This page details the cellranger multi output structure for Flex data, using a Flex v2 (multiplex) experiment as an example.

Upon completion, the cellranger multi pipeline will produce an outs/ directory with a structure similar to the following:

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├── config.csv
├── feature_reference.csv
├── filtered_feature_bc_matrix
│   ├── barcodes.tsv.gz
│   ├── features.tsv.gz
│   └── matrix.mtx.gz
├── filtered_feature_bc_matrix.h5
├── multiplexing_analysis
│   ├── cells_per_tag.json
│   └── frp_gem_barcode_overlap.csv
├── per_sample_outs
│   ├── A-A01_K562_CRL00589_384plex
│   └── A-B01_K562_CRL00589_384plex
├── probe_set.csv
├── qc_library_metrics.csv
├── qc_report.html
├── qc_sample_metrics.csv
├── raw_cloupe.cloupe
├── raw_feature_bc_matrix
│   ├── barcodes.tsv.gz
│   ├── features.tsv.gz
│   └── matrix.mtx.gz
├── raw_feature_bc_matrix.h5
├── raw_molecule_info.h5
└── raw_probe_bc_matrix.h5

The files included at the top level may include:

• config.csv: a duplicate of the input config CSV file.
• feature_reference.csv: a duplicate of the input Feature Reference CSV file, if provided.
• filtered_feature_bc_matrix.h5: filtered feature-barcode matrix (containing only barcodes called as cells), concatenated across all samples, in HDF5 format.
• probe_set.csv: a duplicate of the input probe set CSV.
• qc_library_metrics.csv: quality control report in CSV format for the entire experiment.
• qc_report.html: quality control report in HTML format for the entire experiment.
• qc_sample_metrics.csv: quality control report in CSV format by sample.
• raw_cloupe.cloupe: Loupe Browser file containing all samples and all cell-associated barcodes in the experiment.
• raw_feature_bc_matrix.h5: raw feature-barcode matrix (containing all barcodes) in HDF5 format.
• raw_molecule_info.h5: information about all molecules in the experiment. This file includes background and cell-associated barcodes from all samples in addition to valid barcodes that were not assigned to a sample.
• raw_probe_bc_matrix.h5: UMI counts of each probe for all detected barcodes in HDF5 format.

Continue reading for descriptions of the directories contained within the top-level outputs.

In addition to the HDF5 format listed above, two directories contain the filtered (concatenated across all samples) and raw feature-barcode matrices in Market Exchange (MEX) Format:

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└── filtered_feature_bc_matrix
    ├── barcodes.tsv.gz
    ├── features.tsv.gz
    └── matrix.mtx.gz
...
└── raw_feature_bc_matrix
    ├── barcodes.tsv.gz
    ├── features.tsv.gz
    └── matrix.mtx.gz

For Flex multiplex experiments, a multiplexing_analysis directory is output. The files in this folder are generic to the entire Flex experiment:

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└── multiplexing_analysis
    ├── cells_per_tag.json
    └── frp_gem_barcode_overlap.csv

The cells_per_tag.json lists the cell-associated barcodes that were assigned a given Probe Barcode tag, for each tag, in JSON format. For each Probe Barcode (e.g., BC001) the cell-associated barcodes (e.g., "AACAAGCTCCCTCAAAACTTTAGG-1", etc.) are listed below it:

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{
    "BC001":[
        "AACAAGCTCCCTCAAAACTTTAGG-1",
        "AACATAGTCCCATAGCACTTTAGG-1",
        "AACCAGGTCATGGTCCACTTTAGG-1",
...
    "BC002":[
        "AAACTGTCAGGAGCAAAACGGGAA-1",
        "AAAGGGATCTAATCGTAACGGGAA-1",
        "AACCAAATCGGTCAAGAACGGGAA-1",
...
}

The frp_gem_barcode_overlap.csv file contains the number of shared 10x GEM Barcodes for all pairs of observed Probe Barcode (BC) tags or Probe Barcode and Antibody Multiplexing Barcode (AB) tags assigned to a sample. This file can be used to troubleshoot scenarios where, for example, two different Probe Barcodes were accidentally added to the same hybridization reaction. For multiplexed GEX + Antibody Capture experiments, overlap between incorrect BC+AB barcode pairs may result from contamination or using the same Probe Barcode for two antibody panels. An alert in the web summary will be triggered if the overlap coefficient is ≥ 60%.

