terms to select per community / for the general enrichment.

TRUE or FALSE. Default FALSE. Is the p-value being displayed in the plots the adjusted p-value? This parameter is relevant for KEGG, Disease Ontology, and Reactome enrichments, and does not affect GO enrichments.

Integer (>=10). Default 50. Maximum number of characters for a term before it is truncated.

Character or NULL. Default NULL. If set to NULL, the default output folder as specified when initiating the object in initializeGRN will be used. Otherwise, all output from this function will be put into the specified folder. If a folder is provided, while we recommend specifying an absolute path, a relative one also works.

NULL or character. Default NULL. Basename of the output files that are produced. If set to NULL, a default basename is chosen. If a custom basename is specified, all output files will have the chosen basename as file prefix, be careful with not overwriting already existing files (if forceRerun is set to TRUE)

TRUE or FALSE. Default TRUE.Should the plots be printed to a PDF file? If set to TRUE, a PDF file is generated, the name of which depends on the value of basenameOutput. If set to FALSE, all plots are printed to the currently active device. Note that most functions print more than one plot, which means you may only see the last plot depending on your active graphics device.

Number>0. Default 12. Width of the PDF, in cm.

Number >0. Default 12. Height of the PDF, in cm.

Integer vector or NULL. Default NULL. Page number(s) to plot. Can be used to plot only specific pages to a PDF or the currently active graphics device.

TRUE or FALSE. Default FALSE. Force execution, even if the GRN object already contains the result. Overwrites the old results.

Object of class GRN

Character or NULL. Default NULL. If set to NULL, the default output folder as specified when initiating the object in initializeGRN will be used. Otherwise, all output from this function will be put into the specified folder. If a folder is provided, while we recommend specifying an absolute path, a relative one also works.

NULL or character. Default NULL. Basename of the output files that are produced. If set to NULL, a default basename is chosen. If a custom basename is specified, all output files will have the chosen basename as file prefix, be careful with not overwriting already existing files (if forceRerun is set to TRUE)

TRUE or FALSE. Default TRUE.Should the plots be printed to a PDF file? If set to TRUE, a PDF file is generated, the name of which depends on the value of basenameOutput. If set to FALSE, all plots are printed to the currently active device. Note that most functions print more than one plot, which means you may only see the last plot depending on your active graphics device.

Number>0. Default 12. Width of the PDF, in cm.

Number >0. Default 12. Height of the PDF, in cm.

NULL or Character. Default NULL. Title to be assigned to the plot.

Integer > 0. Default 500. Refers to the maximum number of connections to be plotted. If the network size is above this limit, nothing will be drawn. In such a case, it may help to either increase the value of this parameter or set the filtering criteria for the network to be more stringent, so that the network becomes smaller.

Integer > 0. Default 8. Maximum number of communities that get a distinct coloring. All additional communities will be colored with the same (gray) color.

Character. Default TF-gene. One of: TF-gene, TF-peak-gene. Whether to plot a graph with links from TFs to peaks to gene, or the graph with the inferred TF to gene connections.

Character. Default type. Either type or community. Color the vertices by either type (TF/peak/gene) or community. See calculateCommunitiesStats

Character. Default fr. One of star, fr, sugiyama, kk, lgl, graphopt, mds, sphere

Named list. Default list(h = 10, c = 85, l = c(25, 95)). The list must specify the color in hcl format (hue, chroma, luminence). See the colorspace package for more details and examples

Named list. Default list(h = 135, c = 45, l = c(35, 95)).

Named list. Default list(h = 260, c = 80, l = c(30, 90)).

Numeric. Default 0.4. Font size (multiplication factor, device-dependent)

Numeric. Default 0 vertex. Distance between the label and the vertex.

TRUE or FALSE. Default FALSE. Force execution, even if the GRN object already contains the result. Overwrites the old results.