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Cell Ranger


Loupe

10x Genomics
Chromium Single Cell Gene Expression

3' Cell Multiplexing Outputs

This page describes the output file structure from the cellranger multi subcommand specifically for 3' Cell Multiplexing data. This subcommand was introduced in Cell Ranger 5.0 for joint analysis of 5' gene expression and V(D)J (GEX + VDJ) data, and in Cell Ranger 6.0 for 3' Cell Multiplexing data.

Table of Contents

Overview

Upon completion, the cellranger multi subcommand will produce an outs/ directory with the following structure:

cellranger web summary

Using the tree Linux command, the file structure looks like this:

├── config.csv
├── multi
│   ├── count
│   └── multiplexing_analysis
└── per_sample_outs
    ├── Sample1
    └── Sample2

The first section of the outputs contains the config.csv file, a duplicate of the input config CSV file. The files in the multi folder are generic to the entire Cell Multiplexing experiment, while the files in the per_sample_outs directory have been demultiplexed to single samples.

Cell Multiplexing outputs directory

Within the multi directory, there are count and multiplexing_analysis directories:

└─ multi
   ├── count
   │   ├── feature_reference.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
   │   ├── unassigned_alignments.bam
   │   └── unassigned_alignments.bam.bai
   └── multiplexing_analysis
       ├── assignment_confidence_table.csv
       ├── cells_per_tag.json
       ├── tag_calls_per_cell.csv
       └── tag_calls_summary.csv

The count directory contains raw files that include cells and background data:

Output File Description
feature_reference.csv Feature reference (contains both CMO and Feature Barcode) used for this sample
raw_cloupe.cloupe A Loupe-readable file containing all cell-associated barcodes in the experiment. This cloupe file also contains UMI counts for all tags (prior to tag assignments), which could be useful for troubleshooting Cell Multiplexing library issues.
raw_feature_bc_matrix A matrix of UMI counts associated with a feature (row) and a barcode (column), in MEX format, including both the GEX and CMO feature counts. This matrix contains every barcode from the fixed list of known good barcode sequences that has at least one read. This includes background and cell-associated barcodes.
raw_feature_bc_matrix.h5 A matrix of UMI counts associated with a feature (row) and a barcode (column), in H5 format, including both the GEX and CMO feature counts. This matrix contains every barcode from the fixed list of known good barcode sequences that has at least one read. This includes background and cell-associated barcodes.
raw_molecule_info.h5 Information about all molecules in the experiment. This file includes background and cell-associated barcodes, and cannot be used as input for cellranger aggr pipeline.
unassigned_alignments.bam Alignments from barcodes not assigned to any sample.
unassigned_alignments.bam.bai Alignments from barcodes not assigned to any sample (index). In cases where the reference transcriptome is generated from a genome with very long chromosomes (>512 Mbp), Cell Ranger v7.0+ generates an unassigned_alignments.bam.csi index file instead.


The multiplexing_analysis directory contains:

Output File Description
assignment_confidence_table.csv A table that contains all information from the tag assignment algorithm for each cell-associated barcode, in CSV format. More details below.
cells_per_tag.json Lists the cell-associated barcodes that were assigned a given tag, for each tag, in JSON format.
tag_calls_summary.csv A table that summarizes the multiplexing results, including the number of cells assigned no tag, one tag, and more than one tag, in CSV format. More details below.
tag_calls_per_cell.csv This table summarizes the assigned tags and the UMI counts per tag for all barcodes assigned as singlets or multiplets in a CSV format. Barcodes called as Unassigned or Blanks are not included in this table. More details below.
barcode_sample_assignments.csv If barcode-sample-assignment was specified in the multi config CSV, a duplicate of the input barcode-sample assignment CSV file will be generated.

Assignment confidence table

The assignment_confidence_table.csv table provides a summary of all the information from the tag-assignment algorithm for each cell-associated barcode, including the probability that a given barcode belongs to a given state. The user may modify the confidence threshold for assigning tags to barcodes on their own in a data-science environment like Python or R to enable further downstream analysis.

Row,CMO301,CMO302,Barcode,Multiplet,Blank,Assignment,Assignment_Probability,CMO301_cnts,CMO302_cnts
0,0.999721559521299,3.4331367105764475e-10,AAACCCAAGAGTGTGC-1,0.0002784107425317906,2.939285533268778e-08,CMO301,0.999721559521299,4.0744873049856905,2.444044795918076
1,2.1178952222935904e-09,0.9999759641250832,AAACCCAAGATGCTTC-1,2.3239920572975783e-06,2.1709764964310346e-05,CMO302,0.9999759641250832,2.6020599913279625,3.8196755199942927
2,0.999822627035718,3.287157579467574e-07,AAACCCAAGGTACAGC-1,8.871810165359213e-05,8.83261468704939e-05,CMO301,0.999822627035718,3.630834517828051,2.3404441148401185
3,0.8947739014808398,9.424785606470267e-09,AAACCCAAGTAGCTCT-1,0.10522608718358256,1.910792092383626e-09,Unassigned,0.8947739014808398,4.219767844658398,2.9916690073799486
4,0.033490762078236785,0.004751323355950833,AAACCCACACGGATCC-1,0.9617539697731167,3.944792695789878e-06,Multiplet,0.9617539697731167,3.6148972160331345,3.4896772916636984Column descriptions:

Column descriptions:

Tag calls summary

The tag_calls_summary.csv summarizes multiplexing results by providing statistics about categories including the number of cells assigned no tag, one tag, more than one tag, etc. The category No tag assigned includes both cells that were considered Blanks and cells considered Unassigned.

