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| File | Version | Author | Date | Message |
|---|---|---|---|---|
| Rmd | 0c32e6a | kevinlkx | 2021-01-06 | wflow_publish("analysis/process_data_Buenrostro2018.Rmd") |
| html | 04cb767 | kevinlkx | 2021-01-06 | Build site. |
| Rmd | 97c421d | kevinlkx | 2021-01-06 | put all four data processing options together |
| html | 694fd3b | kevinlkx | 2020-11-19 | Build site. |
| Rmd | 4b7d5fa | kevinlkx | 2020-11-19 | process data with peaks in less than 1% samples filtered out |
| html | d5b411d | kevinlkx | 2020-11-18 | Build site. |
| Rmd | ee608fd | kevinlkx | 2020-11-18 | process counts for aggregated single-cell peaks using chromVAR |
| html | 594fd86 | kevinlkx | 2020-11-10 | Build site. |
| Rmd | 614814b | kevinlkx | 2020-11-10 | process Buenrostro2018 data using Downloaded scATAC-seq processed data from GEO |
| Rmd | c113844 | kevinlkx | 2020-11-10 | wflow_rename("analysis/process_data_Buenrostro2018.Rmd", "analysis/process_data_Buenrostro2018_Chen2019pipeline.Rmd") |
| html | c113844 | kevinlkx | 2020-11-10 | wflow_rename("analysis/process_data_Buenrostro2018.Rmd", "analysis/process_data_Buenrostro2018_Chen2019pipeline.Rmd") |
| html | 1905c58 | kevinlkx | 2020-11-05 | Build site. |
| Rmd | 1da32f8 | kevinlkx | 2020-11-05 | set binarized counts as sparse matrix |
| html | 89b45be | kevinlkx | 2020-11-04 | Build site. |
| Rmd | 857e1e8 | kevinlkx | 2020-11-04 | process data from Buenrostro 2018 paper |
| html | 907fa65 | kevinlkx | 2020-11-04 | Build site. |
| Rmd | 12bf4b3 | kevinlkx | 2020-11-04 | process data from Buenrostro 2018 paper |
Reference: Buenrostro, J. D. et al. Integrated Single-Cell Analysis Maps the Continuous Regulatory Landscape of Human Hematopoietic Differentiation. Cell 173, 1535–1548.e16 (2018).
Data were downloaded from GEO: GSE96772 More details about the samples: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSM2540299 RCC directory: /project2/mstephens/kevinluo/scATACseq-topics/data/Buenrostro_2018/
Here we tried four different options to process the scATAC-seq data.
In option 1, we used the processed counts and peaks from Buenrostro et al (2018) paper. However, the ATAC-seq peaks were defined using the bulk ATAC-seq samples. This may not be ideal as this approach may miss cell-type specific peaks that do not pass the peak calling threshold using all the bulk ATAC-seq samples. Thus, we did not use this option in our topic modeling of the dataset.
In option 2-4, we obtained the processed counts using the BAM reads and peaks from aggregated single cell ATAC-seq data. The processed peaks and BAM files were downloaded from scATAC-benchmarking website from Chen et al
Reference: Chen, H. et al. Assessment of computational methods for the analysis of single-cell ATAC-seq data. Genome Biol. 20, 241 (2019).
In option 2, we used the read count processing pipeline from Chen et al (2019), which uses bedtools to count reads in peaks. But it seems they did not consider the paired-end reads from the ATAC-seq data, i.e. they treated the paired-end reads as independent reads. This would bias the actual read counts, but should not affect the binarized counts (which we used in our topic modeling). The count processing pipeline from Chen et al (2019) also filtered peaks by requring peaks to be accessbile in at least 1% samples.
In option 3 and 4, we used chromVAR to count the reads in peaks, which considers the paired-end reads. In option 4, we further filtered the peaks by requring peaks to be accessbile in at least 1% samples. This filtering was also done using chormVAR.
The Buenrostro et al (2018) paper called ATAC-seq peaks from the bulk ATAC-seq samples.
Using the previously described approach (Corces et al., 2016), we defined a peak list using all bulk hematopoietic data analyzed here, resulting in 491,437 500bp non-overlapping peaks which we use for the remainder of this study.
Downloaded scATAC-seq processed data from GEO: GSE96769
GSE96769_PeakFile_20160207.bed.gzGSE96769_scATACseq_counts.txt.gzlibrary(Matrix)
library(tools)
library(readr)
library(data.table)Load the fragment counts as a 2,953 x 491,437 sparse matrix.
