/multiome_process

Process and integrate 10x Multiome Data

Primary LanguageR

multiome_process

Process and integrate 10x Multiome Data

The following scripts should be used in this order to process the multiome data:

Create signac environment and install packages if needed

Create multiome object on cluster from cellranger output path individually (optional)

Script: 01_multiome_signac.R

Input arguments:

  • argument 1: cellranger out path
  • argument 2: sample name
  • argument 3: species, human or mouse

Example Usage:

Rscript ~/scripts/01_multiome_signac.R /athena/melnicklab/users/scratch/chc2077/ls_multiome/ls_multiome/outs ls_multiome mouse

Object will be saved into the cellranger-arc output folder

Create a csv runsheet that has a unique name for each sample, and a path to cellranger-arc "out" folder

csv runsheet header must contain "Sample" "sobj_path". Additional columns will be added as metadata

Example runsheet included: Example_runsheet.csv

Optional - add velocity data

Script: 21_multiome_velo.R

This script will add the velocity data to multiome objects. Argument is a path to the runsheet

Load and combine single cell data from runsheet, save as RDS file

This script will create multiome objects if 01_multiome_signac.R was not run, but will not contain velocity data

Script: 08_combined_multiome_2.R

Input arguments:

  • argument 1: path to runsheet
  • argument 2: species to use (Human or Mouse)
  • argument 3 (optional): number of cores to use

Example Usage:

Rscript ~/.scripts/08_combine_multiome.R ./ls_msobj_combine_runsheet.csv mouse 32

A work directory will be created within the directory that this script is run from. The combined object will be saved as "combined.multiome.sobj.rds" within this work folder

Use harmony to correct batch effect in both ATAC and RNA assays

Script: 12_multiome_harmony_rds.R

Input arguments:

  • argument 1: path to object - use the "combined.multiome.sobj.rds" file generated by 08_combined_multiome_2.R
  • argument 2: object name - name to give the object
  • argument 3: batch name - name of the metadata column to correct for. orig.ident
  • argument 4 (optional): number of cores to use

Example Usage:

Rscript ~/scripts/12_multiome_harmony_rds.R ./work/integrated.multiome.sobj.celltype.fine.celltype.rds ls_am_dm_multiome orig.ident

Label multiome data by RNA using a reference single cell object

Script: 16_label_singlecell_3.R

  • argument 1: Path to seurat or signac object as an RDS file
  • argument 2: Path to reference seurat or signac object as an RDS file
  • argument 3: Name of assay to use
  • argument 4+: Names of labeles to transfer

Example Usage:

Rscript ~/scripts/16_label_singlecell.R ./work/integrated.multiome.sobj.rds /athena/masonlab/scratch/users/chc2077/references/gcb/20210512_melnick.int.sobj.RDS RNA celltype fine.celltype