User Guide for APEC (v1.1.0)
(Accessibility Pattern based Epigenomic Clustering)
APEC can perform fine cell type clustering on single cell chromatin accessibility data from scATAC-seq, snATAC-seq, sciATAC-seq or any other relevant experiment. It can also be used to evaluate gene score from relevant accesson, search for differential motifs/genes for each cell cluster, find super enhancers, and construct pseudo-time trajectory (by calling Monocle). If users have already obtained the fragment-count-per-peak matrix from other mapping pipelines (such as CellRanger), please run APEC from the first Section "Run APEC from fragment count matrix". If users have only the raw fastq files, please jump to the second Section "Get fragment count matrix from raw data".
Run AEPC from fragment count matrix
1. Requirements and installation
1.1 Requirements
APEC requires Linux system (CentOS 7.3+ or Ubuntu 16.04+), as well as Python (2.7.15+ or 3.6.8+). If users want to build pseudotime trajectory with APEC, please install R (3.4.0+) environment and monocle (2.8.0). Also, the following software are required for APEC:
Bedtools: http://bedtools.readthedocs.io/en/latest/content/installation.html
Meme 4.11.2: http://meme-suite.org/doc/download.html?man_type=web
Homer: http://homer.ucsd.edu/homer/
notes: Users need to download genome reference for Homer by "perl /path-to-homer/configureHomer.pl -install hg19" and "perl /path-to-homer/configureHomer.pl -install mm10".
The files in reference folder are required for APEC. But we didn't upload reference files to GitHub since they are too big. Users can download all reference files from http://galaxy.ustc.edu.cn:30803/APEC/. The reference folder should contains the following files:
hg19_RefSeq_genes.gtf, hg19_chr.fa, hg19_chr.fa.fai,
mm10_RefSeq_genes.gtf, mm10_chr.fa, mm10_chr.fa.fai,
JASPAR2018_CORE_vertebrates_non-redundant_pfms_meme.txt, tier1_markov1.norc.txt
1.2 Install and import APEC
Users can install APEC by:
pip install APEC
Due to the compatibility problem (especially for rpy2), we don't recommend conda environment. Users can use pyenv to build a sub environment for APEC.
In Ipython, Jupyter-notebook or a python script, users can import packages of APEC by:
from APEC import clustering,plot,generate
Users can inquire the manual for each function of APEC by using "help()" in Ipython or Jupyter, for example:
help(clustering.cluster_byAccesson)
2. Input data
Users need to prepare a project folder (termed '$project'), which contains matrix, peak, result and figure folders. Please place "filtered_cells.csv" and "filtered_reads.mtx" in matrix folder, "top_filtered_peaks.bed" in peak folder. Here is the instruction for three input files:
filtered_cells.csv: Two-column (separated by tabs) list of cell information ('name' and 'notes'): the 'name' column stores cell names (or barcodes), the 'notes' column can be cell-type, development stage, batch index or any other cell information (or empty), such as:
name notes
CD4-001 CD4
CD4-002 CD4
CD8-001 CD8
CD8-002 CD8
top_filtered_peaks.bed: Three-column list of peaks, which is a standard bed format file.
It is similar to the "peaks.bed" file in the CellRanger output of a 10X scATAC-seq dataset.
filtered_reads.mtx: Fragment count matrix in mtx format, where each row is a peak and each column represents a cell.
It is similar to the "matrix.mtx" file in the CellRanger output of a 10X scATAC-seq dataset.
The order of cells should be same with "filtered_cells.csv", and the order of peaks should be
same with "filtered_top_peaks.bed".
3. Functions of APEC (step by step)
3.1 Clustering by APEC
Use the following codes to cluster cells by APEC algorithm:
clustering.build_accesson('$project', ngroup=600)
clustering.cluster_byAccesson('$project', nc=0, norm='zscore', filter='yes')
input parameters:
ngroup: Number of accessons, default=600.
nc: Number of cell clusters, set it to 0 if users want to predict cluster number by Louvain algorithm, default=0.
norm: Normalization method for accesson matrix, can be 'zscore' or 'probability', default='zscore'.
filter: Filter high dispersion accessons or not, can be 'yes' or 'no', default='yes'.
