Demuxlet

This workflow runs demuxlet to deconvolute sample identity when multiple samples are pooled by barcoded single-cell sequencing.

  1. Align your single-cell sequencing data (for example using the cellranger or drop_seq workflows).

  2. Create a sample sheet.

    Please note that the columns in the tab separated file must be in the order shown below and does not contain a header line.

    Column Description
    Name Sample name.
    BAM Location of the BAM file in the cloud (gs:// URL).
    Barcodes Location of the valid cellular barcodes file in the cloud (gs:// URL).
    VCF Location of the VCF file to use for this sample in the cloud (gs:// URL).

    Example:

    sample-1,gs://fc-e0000000/sample-1/out/possorted_genome_bam.bam,gs://fc-e0000000/sample-1/out/filtered_feature_bc_matrix/barcodes.tsv.gz,gs://fc-e0000000/sample-1.vcf
    sample-2,gs://fc-e0000000/sample-2/out/possorted_genome_bam.bam,gs://fc-e0000000/sample-2/out/filtered_feature_bc_matrix/barcodes.tsv.gz,gs://fc-e0000000/sample-2.vcf
    
  3. Upload your sample sheet to the workspace bucket.

    Example:

    gsutil cp /foo/bar/projects/sample_sheet.tsv gs://fc-e0000000/
    
  4. Import demuxlet workflow to your workspace.

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

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

  5. In your workspace, open demuxlet in WORKFLOWS tab. Select Process single workflow from files as below

    _images/single_workflow.png

    and click the Save button.


Inputs

Please see the description of important inputs below.

Column Description
tsv_file Four column tab-separated file without a header with name, coordinate sorted bam, barcodes, and vcf
min_MQ Minimum mapping quality to consider (default 20)
alpha Grid of alpha to search for (default [0.1, 0.2, 0.3, 0.4, 0.5]).
min_TD Minimum distance to the tail (default 0)
tag_group Tag representing readgroup or cell barcodes, in the case to partition the BAM file into multiple groups (default “CB”)
tag_UMI Tag representing UMIs (default “UB”“)
field FORMAT field to extract the genotype, likelihood, or posterior from (default “GT”)
geno_error Offset of genotype error rate (default 0.1)

Outputs

The demuxlet output file contains the best guess of the sample identity, with detailed statistics to reach to the best guess.