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barcode1_id,barcode2_id,barcode1_gems,barcode2_gems,common_gems,overlap
BC009,BC010,2333,2608,81,0.034719245606515216
[ … ]
BC012,AB011,2461,4419,188,0.07639171068671272

Column descriptions:

• barcode1_id: First barcode identifier
• barcode2_id: Second barcode identifier
• barcode1_gems: Number of 10x GEM Barcodes for barcode1
• barcode2_gems: Number of 10x GEM Barcodes for barcode2
• common_gems: Number of 10x GEM Barcodes in common
• overlap: The overlap coefficient of these two barcodes (either pair of Probe Barcodes (BC), Probe Barcode (BC) and Antibody Multiplexing Barcode (AB), or AB-AB for Antibody Capture only analysis).

Overlap coefficient = $\frac{{\text{Common GEMS}}} {\text{min(Barcode 1 GEMS, Barcode 2 GEMS)}}$

Outputs demultiplexed per sample (in a multiplexed experiment) are provided in the per_sample_outs directory. In this example, two samples are shown (A-A01_K562_CRL00589_384plex and A-B01_K562_CRL00589_384plex):

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├── A-A01_K562_CRL00589_384plex
│   ├── analysis
│   ├── crispr_analysis
│   ├── metrics_summary.csv
│   ├── sample_cloupe.cloupe
│   ├── sample_filtered_barcodes.csv
│   ├── sample_filtered_feature_bc_matrix
│   ├── sample_filtered_feature_bc_matrix.h5
│   ├── sample_molecule_info.h5
│   ├── sample_raw_feature_bc_matrix
│   ├── sample_raw_feature_bc_matrix.h5
│   ├── sample_raw_probe_bc_matrix.h5
│   └── web_summary.html
├── A-B01_K562_CRL00589_384plex
│   ├── analysis
│   ├── crispr_analysis
│   ├── metrics_summary.csv
│   ├── sample_cloupe.cloupe
│   ├── sample_filtered_barcodes.csv
│   ├── sample_filtered_feature_bc_matrix
│   ├── sample_filtered_feature_bc_matrix.h5
│   ├── sample_molecule_info.h5
│   ├── sample_raw_feature_bc_matrix
│   ├── sample_raw_feature_bc_matrix.h5
│   ├── sample_raw_probe_bc_matrix.h5
│   └── web_summary.html

Each per-sample directory contains the following files and folders:

• analysis: secondary analysis results, including dimensionality reduction, clustering, and differential gene expression.
• crispr_analysis: if the experimental design includes CRISPR, the guide capture outputs are provided here.
• metrics_summary.csv: experimental metrics in CSV format.
• sample_cloupe.cloupe: sample-specific Loupe Browser file
• sample_filtered_barcodes.csv: sample-specific filtered barcodes in CSV format.
• sample_filtered_feature_bc_matrix: sample-specific filtered feature-barcode matrix in Market Exchange (MEX) Format
• sample_filtered_feature_bc_matrix.h5: sample-specific filtered feature-barcode matrix (containing only barcodes called as cells within this sample) in HDF5 format.
• sample_molecule_info.h5: information about all molecules in the sample. This file includes background and cell-associated barcodes.
• sample_raw_feature_bc_matrix: sample-specific raw feature-barcode matrix in Market Exchange (MEX) Format
• sample_raw_feature_bc_matrix.h5: sample-specific raw feature-barcode matrix (containing all barcodes assigned to this sample) in HDF5 format.
• sample_raw_probe_bc_matrix.h5: sample-specific UMI counts of each probe for all detected barcodes in HDF5 format.
• web_summary.html: sample-specific web summary HTML, a starting point for quality control.