hello，大家好，这一篇给大家带来的是10X单细胞和10XATAC联合进行网络调控的分析，也很经典，参考的文献在IReNA: integrated regulatory network analysis of single-cell transcriptomes，我们看看分析的方法和内容,关于10X单细胞联合ATAC分析调控网络的内容，大家可以参考我之前的文章10X单细胞（10X空间转录组）基因调控网络分析之Pando
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IReNA（Integrated Regulatory Network Analysis）是一个用于执行regulatory network analysis的 R 包。 IReNA 包含两种重建基因调控网络的方法。 第一种是单独使用单细胞 RNA 测序 (scRNA-seq) 数据。 第二个是使用测序 (ATAC-seq) 整合 scRNA-seq 数据和来自 Assay for Transposase Accessible Chromatin 的染色质可及性概况。 IReNA 执行模块化调控网络，以揭示模块之间的关键转录因子和重要调控关系，提供有关调控机制的生物学见解。
For users who want to use ATAC-seq data to refine regulatory relationships, Computer or server of linux system and the following software are needed: samtools, bedtools and fimo.
安装
if (!requireNamespace("BiocManager", quietly = TRUE))
install.packages("BiocManager")
BiocManager::install(version = "3.12")
BiocManager::install(c('Rsamtools', 'ChIPseeker', 'monocle',
'RcisTarget', 'RCy3', 'clusterProfiler'))
install.packages("devtools")
devtools::install_github("jiang-junyao/IReNA")
library(IReNA)
Workflow
Inputs of IReNA
如果仅使用 scRNA-seq 或大量 RNA-seq 数据运行 IReNA，则需要以下输入：(2) scRNA-seq 或bulk RNA-seq 数据的Raw counts和 (3) 物种的参考基因组 (4) GTF 文件 (5) 转录因子的Motif information。
If both ATAC-seq data and scRNA-seq data are used to perform IReNA, the following files are needed: (1) Bam, peak and footprint files of ATAC-seq data (2) Raw counts of scRNA-seq or bulk RNA-seq data (3) Reference genome of the species (5) Motif information of transcription factors.
- (1) Bam, peak and footprint files of ATAC-seq data
- (2) Seurat object and monocle object / Raw counts of scRNA-seq or bulk RNA-seq data
对于分析过seurat对象和monocle对象的文件，IReNA提供了将monocle对象的伪时间添加到seurat对象的meta数据中的功能（该功能仅支持monocle2的monocle对象）。 然后，继续下一步。
###Add pseudotime to the Seurat object
seurat_with_time <- add_pseudotime(seurat_object, monocle_object)
对于只有raw counts的分析文件，IReNA 提供了“load_counts”函数来加载 scRNA-seq 数据的raw counts，并返回 seurat 对象。 如果数据是 10X 格式，设置参数‘datatype = 0’。 如果数据为普通计数格式（'txt'为文件名后缀），则设置参数'dayatype =1'。
### load 10X counts
seurat_object <- load_counts('10X_data/sample1/', datatype = 0)
### load normal counts
seurat_object <- load_counts('test_data.txt',datatype = 1)
如果使用bulk RNA-seq 数据来识别基本的调控关系，只需加载bulk RNA-seq 数据的raw counts，然后使用与 scRNA-seq 数据相同的代码进行进一步分析。 建议只加载差异表达基因的表达谱，否则将花费更多的时间来计算每个基因对的相关性。 Deseq2 和 edgeR 可用于识别bulk RNA-seq 数据中差异表达的基因。
- (3) Reference genome of the species
- (4) GTF file
Gene transfer format, you can download it from http://www.ensembl.org/info/data/ftp/index.html - (5) Motif information of transcription factors
IReNA 包含源自 TRANSFAC 版本 201803 的四种物种（智人、Mus musculus、斑马鱼和鸡）的 DNA 模体数据集。以下代码用于从 TRANSFAC 或user定义的模体数据集中获取模体数据集，其格式应与这些相同 来自 TRANSFAC 数据库。
library(IReNA)
###call Mus musculus motif database
motif1 <- Tranfac201803_Mm_MotifTFsF
###call Homo sapiens motif database
motif1 <- Tranfac201803_Hs_MotifTFsF
###call Zebrafish motif database
motif1 <- Tranfac201803_Zf_MotifTFsF
###call Chicken motif database
motif1 <- Tranfac201803_Ch_MotifTFsF
以下内容包含4个部分
IReNA 包含四个主要部分来重建监管网络：
- 第 1 部分：分析 scRNA-seq 或bulk RNA-seq 数据以获得基本的调控关系
- 第 2 部分：使用 Fimo（或替代选项：RcisTarget）refine regulatory relaionships（无 ATAC-seq 数据）
- 第 3 部分：分析 ATAC-seq 数据以refine regulatory relationships（使用 ATAC-seq 数据）
- 第 4 部分：监管网络分析和可视化
Part 1: Analyze scRNA-seq or bulk RNA-seq data to get basic regulatory relationships
IReNA 支持三种输入格式：
- scRNA-seq 或bulk RNA-seq 数据的raw count。 raw count可以通过 IReNA 中的“load_counts”函数加载并转换为 Seurat 对象；
- 包含raw count的 Seurat 对象。 数据加载完毕后，IReNA会使用R包monocle计算pseudotime，并将pseudotime添加到Seurat对象的meta数据中。
- meta数据中带有伪时间的 Seurat 对象。
Parameter ‘gene.use’ in ‘get_pseudotime’ function indicate the genes use to calculate pseudotime, if it’s null, this function will automatically use variable genes calculated by ‘FindVariableFeatures’ function in Seurat package to calculate pseudotime.
