Endothelial cell differentiation — CMO multiome processing

Processing pipelines for the 10x multiome EC differentiation datasets, covering FASTQ reconstruction, CMO/GEX demultiplexing, and CMO tag quantification.

Datasets

10x multiome — 5 timepoints

GEX and CMO reads arrive mixed in the same FASTQs and must be separated with splitcode before quantification. CMO quantification is automated end-to-end via IGVF accessions.

Channel	IGVF analysis set
Channel 1	IGVFDS5477BPOI
Channel 2	IGVFDS3995WHFT

10x multiome — 5 timepoints (McGinnis)

Single-channel CMO library sequenced as a standard 10x multiome run (no splitcode required). CMO tag is in R2 (MULTI-seq barcode, 8 bp); R3 carries cDNA and is not used for CMO quantification.

Channel	IGVF analysis set
Single channel	IGVFDS1612ZNCA

10x multiome — 15 timepoints

GEX, CMO, and ATAC reads arrive pre-demultiplexed (separate sample indices). No splitcode step required.

Repository layout

analyses/
  10x_multi_5_timepoints/          # 5 tp pipeline (manual steps 1–14 + automated CMO quant)
    cmo_quantification/            # end-to-end CMO quantification from IGVF accessions
  10x_multi_5_timepoints_mcginnis/ # McGinnis 5 tp pipeline (single channel, automated CMO quant)
    cmo_quantification/            # end-to-end CMO quantification from IGVF accessions
  10x_multi_15_timepoints/         # 15 tp pipeline scripts
  create_seqspecs/                 # marimo notebook for generating seqspec YAMLs
config/
  conda/                       # conda environment definition and build sbatch
  splitcode/                   # splitcode tag configs for 5 tp and 15 tp datasets
metadata/                      # CMO design sheets (TSV)
scripts/
  bash/                        # exploratory / benchmarking scripts
  python/                      # shared Python utilities (IGVF auth, download, KITE helpers)
templates/                     # Jinja2 templates for seqspec YAML generation

Environment

All tools (kb-python, splitcode, seqspec, igvf-utils) are in the project conda env. Build once:

sbatch config/conda/build_env.sbatch

Activate for interactive use:

source /home/users/emattei/miniforge3/etc/profile.d/conda.sh
conda activate /oak/stanford/groups/engreitz/Users/emattei/git/broad-nnfc-cmo-multiome/env/nnfc-cmo-multiome

Authentication

IGVF downloads require a .env file at the repo root:

IGVF_API_KEY=<your_key>
IGVF_SECRET_KEY=<your_secret>