10X Multiome doublet detection pipeline

This pipeline runs demultiplexing with demuxlet (if VCF file provided), runs ATAC-based doublet detection using AMULET, and runs RNA-based doublet detection using DoubletFinder. It also performs an initial (unoptimized) clustering of libraries, individually and jointly. The joint clustering may be useful for identifying doublet clusters containing nuclei not previously identified as doublets.

Dependencies

Singularity (v. 3) and NextFlow (>= v. 20.10.0). Containers with the software for each step are pulled from the Sylabs cloud library (https://cloud.sylabs.io/library) or Docker hub (https://hub.docker.com/).

Running

You'll need to provide a config.json file like this:

{
    "libraries": {
        "9240-VD-1-hg38": {
            "atac_bam": "/path/to/multiome-atacseq/results/prune/9240-VD-1-hg38.pruned.bam",
            "atac_pass_qc_barcodes": "/path/to/list-of-pass-qc-atac-barcodes.txt",
            "genome": "hg38",
            "rna_bam": "/path/to/multiome-rnaseq/results/prune/9240-VD-1-hg38.before-dedup.bam",
            "rna_cellbender": "/path/to/multiome-rnaseq/results/cellbender/9240-VD-1-hg38.cellbender_FPR_0.05.h5",
            "rna_pass_qc_barcodes": "/path/to/list-of-pass-qc-rna-barcodes.txt",
            "vcf": "/path/to/donors.vcf.gz"
        }
    }
}

You'll also need to update the reference file paths in the nextflow.config file in this directory.

Then run the pipeline:

nextflow run -resume -params-file config.json --atac_barcodes /path/to/snATACseq-NextFlow/737K-arc-v1.txt --rna_barcodes /path/to/snRNAseq-NextFlow/737K-arc-v1.txt --results /path/to/results /path/to/Multiome-Doublet-Detection-NextFlow/main.nf

Where --atac_barcodes and --rna_barcodes are the barcode whitelists for the two modalities.

Output

atac-doublet-detection: Doublet detection results based on AMULET, including lists of singlet RNA/ATAC barcodes (to be used for downstream analysis)
demuxlet/out: Raw demuxlet output files for each library and modality
demuxlet/processed: List of demuxlet --> doublet / singlet (+ individual) assignments, and plots of demuxlet assignments and ATAC / RNA concordance
doubletfinder: Doublet detection results based on DoubletFinder
cluster: Seurat clustering results, with doublets included
merged-counts: Input RNA count matrix for joint clustering
pass-qc-nuclei-counts-with-doublets: Input RNA count matrix for clustering of individual libraries
doublets-in-clustering: Plots of doublet vs singlet nuclei in joint clustering

alicewang19/Multiome-Doublet-Detection-NextFlow

10X Multiome doublet detection pipeline

Dependencies

Running

Output