###Read seurat_object
seurat_object <- readRDS('seurat_object.rds')
###calculate the pseudotime and return monocle object
monocle_object <- get_pseudotime(seurat_object,gene.use)
###Add pseudotime to the Seurat object
###This function only support monocle object from monocle2
seurat_with_time <- add_pseudotime(seurat_object, monocle_object)
Next, use differentially expressed genes (DEGs) across the pseudotime to refine the seurat object, if you already have identified DEGs, you just need to run subset function in seurat:
### DEGs used here is the character class
seurat_with_time <- subset(seurat_with_time, features = DEGs)
I also recommend ‘differentialGeneTest’ function in monocle to calculate DEGs across pseudotime. DEGs will be used to make expression profile in further analysis. (If you use our test data, you can skip this part, because our test data only contains differentially expressed genes)
### identify DEGs across pseudotime (qvalue < 0.05 and num_cells_expressed > 0.1)
library(monocle)
monocle_object <- detectGenes(monocle_object, min_expr = 3)
monocle_object <- estimateDispersions(monocle_object)
diff1 <- monocle::differentialGeneTest(mo,fullModelFormulaStr = "~Pseudotime",relative_expr = TRUE)
sig_genes <- subset(diff1, qval < 0.05)
sig_genes <- subset(sig_genes, num_cells_expressed > 0.1)
### Use DEGs to refine seurat object
seurat_with_time <- subset(seurat_with_time, features = rownames(sig_genes))
Then, cells are divided into 50 bins across pseudotime. The bin is removed if all genes in this bin have no expression. The gene is filtered if absolute fold change < 0.01 (set by the parameter ‘FC’). Then, genes will be clustered through K-means algorithm (the number of clusters ‘K’ is set by the parameter ‘K1’).
If bulk RNA-seq data are used to identify regulatory relationships, load your expression matrix as expression_profile that generated by get_SmoothByBin_PseudotimeExp(), then run the following codes. I suggest to input expression profile that only contains differentially expressed genes, or you will a huge of time to calculate correlation of each gene pair.