For large datasets (cell number over 10000), users should set filter='no'.
output files:
$project/matrix/Accesson_peaks.csv
$project/matrix/Accesson_reads.csv
$project/result/louvain_cluster_by_APEC.csv
Then users can plot tSNE, UMAP or corrlation heatmap for cells:
plot.plot_tsne('$project', matrix_type='APEC', cell_label='notes', cluster='louvain')
plot.plot_umap('$project', matrix_type='APEC', cell_label='notes', cluster='louvain')
plot.correlation('$project', matrix_type='APEC', cell_label='notes', cluster='louvain')
input parameters:
matrix_type: Type of input matrix, can be 'APEC' or 'chromVAR', default='APEC'.
If matrix_type='APEC', it will use accesson matrix yielded by clustering.cluster_byAccesson();
if matrix_type='chromVAR', it will use deviation matrix yielded by clustering.cluster_byMotif().
cell_label: Color labels for cells, can be 'notes' or 'cluster', default='notes'.
cluster: Clustering algorithm used in clustering.cluster_byXXX(), default='louvain'.
output files:
$project/result/TSNE_by_APEC.csv
$project/figure/TSNE_by_APEC_with_notes_label.pdf
$project/result/UMAP_by_APEC.csv
$project/figure/UMAP_by_APEC_with_notes_label.pdf
$project/figure/cell_cell_correlation_by_APEC_with_louvain_clustering.png
3.2 Clustering by chromVAR
Use the following codes to cluster cells by chromVAR algorithm:
generate.motif_matrix('$project', genome_fa='$reference/hg19_chr.fa',
background='$reference/tier1_markov1.norc.txt',
meme='$reference/JASPAR2018_CORE_vertebrates_redundant_pfms_meme.txt',
np=4)
clustering.cluster_byMotif('$project', nc=0, ns=50, np=4)
input parameters:
genome_fa: Path to hg19_chr.fa or mm10_chr.fa in $reference folder.
background: Path to tier1_markov1.norc.txt in $reference folder.
meme: Path to JASPAR2018_CORE_vertebrates_redundant_pfms_meme.txt in $reference folder.
np: Number of CPU cores used for parallel calculation, default=4.
nc: Number of cell clusters, set it to 0 if users want to predict cluster number by Louvain algorithm, default=0.
ns: Number of permuted sampling, default=50.
output files:
$project/matrix/Accesson_peaks.csv
$project/matrix/Accesson_reads.csv
$project/result/louvain_cluster_by_APEC.csv
3.3 Evaluate ARI, NMI and AMI for clustering result
If users have the real cell type in the 'notes' column of '$project/matrix/filtered_cells.csv', please use the following code to calculate ARI, NMI and AMI to estimate the accuracy of the clustering algorithm.
clustering.cluster_comparison('$project/matrix/filtered_cells.csv',
'$project/result/louvain_cluster_by_APEC.csv')
The output ARI, NMI and AMI values will present on the screen directly.
3.4 Generate pseudotime trajectory
generate.monocle_trajectory('$project', npc=5)
plot.plot_trajectory('$project', cell_label='notes', cluster='louvain', angle=[30,30])
input parameters:
npc: Number of principal components used to build trajectory, default=5.
cell_label: Color labels for cells, can be 'notes' or 'cluster', default='notes'.
cluster: Clustering algorithm used in clustering.cluster_byXXX(), default='louvain'.
angles: Rotation angles for 3D trajectory, e.g. [100,20], default=[30,30].
output files:
$project/result/monocle_trajectory.csv
$project/result/monocle_reduced_dimension.csv
$project/figure/pseudotime_trajectory_with_notes_label.pdf
3.5 Generate gene scores
generate.gene_score('$project', genome='hg19', width=1000000, pvalue=0.01):
input parameters:
genome: Genome reference for Homer, can be "hg19" or "mm10", default="hg19".
width: Width of Genome region for fisher exact test, default=1000000.
pvalue: P-value threshold for fisher exact test, default=0.01.
output files:
$project/matrix/Accesson_annotated.csv
$project/matrix/gene_annotated.csv
$project/matrix/gene_score.csv
3.6 Generate differential feature for a cell cluster
generate.differential_feature('$project', feature='motif', target='0', vs='all')
generate.differential_feature('$project', feature='gene', target='0', vs='all')
input parameters:
feature: Type of feature, can be 'accesson' or 'motif' or 'gene', default='accesson'.
