#### Source Code for TRIGR paper ###

Note that the code is provided as-is, and is meant to be used to demonstrate the methods used to perform tertiary analysis on the sequencing data which is archived on EGA as described in the TRIGR paper. 

Before running the code,  initial sample processing using cellranger and cellranger aggr as appropriate will be needed to generate the files data files needed by Seurat and Signac for the initial data preparation stages.

YOU WILL NEED TO ADJUST THE SOURCE CODE TO REFLECT THE LOCATIONS OF THE INPUT FILES AND THE OUTPUT FILES FOR YOUR FILESYSTEM.  You will also likely need to install the Seurat and Signac libraries and their dependencies.

https://satijalab.org/seurat/
https://stuartlab.org/signac/

Genotype demultiplexing of the cell barcodes was done using popscle implementation of demuxlet

https://github.com/statgen/popscle

### Contents:
## SRC/ : Source code directory

# utility
SRC/knitr_common.r : shared Rmarkdown setup script

# Initial data preparation (create Seurat objects)
SRC/demuxlet.atac.trigr.b38.sh : example code to regenerate scATAC demultiplexing results
SRC/TRIGR_scATAC_so_prep.Rmd : seurat object prep for single cell ATAC
SRC/TRIGR_scMulti_so_prep.Rmd : seurat object prep for single cell multiome
SRC/TRIGR_scRNA_so_prep.Rmd : seurat object prep for single cell RNA

SRC/TRIGR_scATAC_cell_type_curation.Rmd : cell type for scATAC
SRC/TRIGR_scMulti_cell_type_curation.Rmd : cell type for multiome
SRC/TRIGR_scRNA_cell_type_projection.Rmd : cell type for scRNA

# merge the ATAC from multiome with scATAC
SRC/TRIGR_scATAC_macs2_peaks_from_scMulti_quantification.Rmd : use multiome peaks for scATAC
SRC/TRIGR_scATAC_scMulti_merging.Rmd : merged ATAC prep (and celltypes)

# Extract average RNA expression and ATAC accessibility
SRC/TRIGR_scRNA_average_expression.Rmd : average expression scRNA
SRC/TRIGR_scATAC_average_accessibility.Rmd : average accessibility scATAC

# examples of analyses described in methods
SRC/TRIGR_scRNA_scATAC_link_correlation.Rmd : scRNA to scATAC correlation
SRC/TRIGR_scRNA.diff_oe_new_groups.R : example of observed/expected analysis on scRNA
SRC/TRIGR_mergedCells_OE_new_groups.R : example of observed/expected analysis for multiome and merged ATAC datasets

# scripts to generate figures
SRC/paper_figs_2024_12.mergedCells_OE.R : Supp Fig. 1-3
SRC/mk-figs.R : Supp Fig 4a,b

## DATA/ : Supporting Data files
DATA/blood_peaks.rds : reference ATAC peaks from blood cells
DATA/demuxlet.atac.trigr.b38.sh : example code to generate scATAC demultiplexing results
DATA/multiome_libraries.csv : list of the multiome libraries
DATA/TRIGR_scRNA_cell_types.tomi.tsv : mapping of predicted cell types to merged cell types for scRNA
DATA/lymphoid_DHS.bed : lymphoid cell DHS sites
DATA/myeloid_erythroid_DHS.bed : myeloid and erythroid cell DHS sites
DATA/gencode.v32.annotation.genes.bed : genes from gencode v32 (for scRNA analysis)
DATA/gencode.v38.annotation.genes.bed : genes from gencode v38 (for multiome and merged ATAC analysis)

# Download cite-seq multimodal reference data to DATA/ from:
https://atlas.fredhutch.org/data/nygc/multimodal/pbmc_multimodal.h5seurat

# Data files containing PHI that must be requested via EGA (these are expected to be in the DATA/ directory).  These are in the archive trigr-sample-info.zip
HLA_assignments.xlsx
TRIGR_2024_new_groups.filtered.geno.tsv
TRIGR_2024_new_groups.filtered.tsv
calendar_age_TRIGR.csv
sample_info.csv
sample_info_multiome.csv

### License

All files are provided as-is and all original code is provided under the terms of the MIT license.