Demultiplex cell-hashing/nuclei-hashing data using demuxEM or prepare for CITE-Seq analysis

Follow the steps below to run cumulus for cell-hashing/nuclei-hashing/CITE-Seq data on Terra.

  1. Run Cell Ranger tool to generate raw gene count matrices and antibody hashtag data.

    Please refer to the cellranger_workflow tutorial for details.

    When finished, you should be able to find the raw gene count matrix (e.g. raw_gene_be_matrices_h5.h5) and ADT count matrix (e.g. sample_1_ADT.csv) for each sample.

  2. Create a sample sheet, sample_sheet_hashing.csv, which describes the metadata for each pair of RNA and antibody hashtag data. The sample sheet should contain 4 columns — OUTNAME, RNA, ADT, and TYPE. OUTNAME is the output name for one pair of RNA and ADT data. RNA and ADT are the raw gene count matrix and the ADT count matrix generated in Step 1, respectively. TYPE is the assay type, which can be cell-hashing, nuclei-hashing, or cite-seq.



    Note that in the example above, sample_2 is 10x genomics’ v3 chemistry. Cumulus can automatically detect v2/v3 chemistry when loading hdf5 files.

  3. (Optional) Create an additional antibody-control sheet antibody_control.csv if you have CITE-Seq data and IgG controls for each antibody. This sheet contains 2 columns — Antibody and Control.


  4. Upload your sample sheets to the Google bucket of your workspace.


    gsutil cp /foo/bar/projects/my_sample_sheet_hashing.csv gs://fc-e0000000-0000-0000-0000-000000000000/
    gsutil cp /foo/bar/projects/antibody_control.csv gs://fc-e0000000-0000-0000-0000-000000000000/
  5. Import cumulus_hashing_cite_seq to your workspace.

    See the Terra documentation for adding a workflow. The cumulus_hashing_cite_seq workflow is under Broad Methods Repository with name “cumulus/cumulus_hashing_cite_seq”.

    Moreover, in the workflow page, click the Export to Workspace... button, and select the workspace to which you want to export cumulus_hashing_cite_seq workflow in the drop-down menu.

  6. In your workspace, open cumulus_hashing_cite_seq in WORKFLOWS tab. Select Run workflow with inputs defined by file paths as below


    and click the SAVE button.

cumulus_hashing_cite_seq inputs:

Name Description Example Default
input_sample_sheet Input CSV file describing metadata of RNA and ADT data pairing “gs://fc-e0000000-0000-0000-0000-000000000000/sample_sheet_hashing.csv”  
output_directory This is the output directory (gs url + path) for all results. There will be one folder per RNA-ADT data pair under this directory “gs://fc-e0000000-0000-0000-0000-000000000000/my_demux_dir”  
genome Reference genome name. If not provided, cumulus will infer the genome name from data “GRCh38”  
demuxEM_min_num_genes demuxEM parameter. Only demultiplex cells/nuclei with at least <demuxEM_min_num_genes> expressed genes 200 100
demuxEM_alpha_on_samples demuxEM parameter. The Dirichlet prior concentration parameter (alpha) on samples. An alpha value < 1.0 will make the prior sparse. 2.0 0.0
demuxEM_min_num_umis demuxEM parameter. Only demultiplex cells/nuclei with at least <demuxEM_min_num_umis> of UMIs. 200 100
demuxEM_min_signal_hashtag demuxEM parameter. Any cell/nucleus with less than <count> hashtags from the signal will be marked as unknown. [default: 10.0] 10.0 10.0
demuxEM_random_state demuxEM parameter. The random seed used in the KMeans algorithm to separate empty ADT droplets from others 0 0
demuxEM_generate_diagnostic_plots demuxEM parameter. If generate a series of diagnostic plots, including the background/signal between HTO counts, estimated background probabilities, HTO distributions of cells and non-cells etc true true
demuxEM_generate_gender_plot demuxEM parameter. If generate violin plots using gender-specific genes (e.g. Xist). <demuxEM_generate_gender_plot> is a comma-separated list of gene names “XIST”  
antibody_control_csv Optional merge_rna_adt parameter. If there is no IgG control information, leave this option blank. Otherwise, specify a CSV file containing the IgG control information for each antibody. “gs://fc-e0000000-0000-0000-0000-000000000000/antibody_control.csv”  
cumulus_version cumulus version to use. Versions available: 0.13.0, 0.12.0, 0.11.0, 0.10.0. “0.13.0” “0.13.0”

Docker registry to use. Options:

  • “cumulusprod” for Docker Hub images;
  • “” for backup images on Red Hat registry.
“cumulusprod” “cumulusprod”
zones Google cloud zones “us-east1-d us-west1-a us-west1-b” “us-central1-a us-central1-b us-central1-c us-central1-f us-east1-b us-east1-c us-east1-d us-west1-a us-west1-b us-west1-c”
num_cpu Number of CPUs per cumulus_hashing_cite_seq job 8 8
memory Memory size string “10G” “10G”
disk_space Total disk space in GB 20 20
preemptible Number of preemptible tries 2 2

cumulus_hashing_cite_seq outputs

See the table below for important cumulus_hashing_cite_seq outputs:

Name Type Description
output_folder Array[String] A list of google bucket urls containing results for every RNA-ADT data pairs.

In the output folder of each cell-hashing/nuclei-hashing RNA-ADT data pair, you can find the following files:

Name Description
output_name_demux.h5ad Demultiplexed RNA count matrix in h5ad format.
output_name_demux.h5sc RNA expression matrix with demultiplexed sample identities in cumulus hdf5 (h5sc) format.
output_name_ADTs.h5ad Antibody tag matrix in h5ad format.
output_name.ambient_hashtag.hist.png Optional output. A histogram plot depicting hashtag distributions of empty droplets and non-empty droplets. Optional output. A bar plot visualizing the estimated hashtag background probability distribution.
output_name.real_content.hist.png Optional output. A histogram plot depicting hashtag distributions of not-real-cells and real-cells as defined by total number of expressed genes in the RNA assay.
output_name.rna_demux.hist.png Optional output. A histogram plot depicting RNA UMI distribution for singlets, doublets and unknown cells.
output_name.gene_name.violin.png Optional outputs. Violin plots depicting gender-specific gene expression across samples. We can have multiple plots if a gene list is provided in demuxEM_generate_gender_plot field of cumulus_hashing_cite_seq inputs.

In the output folder of each CITE-Seq RNA-ADT data pair, you can find the following file:

Name Description
output_name.h5sc A Cumulus hdf5 format (h5sc) file containing both RNA and ADT count matrices.

Load demultiplexing results into Python and R

To load demultiplexing results into Python, you need to install Python package anndata first. Then follow the codes below:

import anndata
data = anndata.read_h5ad('output_name_demux.h5ad')

Once you load the data object, you can find predicted droplet types (singlet/doublet/unknown) in data.obs['demux_type']. You can find predicted sample assignments in data.obs['assignment']. You can find estimated sample fractions (sample1, sample2, …, samplen, background) for each droplet in data.obsm['raw_probs'].

To load the results into R, you need to install R package reticulate in addition to Python package anndata. Then follow the codes below:

ad <- import("anndata", convert = FALSE)
data <- ad$read_h5ad("output_name_demux.h5ad")

Results are in data$obs['demux_type'], data$obs['assignment'], and data$obsm['raw_probs'].