/vmrseq-workflow-vignette

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An Example Workflow of Single-cell Bisulfite Sequencing Data Analysis

This is an auxillary repository that demonstrate a workflow that analyzes a real-world dataset using the vmrseq package.

Dataset

We took chromosome 1 of a subset of the single-cell bisulfite sequencing data published in Luo et al [1] as the example dataset in this repository. Specifically, we took cells from 4 subtypes: the excitatory subtype mDL-2 and inhibitory subtypes mPv, mSst-1 and mSst-2. The CpG methylation levels of these files has been packed up into a SummarizedExperiment object (stored in data/example/chr1) using data.pool function in the vmrseq package.

[1] Luo, Chongyuan et al. “Single-cell methylomes identify neuronal subtypes and regulatory elements in mammalian cortex.” Science (New York, N.Y.) vol. 357,6351 (2017): 600-604. doi:10.1126/science.aan3351

Workflow

We have organized the scripts into the order of the analysis pipeline in the scripts/ folder：

  • 01_run_vmrseq: apply the vmrseq methodology to the example dataset.
  • 02_run_region_summary: summarize the regional methylation information per cell on detected VMRs.
  • 03_downstream_analysis: downstream analysis including gene/CpG context annotation and unsupervised cell clustering.

Important notes:

  • We strongly recommend the user to use high-performance computing to run this workflow on large-scale single-cell datasets (e.g., > 500 cells) due to the extensive demand on memory and parallelism.
  • We also recommend to process one chromosome at a time or in parallel to facilitate a manageable computation time.

Required packages

These packages are required to run the scripts in this repo:

# # install.packages("devtools")
# # install.packages("BiocManager")
#
# devtools::install_github("nshen7/vmrseq")
# BiocManager::install("here")
# BiocManager::install("SummarizedExperiment")
# BiocManager::install("HDF5Array")
# BiocManager::install("BiocParallel")
# BiocManager::install("annotatr")
# BiocManager::install("TxDb.Mmusculus.UCSC.mm10.knownGene")
# BiocManager::install("org.Mm.eg.db")
#
# install.packages("uwot")
# install.packages("data.table")
# install.packages("tidyverse")

Citation

If you applied the functions in vmrseq package, please cite our paper:

Ning Shen and Keegan Korthauer. 2023. “Vmrseq: Probabilistic Modeling of Single-Cell Methylation Heterogeneity.” bioRxiv. https://doi.org/10.1101/2023.11.20.567911.