# The first row has the sample names
file_counts <- "/project2/mstephens/kevinluo/scATACseq-topics/data/Buenrostro_2018/GEO_data/GSE96769_scATACseq_counts.txt.gz"
sample_names <- readLines(file_counts,n = 1)
sample_names <- unlist(strsplit(sample_names,"\t",fixed = TRUE))
sample_names <- unlist(strsplit(sample_names,";",fixed = TRUE))
sample_names <- sample_names[-1]
# Load the fragment counts as sparse matrix.
dat <- fread(file_counts,sep = "\t",skip = 1)
class(dat) <- "data.frame"
names(dat) <- c("i","j","x")
counts <- sparseMatrix(i = dat$i,j = dat$j,x = dat$x)
counts <- t(counts)
rownames(counts) <- sample_names
dim(counts)peaks (from bulk ATAC-seq)
peaks <- fread("/project2/mstephens/kevinluo/scATACseq-topics/data/Buenrostro_2018/GEO_data/GSE96769_PeakFile_20160207.bed.gz")
peak_names <- paste(peaks$V1,peaks$V2,peaks$V3,sep = "_")
rownames(peaks) <- peak_names
cat(sprintf("Number of peaks: %d\n",nrow(peaks)))
colnames(counts) <- peak_namesThe supplementary Table S1 provides more details about these samples. https://ars.els-cdn.com/content/image/1-s2.0-S009286741830446X-mmc1.xlsx
Select the 2,034 samples (cells) passing quality filtering
Single-cell profiles were of consistent high-quality with 2,034 cells passing stringent quality filtering, yielding a median of 8,268 fragments per cell with 76% of those fragments mapping to peaks, resulting in a median of 6,442 fragments in peaks per cell (figure 1E).
Single-cells were filtered for quality requiring at least 60% of fragments in peaks and requiring greater than 1,000 fragments passing quality filters, quality filters are previously described (Corces et al., 2016) which includes removal of mitochondrial reads and low alignment quality (Q30).
The 2,034 samples (pass filter) were included in the metadata.tsv file on the scATAC-benchmarking website from Chen et al.
samples_filtered <- read.table('/project2/mstephens/kevinluo/scATACseq-topics/data/Buenrostro_2018/data/input_Chen_2019/metadata.tsv', header = TRUE, stringsAsFactors=FALSE, quote="",row.names=1)
samples_filtered$name <- rownames(samples_filtered)
idx_samples_filtered <- match(samples_filtered$name, sample_names)
sample_names_filtered <- sample_names[idx_samples_filtered]
counts <- counts[idx_samples_filtered,]
samples <- samples_filtered[,c("name", "label")]
dim(counts)Remove peaks not exist in any of the cells
j <- which(colSums(counts > 0) >= 1)
peaks <- peaks[j,]
counts <- counts[,j]
cat(length(j), "peaks after filtering. \n")Binarize counts
binarized_counts <- as.matrix((counts > 0) + 0)
binarized_counts <- Matrix(binarized_counts, sparse = TRUE)
dim(binarized_counts)Save processed data
data.dir <- "/project2/mstephens/kevinluo/scATACseq-topics/data/Buenrostro_2018/processed_data/"
dir.create(data.dir, showWarnings = FALSE, recursive = TRUE)
saveRDS(counts, file.path(data.dir, "counts_Buenrostro_2018.rds"))
saveRDS(binarized_counts, file.path(data.dir, "binarized_counts_Buenrostro_2018.rds"))
save(list = c("samples", "peaks", "counts"),
file = file.path(data.dir, "Buenrostro_2018.RData"))
counts <- binarized_counts
save(list = c("samples", "peaks", "counts"),
file = file.path(data.dir, "Buenrostro_2018_binarized.RData"))data.dir <- "/project2/mstephens/kevinluo/scATACseq-topics/data/Buenrostro_2018/processed_data/"
load(file.path(data.dir, "Buenrostro_2018.RData"))
cat(sprintf("Loaded %d x %d counts matrix.\n",nrow(counts),ncol(counts)))
cat(sprintf("Number of samples (cells): %d\n",nrow(counts)))
cat(sprintf("Number of peaks: %d\n",ncol(counts)))
cat(sprintf("Proportion of counts that are non-zero: %0.1f%%.\n",
100*mean(counts > 0)))Processed peaks (from aggregated single cell ATAC-seq data) and BAM files were downloaded from the scATAC-benchmarking website (Chen et al 2019) (https://github.com/pinellolab/scATAC-benchmarking/)