###Get expression profiles ordered by pseudotime
expression_profile <- get_SmoothByBin_PseudotimeExp(seurat_with_time, Bin = 50)
###Filter noise and logFC in expression profile
expression_profile_filter <- fileter_expression_profile(expression_profile, FC=0.01)
###K-means clustering
clustering <- clustering_Kmeans(expression_profile_filter, K1=4)
clustering[1:5,1:5]
## KmeansGroup FoldChangeQ95 SmExp1 SmExp2 SmExp3
## TCEB3 1 2.395806 -0.2424532 -0.8964990 -0.9124960
## CLK1 1 2.508335 -0.1819044 0.7624798 0.4867972
## MATR3 1 2.700294 -1.4485729 0.7837425 0.3028892
## AKAP11 1 2.415084 -0.6120681 -0.3849580 0.3898393
## HSF2 1 2.528111 -0.8125698 -0.6166004 0.8533309
Visualize the clustering of gene expression profiles through the heatmap
plot_kmeans_pheatmap(clustering,ModuleColor1 = c('#67C7C1','#5BA6DA','#FFBF0F','#C067A9'))
由于Kmeans算法的特点，每次聚类都会得到不同的结果。
在第一列中添加基因的Ensmble ID，然后计算每个基因对的相关性并选择包含至少一个转录因子且绝对相关性> 0.4（由参数'correlation_filter'设置）的基因对。 ‘correlation_filter’的值取决于数据的噪声，数据噪声越大，需要越大的‘correlation_filter’来获得可信的相关关系。 建议将 p 值 < 0.05 的相关性用于参数“correlation_filter”。
###Add Ensembl ID as the first column of clustering results
Kmeans_clustering_ENS <- add_ENSID(clustering, Spec1='Hs')
Kmeans_clustering_ENS[1:5,1:5]
## Symbol KmeansGroup FoldChangeQ95 SmExp1 SmExp2
## ENSG00000011007 TCEB3 1 2.395806 -0.2424532 -0.8964990
## ENSG00000013441 CLK1 1 2.508335 -0.1819044 0.7624798
## ENSG00000015479 MATR3 1 2.700294 -1.4485729 0.7837425
## ENSG00000023516 AKAP11 1 2.415084 -0.6120681 -0.3849580
## ENSG00000025156 HSF2 1 2.528111 -0.8125698 -0.6166004
### Caculate the correlation
motif1 <- Tranfac201803_Hs_MotifTFsF
### for
regulatory_relationships <- get_cor(Kmeans_clustering_ENS, motif = motif1, correlation_filter = 0.6, start_column = 4)
Part 2: Analyze binding motifs of target genes to refine regulatory relaionships (without ATAC-seq data)
For ATAC-seq data is not available. IReNA uses fimo to check whether motifs of transcription factors that regulate the target gene occur in the upstream of target genes. If motifs of transcription factors that regulate the target gene exist, this gene pair will be retained.
First, get TSS (transcription start site) regions (default is upstream 1000 to downstream 500) of target genes. Gtf file used here is available from http://www.ensembl.org/info/data/ftp/index.html
gtf <- read.delim("D:/Homo_sapiens.GRCh38.104.gtf", header=FALSE, comment.char="#")
gene_tss <- get_tss_region(gtf,rownames(Kmeans_clustering_ENS))
head(gene_tss)
## gene chr start end
## 1 ENSG00000237973 chr1 630074 631574
## 2 ENSG00000116285 chr1 8027309 8025809
## 3 ENSG00000162441 chr1 9944407 9942907
## 4 ENSG00000116273 chr1 6612731 6614231
## 5 ENSG00000177674 chr1 11735084 11736584
## 6 ENSG00000171608 chr1 9650731 9652231
Then, get the sequence of these TSS regions based on reference genome (reference genome can be download from UCSC database), and use fimo to scan these sequence to check whether the motif of transcription factor that regulates the target gene occur in the promoter regions of the target gene. Because fimo software only supports linux environment, we generate some shell script to run Fimo software.
应该设置以下四个参数：
- refdir：参考基因组路径
- fimodir：Fimo 软件的路径。 如果 Fimo 已经设置为全局环境变量，只需设置‘fimodir <- fimo’。
- outputdir1：shell脚本的输出路径和目标基因tss区域的序列。（函数'find_motifs_targetgenes'会在'outputdir1'路径下自动生成两个文件夹（'fasta'和'fimo'），并存储目标基因tss区域的序列 在“fimo”中的“fasta”和shell脚本中）
- Motifdir：motif 文件的路径，可从 https://github.com/jiang-junyao/MEMEmotif 或 TRANSFAC 数据库下载。
Please note that, at the end of ‘outputdir1’,‘motifdir’ and ‘sequencedir’, the symbol ‘/’should be contained. What’s more, the chromosome name of your reference genome used here should be the same as the chromosome name in the gene_tss
### run the following codes in the R under linux environment
refdir='/public/home/user/genome/hg38.fa'
fimodir <- 'fimo'
outputdir1 <- '/public/home/user/fimo/'
motifdir <- '/public/home/user/fimo/Mememotif/'
find_motifs_targetgenes(gene_tss,motif1,refdir,fimodir,outputdir1,motifdir)
Then run the following shell codes to activate fimo
### run the following codes in the shell
cd /public/home/user/fimo/fimo
sh fimoall.sh
Then, Fimo result are stored in the ‘fimo’ folder under outputdir1, and we run the following R codes to refine regulatory relationships
motif1 <- Tranfac201803_Hs_MotifTFsF
outputdir <- paste0(outputdir1,'fimo/')
fimo_regulation <- generate_fimo_regulation(outputdir,motif1)
filtered_regulatory_relationships <- filter_regulation_fimo(fimo_regulation, regulatory_relationships)
Part 3: Analyze ATAC-seq data to refine regulatory relationships (with ATAC-seq data)
For ATAC-seq data is available, IReNA calculates the footprints of transcription factors and footprint occupancy score (FOS) to refine regulatory relationships. The footprints whose distance is less than 4 are merged and then the sequence of each footprint is obtained from the reference genome through the function ‘get_merged_fasta’. The reference genome should be in fasta/fa format which can be downloaded from UCSC or other genome database.