If feature='accesson', run clustering.cluster_byAccesson() first;
if feature='motif', run clustering.cluster_byMotif() first;
if feature='gene', run generate.gene_score() first.
cell_label: Cell labels used for differential analysis, can be 'notes' or 'cluster', default='cluster'.
cluster: Clustering algorithm used in clustering.cluster_byXXX(), default='louvain'.
target: The target cluster that users search for differential features, default='1'.
If cell_label='cluster', target is one element in the 'cluster' column of XXX_cluster_by_XXX.csv file;
if cell_label='notes', target is one element in the 'notes' column of filtered_cells.csv file.
vs: Versus which clusters, can be '2,3,4' or 'all', default='all' (means all the rest clusters).
pvalue: P-value for student-t test, default=0.01.
log2_fold: Cutoff for log2(fold_change), default=1.
The differential motifs/genes of cell cluster '0' will presents on the screen directly. Notes: Differential motif search requires the running of clustering.cluster_byMotif() beforehand (see 3.2), and differential gene search requires the running of generate.gene_score() beforehand (see 3.5).
3.7 Plot motif/gene on tSNE/trajectory diagram
plot.plot_feature('$project', space='tsne', feature='gene', name='FOXO1')
plot.plot_feature('$project', space='trajectory', feature='motif', name='GATA1')
input parameters:
space: In which space we draw the feature, can be 'tsne' or 'umap' or 'trajectory', default='tsne'.
If space='tsne', run plot.plot_tsne() first;
if space='umap', run plot.plot_umap() first;
if space='trajectory', run generate.monocle_trajectory() first.
feature: Type of the feature, can be 'accesson' or 'motif' or 'gene', default='accesson'.
If feature='accesson', run clustering.cluster_byAccesson() first;
if feature='motif', run clustering.cluster_byMotif() first;
if feature='gene', run generate.gene_score() first.
matrix_type: Type of input matrix for tSNE/UMAP, can be 'APEC' or 'chromVAR', default='APEC'.
If matrix_type='APEC', it will use tSNE/UMAP result of APEC;
if matrix_type='chromVAR', it will use tSNE/UMAP result of chromVAR.
name: Name of the feature.
If feature='accesson', name=accesson number, i.e. '1';
if feature='motif', name=motif symbol, i.e. 'GATA1';
if feature='gene', name=gene symbol, i.e. 'CD36'.
clip: Clip range for the input matrix, can be [min, max] or 'none', default='none'.
angles: Rotation angles for 3D trajectory, e.g. [100,20], default=[30,30].
output files:
$project/figure/gene_FOXO1_on_tsne_by_APEC.pdf
$project/figure/motif_GATA1_on_trajectory_by_APEC.pdf
Notes: Plotting feature on tSNE diagram requires the running of plot.plot_tsne() beforehand (see 3.1), and plotting feature on trajectory requires the running of generate.monocle_trajectory() beforehand (see 3.4).
3.8 Generate potential super enhancer
generate.search_super_enhancer('$project', super_range=1000000)
input parameter:
super_range: Genome range to search for super enhancer, default=1000000.
output file:
$project/result/potential_super_enhancer.csv
Get fragment count matrix from raw data
(this part is only available on GitHub:https://github.com/QuKunLab/APEC)
1. Requirements and installation
All of the following software needs to be placed in the global environment of the Linux system to ensure that they can be called in any path/folder. Picard is also required, but we have placed it into $APEC/reference folder, and users don't need to install it. We recommend that users adopt the latest version of these software, except Meme (version 4.11.2).