Process counts using Chen et al pipeline
library(Matrix)
library(tools)
library(readr)
library(data.table)count scATAC-seq reads in peaks
sbatch ~/projects/scATACseq-topics/scripts/count_reads_peaks_Buenrostro_2018.sbatchcombine counts from all samples
dir_readcount_output <- '/project2/mstephens/kevinluo/scATACseq-topics/data/Buenrostro_2018/data/count_reads_peaks_output/'
files <- list.files(dir_readcount_output, pattern = "\\.txt$")
sample_names <- sapply(strsplit(files,'\\.'),'[', 1)
cat(length(sample_names), "samples. \n")datalist <- lapply(files, function(x)fread(file.path(dir_readcount_output,x))$V4)
counts <- do.call("cbind", datalist)data.dir <- "/project2/mstephens/kevinluo/scATACseq-topics/data/Buenrostro_2018/processed_data/"
dir.create(data.dir, showWarnings = FALSE, recursive = TRUE)
# saveRDS(counts, file.path(data.dir, "raw_counts_Buenrostro_2018.rds"))
counts <- readRDS(file.path(data.dir, "raw_counts_Buenrostro_2018.rds"))Peaks
peaks <- read.csv("/project2/mstephens/kevinluo/scATACseq-topics/data/Buenrostro_2018/data/input_Chen_2019/combined.sorted.merged.bed",
sep = '\t',header=FALSE,stringsAsFactors=FALSE)
peaknames <- paste(peaks$V1,peaks$V2,peaks$V3,sep = "_")
rownames(peaks) <- peaknames
cat(sprintf("Number of peaks: %d\n",nrow(peaks)))
head(peaknames)
colnames(counts) <- sample_names
rownames(counts) <- peaknames
dim(counts)Samples metadata
samples <- read.table('/project2/mstephens/kevinluo/scATACseq-topics/data/Buenrostro_2018/data/input_Chen_2019/metadata.tsv', header = TRUE, stringsAsFactors=FALSE, quote="",row.names=1)
head(samples)Filter peaks (filter out peaks with counts in < 1% samples)
# adapted from Chen et al. Genome Biology 2019 paper https://github.com/pinellolab/scATAC-benchmarking/blob/master/Real_Data/Buenrostro_2018/run_methods/Control/Control_buenrostro2018.ipynb
# filter peaks with counts in at least 1% samples
filter_peaks <- function (datafr,cutoff = 0.01){
binary_mat = as.matrix((datafr > 0) + 0)
binary_mat = Matrix(binary_mat, sparse = TRUE)
num_cells_ncounted = Matrix::rowSums(binary_mat)
ncounts = binary_mat[num_cells_ncounted >= dim(binary_mat)[2]*cutoff,]
ncounts = ncounts[rowSums(ncounts) > 0,]
options(repr.plot.width=4, repr.plot.height=4)
hist(log10(num_cells_ncounted),main="No. of Cells Each Site is Observed In",breaks=50)
abline(v=log10(min(num_cells_ncounted[num_cells_ncounted >= dim(binary_mat)[2]*cutoff])),lwd=2,col="indianred")
# hist(log10(new_counts),main="Number of Sites Each Cell Uses",breaks=50)
peaks_selected = rownames(ncounts)
return(peaks_selected)
}peaks_selected <- filter_peaks(counts)
counts <- counts[peaks_selected,]
peaks <- peaks[peaks_selected, ]
counts <- t(counts)
counts <- Matrix(counts, sparse = TRUE)
dim(counts)Binarize counts
binarized_counts <- as.matrix((counts > 0) + 0)
binarized_counts <- Matrix(binarized_counts, sparse = TRUE)
dim(binarized_counts)Save processed data
saveRDS(counts, file.path(data.dir, "counts_Buenrostro_2018.rds"))
saveRDS(binarized_counts, file.path(data.dir, "binarized_counts_Buenrostro_2018.rds"))
save(list = c("samples", "peaks", "counts"),
file = file.path(data.dir, "Buenrostro_2018_counts.RData"))
counts <- binarized_counts
save(list = c("samples", "peaks", "counts"),
file = file.path(data.dir, "Buenrostro_2018_binarized_counts.RData"))data.dir <- "/project2/mstephens/kevinluo/scATACseq-topics/data/Buenrostro_2018/processed_data/"
load(file.path(data.dir, "Buenrostro_2018_counts.RData"))
cat(sprintf("Loaded %d x %d counts matrix.\n",nrow(counts),ncol(counts)))
cat(sprintf("Number of samples (cells): %d\n",nrow(counts)))
cat(sprintf("Number of peaks: %d\n",ncol(counts)))