###merge footprints whose distance is less than 4
filtered_footprints <- read.table('footprints.bed',sep = '\t')
fastadir <- 'Genome/hg38.fa'
merged_fasta <- get_merged_fasta(filtered_footprints,fastadir)
write.table(merged_fasta,'merged_footprints.fasta',row.names=F,quote=F,col.names=F)
After obtaining the motif sequences, use fimo software to identify binding motifs in the footprints. Because fimo software only supports linux environment, we generate a shell script to run Fimo software.
First, identify differentially expressed genes related motifs through the function ‘motif_select’, which will reduce the running time of the subsequent analysis process.
Then, you should set the following five parameters:
- fimodir: path of Fimo software. If Fimo has been set to the global environment variable, just set ‘fimodir <- fimo’.
- outputdir1: output path of the shell scripts.
- outputdir: output path of Fimo result.
- motifdir: path of the motif file, which can be downloaded from https://github.com/jiang-junyao/MEMEmotif or TRANSFAC database.
- sequencedir: path of the sequence which is generated by the function ‘get_merged_fasta’.
Please note that, at the end of ‘outputdir’ and ‘sequencedir’ the symbol ‘/’should be contained.
### Identify differentially expressed genes related motifs
motif1 <- motifs_select(Tranfac201803_Hs_MotifTFsF, rownames(Kmeans_clustering_ENS)) ###Kmeans_clustering_ENS was obtained in part1
### run find_motifs()
fimodir <- 'fimo'
outputdir1 <- '/public/home/user/fimo/'
outputdir <- '/public/home/user/fimo/output/'
motifdir <- '/public/home/user/fimo/Mememotif/'
sequencedir <- '/public/home/user/fimo/merged_footprints.fasta'
find_motifs(motif1,step=20,fimodir, outputdir1, outputdir, motifdir, sequencedir)
Then run the following shell codes to activate fimo
### run the following commands in the shell
cd /public/home/user/fimo/
mkdir output
sh ./fimo_all.sh
Then, we combine these Fimo consequence in ‘outputdir’. Notably outputdir folder should only contain fimo result files. Next, we load the peaks file and overlap differential peaks and motif footprints through overlap_footprints_peaks() function
###Combine all footprints of motifs
combined <- combine_footprints(outputdir)
peaks <- read.delim('differential_peaks.bed')
overlapped <- overlap_footprints_peaks(combined,peaks)
However, the running time of overlap_footprints() is too long, so it’s highly recommanded to use bedtools to do overlap in linux system. If you want to use bedtools to do overlap, you need to output ‘combined_footprints’ dataframe, and transfer it to shell.(If you make analysis in linux system, ignore transfer part)
### output combined_footprints
write.table(combined,'combined.txt',quote = F,row.names = F,col.names = F,sep = '\t')
### Transfer combied.txt and differential_peaks.bed to linux system, and then run the following commands in the shell
bedtools intersect -a combined.txt -b differential_peaks.bed -wa -wb > overlappd.txt
Next, we intergrate bioconductor package ChIPseeker to get footprint-related genes. Before we run get_related_genes(), we need to specify TxDb, which can be download from: Bioconductor TxDb list. Kmeans_clustering_ENS used here was obtained in part1.
