Last updated: 2020-11-04

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Rmd 12bf4b3 kevinlkx 2020-11-04 process data from Buenrostro 2018 paper

About Buenrostro 2018 dataset

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

RCC directory: /project2/mstephens/kevinluo/scATACseq-topics/data/Buenrostro_2018/

Processed data

Downloaded scATAC-seq processed data from GEO: GSE96769

  • GSE96769_PeakFile_20160207.bed.gz
  • GSE96769_scATACseq_counts.txt.gz

Downloaded processed data from the scATAC-benchmarking website (Chen et al. Genome Biology 2019) (https://github.com/pinellolab/scATAC-benchmarking/)

Prepare data for topic modeling

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.sbatch

combine 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$")
sample_names <- sapply(strsplit(files,'\\.'),'[', 1)
cat(length(sample_names), "samples. \n")
2034 samples. 
datalist <- lapply(files, function(x)fread(file.path(dir_readcount_output,x))$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"))

Processing count files (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)

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)))
Number of peaks: 237450
head(peaknames)
[1] "chr1_10413_10625"   "chr1_13380_13624"   "chr1_16145_16354"  
[4] "chr1_96388_96812"   "chr1_115650_115812" "chr1_237625_237888"
colnames(counts) <- sample_names
rownames(counts) <- peaknames

dim(counts)
[1] 237450   2034

Sample labels

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)
                            label
BM1077-CLP-Frozen-160106-13   CLP
BM1077-CLP-Frozen-160106-14   CLP
BM1077-CLP-Frozen-160106-2    CLP
BM1077-CLP-Frozen-160106-21   CLP
BM1077-CLP-Frozen-160106-27   CLP
BM1077-CLP-Frozen-160106-3    CLP

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)
[1]   2034 101172

Binarize counts

binarized_counts <- as.matrix((counts > 0) + 0)
dim(binarized_counts)
[1]   2034 101172
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"))

save(list = c("samples", "peaks", "binarized_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)))
Loaded 2034 x 101172 counts matrix.
cat(sprintf("Number of samples (cells): %d\n",nrow(counts)))
Number of samples (cells): 2034
cat(sprintf("Number of peaks: %d\n",ncol(counts)))
Number of peaks: 101172
cat(sprintf("Proportion of counts that are non-zero: %0.1f%%.\n",
            100*mean(counts > 0)))
Proportion of counts that are non-zero: 6.2%.

sessionInfo()
R version 3.6.1 (2019-07-05)
Platform: x86_64-pc-linux-gnu (64-bit)
Running under: Scientific Linux 7.4 (Nitrogen)

Matrix products: default
BLAS/LAPACK: /software/openblas-0.2.19-el7-x86_64/lib/libopenblas_haswellp-r0.2.19.so

locale:
 [1] LC_CTYPE=en_US.UTF-8       LC_NUMERIC=C              
 [3] LC_TIME=en_US.UTF-8        LC_COLLATE=en_US.UTF-8    
 [5] LC_MONETARY=en_US.UTF-8    LC_MESSAGES=en_US.UTF-8   
 [7] LC_PAPER=en_US.UTF-8       LC_NAME=C                 
 [9] LC_ADDRESS=C               LC_TELEPHONE=C            
[11] LC_MEASUREMENT=en_US.UTF-8 LC_IDENTIFICATION=C       

attached base packages:
[1] tools     stats     graphics  grDevices utils     datasets  methods  
[8] base     

other attached packages:
[1] data.table_1.12.8 readr_1.3.1       Matrix_1.2-18     workflowr_1.6.2  

loaded via a namespace (and not attached):
 [1] Rcpp_1.0.5       knitr_1.28       whisker_0.4      magrittr_1.5    
 [5] hms_0.5.3        lattice_0.20-38  R6_2.5.0         rlang_0.4.8     
 [9] stringr_1.4.0    grid_3.6.1       xfun_0.14        git2r_0.27.1    
[13] ellipsis_0.3.1   htmltools_0.4.0  yaml_2.2.0       digest_0.6.27   
[17] rprojroot_1.3-2  lifecycle_0.2.0  tibble_3.0.4     crayon_1.3.4    
[21] later_1.0.0      vctrs_0.3.4      promises_1.1.0   fs_1.3.1        
[25] glue_1.4.2       evaluate_0.14    rmarkdown_2.1    stringi_1.4.6   
[29] pillar_1.4.6     compiler_3.6.1   backports_1.1.10 httpuv_1.5.3.1  
[33] pkgconfig_2.0.3