cat(sprintf("Proportion of counts that are non-zero: %0.1f%%.\n",
100*mean(counts > 0)))Processed peaks (from aggregated scATAC-seq) and BAM files were downloaded from the scATAC-benchmarking website (Chen et al 2019) (https://github.com/pinellolab/scATAC-benchmarking/)
Count paired-end scATAC-seq reads in peaks using chromVAR, following the pipeline
Load chromVAR and related packages
# if (!requireNamespace("BiocManager", quietly = TRUE))
# install.packages("BiocManager")
# BiocManager::install("chromVAR")
# BiocManager::install("motifmatchr")
# BiocManager::install("BSgenome.Hsapiens.UCSC.hg19")
# BiocManager::install("JASPAR2016")library(chromVAR)
library(motifmatchr)
library(Matrix)
library(SummarizedExperiment)
library(BiocParallel)
library('JASPAR2016')
library(BSgenome.Hsapiens.UCSC.hg19)
register(MulticoreParam(10))
data.dir <- "/project2/mstephens/kevinluo/scATACseq-topics/data/Buenrostro_2018/processed_data_Chen2019pipeline/chromVAR/"
dir.create(data.dir, showWarnings = FALSE, recursive = TRUE)samples metadata
samples <- read.table('/project2/mstephens/kevinluo/scATACseq-topics/data/Buenrostro_2018/data/input_Chen_2019/metadata.tsv', header = TRUE, stringsAsFactors=FALSE, quote="",row.names=1)peaks (from aggregated scATAC-seq)
peakfile <- "/project2/mstephens/kevinluo/scATACseq-topics/data/Buenrostro_2018/data/input_Chen_2019/combined.sorted.merged.bed"
peaks <- getPeaks(peakfile, sort_peaks = TRUE)
peaks <- resize(peaks, width = 500, fix = "center")
cat(length(peaks), "peaks \n")BAM files of scATAC-seq reads
dir_bamfiles <- "/project2/mstephens/kevinluo/scATACseq-topics/data/Buenrostro_2018/data/input_Chen_2019/sc-bams_nodup/"
bamfiles <- list.files(path = dir_bamfiles, pattern = "\\.bam$")
cellnames <- sapply(strsplit(bamfiles,'.',fixed = TRUE), "[[", 1)
sum(cellnames == rownames(samples))count scATAC-seq paired-end fragments in the peaks using chromVAR
fragment_counts <- getCounts(file.path(dir_bamfiles,bamfile),
peaks,
paired = TRUE,
by_rg = TRUE,
format = "bam",
colData = data.frame(celltype = cellnames))
saveRDS(fragment_counts, file.path(data.dir, "fragment_counts_scPeaks_chromVAR_Buenrostro_2018.rds"))fragment_counts <- readRDS(file.path(data.dir, "fragment_counts_scPeaks_chromVAR_Buenrostro_2018.rds"))
fragment_countscounts <- assay(fragment_counts)
counts <- t(counts)
peaks <- as.data.frame(peaks)[,1:3]
colnames(peaks) <- c("chr", "start", "end")
peak_names <- paste(peaks$chr, peaks$start, peaks$end, sep = "_")
colnames(counts) <- peak_namesFilter peaks (accessible in at least 1 sample) using filterPeaks in chromVAR
idx_peaks_filtered <- filterPeaks(fragment_counts, min_fragments_per_peak = 1, non_overlapping = TRUE, ix_return = TRUE)
peaks <- peaks[idx_peaks_filtered,]
counts <- counts[,idx_peaks_filtered]
cat(length(idx_peaks_filtered), "peaks after filtering. \n")Binarize counts
binarized_counts <- as.matrix((counts > 0) + 0)
binarized_counts <- Matrix(binarized_counts, sparse = TRUE)
dim(binarized_counts)Save processed data
saveRDS(counts, file.path(data.dir, "counts_scPeaks_Buenrostro_2018.rds"))
saveRDS(binarized_counts, file.path(data.dir, "binarized_counts_scPeaks_Buenrostro_2018.rds"))
save(list = c("samples", "peaks", "counts"),
file = file.path(data.dir, "Buenrostro_2018_scPeaks.RData"))
counts <- binarized_counts
save(list = c("samples", "peaks", "counts"),
file = file.path(data.dir, "Buenrostro_2018_binarized_scPeaks.RData"))data.dir <- "/project2/mstephens/kevinluo/scATACseq-topics/data/Buenrostro_2018/processed_data_Chen2019pipeline/chromVAR/"
load(file.path(data.dir, "Buenrostro_2018_binarized_scPeaks.RData"))
cat(sprintf("Loaded %d x %d counts matrix.\n",nrow(counts),ncol(counts)))
cat(sprintf("Number of samples (cells): %d\n",nrow(counts)))