### If you make overlap by bedtools, read 'overlapped.txt' to R
overlapped <- read.table('overlapped.txt')
###get footprint-related genes
library(TxDb.Hsapiens.UCSC.hg38.knownGene)
txdb <- TxDb.Hsapiens.UCSC.hg38.knownGene
list1 <- get_related_genes(overlapped,txdb = txdb,motif=Tranfac201803_Mm_MotifTFsF,Species = 'Hs')
###Get candidate genes/TFs-related peaks
list2 <- get_related_peaks(list1,Kmeans_clustering_ENS)
### output filtered footprints
write.table(list2[[1]],'filtered_footprints.bed', quote = F, row.names = F, col.names = F, sep = '\t')
Then, because of the size of original BAM file is too large, so we need to use samtools to extract footprints realated regions in BAM to reduce running time of function which analyze bam files in IReNA. (If you use our test data, just skip this step)
### transfer filtered_footprints.bed to linux system and run the following codes
samtools view -hb -L filtered_footprint.bed SSC_patient1.bam > SSC1_filter.bam
samtools view -hb -L filtered_footprint.bed SSC_patient2.bam > SSC2_filter.bam
samtools view -hb -L filtered_footprint.bed esc.bam > esc_filter.bam
此外，通过 wig_track() 计算足迹中每个位置的切割，并使用这些切割来计算足迹的 FOS，以识别决定监管关系的丰富 TF。 此处使用的regulatory_relationships 是在第1 部分中计算的。 这里使用的参数 FOS_threshold 是过滤低质量足迹的阈值，您可以增加它以减少导出足迹的数量。
### calculate cuts of each each position in footprints
bamfilepath1 <- 'SSC1_filter.bam'
bamfilepath2 <- 'SSC2_filter.bam'
bamfilepath3 <- 'esc_filter.bam'
cuts1 <- wig_track(bamfilepath = bamfilepath1,bedfile = list2[[1]])
cuts2 <- wig_track(bamfilepath = bamfilepath2,bedfile = list2[[1]])
cuts3 <- wig_track(bamfilepath = bamfilepath3,bedfile = list2[[1]])
wig_list <- list(cuts1,cuts2,cuts3)
### get related genes of footprints with high FOS
potential_regulation <- Footprints_FOS(wig_list,list2[[2]], FOS_threshold = 0.1)
### Use information of footprints with high FOS to refine regulatory relationships
filtered_regulatory <- filter_ATAC(potential_regulation,regulatory_relationships)
Part 4: Regulatory network analysis and visualization
After we get ‘filtered_regulatory_relationships’ and ‘Kmeans_clustering_ENS’, we can reconstruct regulatory network. Run network_analysis() to get regulatory, this function will generate a list which contain the following 9 elements:
- (1)Cor_TFs.txt: list of expressed TFs in the gene networks.
- (2)Cor_EnTFs.txt: list of TFs which significantly regulate gene modules (or enriched TFs).
- (3)FOSF_RegMTF_Cor_EnTFs.txt: regulatory pairs in which the source gene is enriched TF.
- (4)FOSF_RegMTF_Cor_EnTFs.txt: regulatory pairs in which both source gene and target gene are enriched TFs.
- (5)FOSF_RegMTF_Cor_EnTFs.txt: regulatory pairs only including regulations within each module but not those between modules, in this step
- (6)TF_list: enriched TFs which significantly regulate gene modules
- (7)TF_module_regulation: details of enriched TFs which significantly regulate gene modules
- (8)TF_network: regulatory network for enriched transcription factors of each module
- (9)intramodular_network: intramodular regulatory network
Here, we use refined regulatory relationships from part2 to reconstruct regulatory networks
TFs_list <- network_analysis(filtered_regulatory_relationships,Kmeans_clustering_ENS,TFFDR1 = 10,TFFDR2 = 10)
We can also make enrichment analysis for differentially expressed genes in each module. Before you run this function, you need to download the org.db for your species through BiocManager.