Bowtie2: https://sourceforge.net/projects/bowtie-bio/files/bowtie2/2.2.9/
Samtools: https://github.com/samtools/samtools
Bedtools: http://bedtools.readthedocs.io/en/latest/content/installation.html
Macs2: https://github.com/taoliu/MACS.git
Meme 4.11.2: http://meme-suite.org/doc/download.html?man_type=web
pysam for python: set up by "pip install pysam"
1.2 Installation
Users can simply install this part by copying the code_v1.1.0 folder and reference folder into a same path. Users must run APEC_prepare_steps.sh directly in code_v1.1.0/, since each program will invoke the reference files automatically. The reference folder is required, but we didn't upload reference files to GitHub since they are too big. Users can download all reference files from http://galaxy.ustc.edu.cn:30803/APEC/. The reference folder should contains the following files:
hg19_refseq_genes_TSS.txt, hg19_RefSeq_genes.gtf, hg19_blacklist.JDB.bed,
hg19_chr.fa, hg19_chr.fa.fai, hg19.chrom.sizes,
hg19.1.bt2, hg19.2.bt2, hg19.3.bt2, hg19.4.bt2, hg19.rev.1.bt2, hg19.rev.2.bt2,
mm10_refseq_genes_TSS.txt, mm10_RefSeq_genes.gtf, mm10_blacklist.BIN.bed,
mm10_chr.fa, mm10_chr.fa.fai, mm10.chrom.sizes,
mm10.1.bt2, mm10.2.bt2, mm10.3.bt2, mm10.4.bt2, mm10.rev.1.bt2, mm10.rev.2.bt2,
JASPAR2018_CORE_vertebrates_non-redundant_pfms_meme.txt, tier1_markov1.norc.txt, picard.jar
2. Fragment count matrix
2.1 Arrangement of raw data
The raw_data folder should contain all raw sequencing fastq files into the. All these pair-end fastq files should be named as:
type1-001_1.fastq, type1-001_2.fastq, type1-002_1.fastq, type1-002_2.fastq, ……;
type2-001_1.fastq, type2-001_2.fastq, type2-002_1.fastq, type2-002_2.fastq, ……;
……
where "_1" and "_2" indicate forward and backward reads for pair-end sequencing. {type1, type2, ...} can be cell-types or batches of samples, such as {GM, K562, ...}, or {batch1, batch2, ...}, or any other words without underline "_" or dash "-". Users need to build a project folder to store the result. The work, matrix, peak and figure folders will be automatically built by subsequent steps, and placed in project folder.
2.2 Easy-run of matrix preparation
Users can use the script APEC_prepare_steps.sh to finish the process from raw data to fragment count matrix. This script includes steps of "trimming", "mapping", "peak calling", "aligning read counts matrix", "quality contral", "estimating gene score". Running this step on our example project (i.e. project01 with 672 cells) will take 10~20 hours on an 8-core 32 GB computer, since the sequence mapping step is the slowest step.
Example:
bash APEC_prepare_steps.sh -r $raw_data -s $project -g hg19 -n 4 -l 3 -p 0.2 -f 2000
Input parameters:
-r: The raw_data folder
-s: The project folder.
-g: "hg19" or "mm10".
-n: Number of CPU cores.
-l: Threshold for the –log(Q-value) of peaks, used to filter peaks.
-p: Threshold of the percentage of fragments in peaks, used to filter cells.
-f: Threshold of the fragment number of each cell, used to filter cells.
Output files:
The script APEC_prepare_steps.sh will generate work, peak, matrix, and figure folders with many output files. Here, we only introduce files that are useful to users. For our example projects, all of these results can be reproduced on a general computer system.
(1) In work folder:
For each cell, the mapping step can generate a subfolder (with cell name) in the work folder. There are several useful files in each subfolder:
cell_name.hist.pdf: A histogram of fragment length distribution of each cell.
cell_name.RefSeqTSS.pdf: Insert enrichment around TSS regions of each cell.
(2) In peak folder:
mergeAll.hist.pdf: A histogram of fragment length distribution of all cells.
mergeAll.RefSeqTSS.pdf: Insert enrichment around TSS regions of all cells.
top_filtered_peaks.bed: Filtered top peaks, ranked by Q-value.
genes_scored_by_peaks.csv: Gene scores evaluated by TSS peaks.
(3) In matrix folder:
reads.csv: Fragment count matrix.
cell_info.merged.csv: Data quality report of each cell.
filtered_cells.csv: Filtered cells information in csv format.
filtered_reads.mtx: Filtered fragment count matrix in mtx format.
(4) In figure folder:
cell_quality.pdf: A scatter plot of the fragment number and the percentage of fragments in peaks.