cat(sprintf("Number of peaks: %d\n",ncol(counts)))
cat(sprintf("Proportion of counts that are non-zero: %0.1f%%.\n",
100*mean(counts > 0)))Processed peaks (from aggregated scATAC-seq) and BAM files were downloaded from the scATAC-benchmarking website (Chen et al 2019) (https://github.com/pinellolab/scATAC-benchmarking/)
Count paired-end scATAC-seq reads in peaks using chromVAR, following the pipeline
Load chromVAR and related packages
library(chromVAR)
library(motifmatchr)
library(Matrix)
library(SummarizedExperiment)
library(BiocParallel)
library('JASPAR2016')
library(BSgenome.Hsapiens.UCSC.hg19)
register(MulticoreParam(10))
data.dir <- "/project2/mstephens/kevinluo/scATACseq-topics/data/Buenrostro_2018/processed_data_Chen2019pipeline/chromVAR/"
dir.create(data.dir, showWarnings = FALSE, recursive = TRUE)samples metadata
samples <- read.table('/project2/mstephens/kevinluo/scATACseq-topics/data/Buenrostro_2018/data/input_Chen_2019/metadata.tsv', header = TRUE, stringsAsFactors=FALSE, quote="",row.names=1)peaks (from aggregated scATAC-seq)
peakfile <- "/project2/mstephens/kevinluo/scATACseq-topics/data/Buenrostro_2018/data/input_Chen_2019/combined.sorted.merged.bed"
peaks <- getPeaks(peakfile, sort_peaks = TRUE)
peaks <- resize(peaks, width = 500, fix = "center")
cat(length(peaks), "peaks \n")BAM reads
dir_bamfiles <- "/project2/mstephens/kevinluo/scATACseq-topics/data/Buenrostro_2018/data/input_Chen_2019/sc-bams_nodup/"
bamfiles <- list.files(path = dir_bamfiles, pattern = "\\.bam$")
cellnames <- sapply(strsplit(bamfiles,'.',fixed = TRUE), "[[", 1)
sum(cellnames == rownames(samples))count scATAC-seq paired-end fragments in the peaks
fragment_counts <- getCounts(file.path(dir_bamfiles,bamfile),
peaks,
paired = TRUE,
by_rg = TRUE,
format = "bam",
colData = data.frame(celltype = cellnames))
saveRDS(fragment_counts, file.path(data.dir, "fragment_counts_scPeaks_chromVAR_Buenrostro_2018.rds"))fragment_counts <- readRDS(file.path(data.dir, "fragment_counts_scPeaks_chromVAR_Buenrostro_2018.rds"))
fragment_countscounts <- assay(fragment_counts)
counts <- t(counts)
peaks <- as.data.frame(peaks)[,1:3]
colnames(peaks) <- c("chr", "start", "end")
peak_names <- paste(peaks$chr, peaks$start, peaks$end, sep = "_")
colnames(counts) <- peak_namesFilter peaks (filter out peaks with fragments in < 1% samples)
fragment_binarized_counts <- fragment_counts
assay(fragment_binarized_counts) <- Matrix(as.matrix((assay(fragment_binarized_counts) > 0) + 0), sparse = TRUE)
idx_peaks_filtered <- filterPeaks(fragment_binarized_counts, min_fragments_per_peak = floor(nrow(samples)*0.01), non_overlapping = TRUE, ix_return = TRUE)
cat(length(idx_peaks_filtered), "peaks selected after filtering. \n")
peaks <- peaks[idx_peaks_filtered,]
counts <- counts[,idx_peaks_filtered]Binarize counts
binarized_counts <- as.matrix((counts > 0) + 0)
binarized_counts <- Matrix(binarized_counts, sparse = TRUE)
dim(binarized_counts)Save processed data
save(list = c("samples", "peaks", "counts"),
file = file.path(data.dir, "Buenrostro_2018_scPeaks_filtered.RData"))
counts <- binarized_counts
save(list = c("samples", "peaks", "counts"),
file = file.path(data.dir, "Buenrostro_2018_binarized_scPeaks_filtered.RData"))data.dir <- "/project2/mstephens/kevinluo/scATACseq-topics/data/Buenrostro_2018/processed_data_Chen2019pipeline/chromVAR/"
load(file.path(data.dir, "Buenrostro_2018_binarized_scPeaks_filtered.RData"))
cat(sprintf("Loaded %d x %d counts matrix.\n",nrow(counts),ncol(counts)))
cat(sprintf("Number of samples (cells): %d\n",nrow(counts)))
cat(sprintf("Number of peaks: %d\n",ncol(counts)))
cat(sprintf("Proportion of counts that are non-zero: %0.1f%%.\n",
100*mean(counts > 0)))
sessionInfo()