### Download Homo sapiens org.db
#BiocManger::install('org.Hs.eg.db')
library(org.Hs.eg.db)
### Enrichment analysis (KEGG)
enrichment_KEGG <- enrich_module(Kmeans_clustering_ENS, org.Hs.eg.db, enrich.db = 'KEGG',organism = 'hsa')
#enrichment_GO <- enrich_module(Kmeans_cluster_ENS, org.Hs.eg.db, 'GO')
head(enrichment_KEGG)
## ID Description module -log10(q-value) GeneRatio
## hsa03010 hsa03010 Ribosome 1 6.500237 13/101
## hsa03040 hsa03040 Spliceosome 1 1.964315 9/101
## hsa03022 hsa03022 Basal transcription factors 1 1.964315 5/101
## hsa05016 hsa05016 Huntington's disease 1 1.126355 9/101
## hsa03013 hsa03013 RNA transport 1 1.126355 8/101
## hsa05200 hsa05200 Pathways in cancer 2 4.866522 38/276
## BgRatio pvalue p.adjust qvalue
## hsa03010 92/5894 3.661614e-09 3.258836e-07 3.160551e-07
## hsa03040 128/5894 3.138210e-04 1.119399e-02 1.085638e-02
## hsa03022 37/5894 3.773255e-04 1.119399e-02 1.085638e-02
## hsa05016 183/5894 3.932733e-03 7.708051e-02 7.475579e-02
## hsa03013 152/5894 4.330366e-03 7.708051e-02 7.475579e-02
## hsa05200 327/5894 1.153409e-07 1.949262e-05 1.359809e-05
## geneID
## hsa03010 6122/6143/6206/6194/51187/6135/6161/6167/6159/6166/9045/6136/6191
## hsa03040 10992/3312/9775/10569/83443/5356/10594/8449/494115
## hsa03022 9519/2962/6877/2957/6879
## hsa05016 9519/5978/7019/51164/498/27089/54205/4512/2876
## hsa03013 11218/8761/1983/9775/5042/50628/6612/10921
## hsa05200 8312/2263/2260/6932/7188/3912/6655/331/5595/7976/5296/1021/7473/4790/329/1027/2335/6772/6654/5290/5335/2258/3845/5156/54583/5899/3688/26060/3716/836/6774/8313/5293/3913/6777/5727/5154/112398
## Count
## hsa03010 13
## hsa03040 9
## hsa03022 5
## hsa05016 9
## hsa03013 8
## hsa05200 38
Moreover, you can do GO analysis
library(org.Hs.eg.db)
### Enrichment analysis (GO)
enrichment_GO <- enrich_module(Kmeans_clustering_ENS, enrich.db = 'GO',org.Hs.eg.db)
You can visualize regulatory networks for enriched transcription factors of each module through plot_network() function by setting type parameter as ‘TF’. This plot shows regulatory relationships between transcription factors in different modules that significantly regulating other modules. The size of each vertex determine the significance of this transcription factor. Yellow edges are positive regulation, grey edges are negative regulation.
plot_tf_network(TFs_list)
可以通过 plot_intramodular_network() 函数可视化具有丰富功能的模块内网络。 在运行此功能之前，您可以选择要在图中显示的每个模块的丰富功能。 如果输入所有丰富的函数，该函数将自动选择每个模块中-log10(qvalue) 最高的函数呈现在图中。 此外，每个模块中边数最多的转录因子也将显示在图中。
### select functions that you want to present in the figure
enrichment_KEGG_select <- enrichment_KEGG[c(3,7,11),]
### plotting
plot_intramodular_network(TFs_list,enrichment_KEGG_select,layout = 'circle')
It is strongly recommended to use Cytoscape to display the regulatory networks. We provide a function that can provide different Cytoscape styles. You need to install and open Cytoscape before running the function.
###optional: display the network in cytoscape, open cytoscape before running this function
initiate_cy(TFs_list, layout1='degree-circle', type='TF')
initiate_cy(TFs_list, layout1='grid', type='module')
These are the picture we processed through Cytoscape, which can show the regulatory relationship of modularized transcription factors.
Use Cytoscape to visualize regulatory network for enriched transcription factors of each module
Use Cytoscape to visualize intramodular network
Option: Use RcisTarget to refine regulatory relaionships (without ATAC-seq data)
IReNA also provides filter_regulation function (Based on RcisTarget) to refine regulation relationships. Due to the limitations of RcisTarget, this function currently only supports three species (Hs, Mm and Fly). So if the species of your data is not included, and you don’t have ATAC-seq data, you can use unrefined regulatory relaionships to perform part4 analysis directly.
Before run this function, you need to download Gene-motif rankings database from https://resources.aertslab.org/cistarget/ and set the Rankingspath1 as the path of the downloaded Gene-motif rankings database. If you don’t know which database to choose, we suggest that using ‘hg19-500bp-upstream-7species.mc9nr’ for human, using ‘mm9-500bp-upstream-10species.mc9nr’ for mouse, using ‘dm6-5kb-upstream-full-tx-11species.mc8nr’ for fly. You can download it manually, or use R code (If you are in mainland china, i suggest you to download database from website):
### Download Gene-motif rankings database
featherURL <- "https://resources.aertslab.org/cistarget/databases/homo_sapiens/hg19/refseq_r45/mc9nr/gene_based/hg19-tss-centered-10kb-7species.mc9nr.feather"
download.file(featherURL, destfile=basename(featherURL)) # saved in current dir
### Refine regulatory relaionships
Rankingspath1 <- 'hg19-500bp-upstream-7species.mc9nr1.feather' # download from https://resources.aertslab.org/cistarget/
filtered_regulation_Rcis <- filter_regulation(Kmeans_clustering_ENS, regulatory_relationships, 'Hs', Rankingspath1)
生活很好，有你更好