Category,num_cells,pct_cells,median_umis,stddev_umis
No tag molecules,0,0.0,None,None
No tag assigned,386,2.9,None,None
1 tag assigned,12465,93.6,None,None
More than 1 tag assigned,472,3.5,None,None
CMO301,6437,48.3,3442.0,7988.3
CMO302,6028,45.2,3515.5,5167.2
CMO301|CMO302,472,3.5,12414.0,9696.0

Column descriptions:

Tag calls per cell

The tag_calls_per_cell.csv file contains tag calls per cell, one line for each barcode. It contains all singlet and multiplet cells; Unassigned or Blanks are not included.

cell_barcode,num_features,feature_call,num_umis
AAACCCAAGCAACAGC-1,1,CMO301,16778
AAACCCAAGCTCGTGC-1,1,CMO301,1735
AAACCCACATGACTGT-1,1,CMO301,1625
AAACCCAGTCCACAGC-1,1,CMO301,19323
AAACCCAGTCGCGGTT-1,1,CMO301,1678

Column descriptions:

Demultiplexed sample outputs directory

The per_sample_outs directory contains sample-level files with data from cells only (background data filtered out):

├── count
│   ├── analysis
│   │   ├── clustering
│   │   ├── diffexp
│   │   ├── pca
│   │   ├── tsne
│   │   └── umap
│   ├── sample_cloupe.cloupe
│   ├── feature_reference.csv
│   ├── sample_alignments.bam
│   ├── sample_alignments.bam.bai
│   ├── sample_filtered_barcodes.csv
│   ├── sample_filtered_feature_bc_matrix
│   │   ├── barcodes.tsv.gz
│   │   ├── features.tsv.gz
│   │   └── matrix.mtx.gz
│   ├── sample_filtered_feature_bc_matrix.h5
│   └── sample_molecule_info.h5
├── metrics_summary.csv
└── web_summary.html

The per_sample_outs directory contains:

Output File Description
count/ Folder containing the results of any gene expression and Feature Barcode analysis, see table below.
metrics_summary.csv Run summary metrics file in CSV format, described in the Cell Multiplexing metrics page.
web_summary.html Run summary metrics and charts in HTML format, described in the multi web summary page.


The count directory contains:

Output File Description
analysis/ Folder containing the results of graph-based clusters and K-means clustering 2-10; differential gene expression analysis between clusters; and PCA, t-SNE, and UMAP dimensionality reduction. Learn more
sample_cloupe.cloupe A Loupe Browser visualization and analysis file, containing only cells assigned to this sample.
feature_reference.csv A duplicate of the input Cell Multiplexing feature reference CSV file
sample_alignments.bam Indexed BAM file containing position-sorted reads aligned to the genome and transcriptome, as well as unaligned reads, annotated with barcode information. Learn more
sample_alignments.bam.bai Index file for the sample_alignments.bam. In cases where the reference transcriptome is generated from a genome with very long chromosomes (>512 Mbp), Cell Ranger v7.0+ generates a sample_alignments.bam.csi index file instead.
sample_filtered_barcodes.csv File containing a list of only cell-associated barcodes.
sample_filtered_feature_bc_matrix/ Contains only detected cell-associated barcodes in MEX format. Each element of the matrix is the number of UMIs associated with a feature (row) and a barcode (column), as described in the feature-barcode matrix page, including both the GEX and CMO feature counts. This file can be input into third-party packages and allows users to wrangle the feature-barcode matrix (e.g. to filter outlier cells, run dimensionality reduction, normalize gene expression).
sample_filtered_feature_bc_matrix.h5 Same information as sample_filtered_feature_bc_matrix in HDF5 format, including both the GEX and CMO feature counts.
sample_molecule_info.h5 Contains per-molecule information for all molecules that contain a valid barcode, valid UMI, and were assigned with high confidence to a gene or Feature Barcode. This file is a required input to run cellranger aggr. Learn more

Antibody Capture/CRISPR Guide Capture outputs

For Cell Multiplexing experiments with Antibody Capture libraries, there will be additional outputs. These files are described in the Antibody outputs overview. The output structure will include an additional antibody_analysis directory in the per_sample_outs/count directory:

├── count
│   ├── analysis
│   ├── sample_cloupe.cloupe
│   ├── antibody_analysis
│   │     └── aggregate_barcodes.csv
│   ├── feature_reference.csv
│   ├── sample_alignments.bam
│   ├── sample_alignments.bam.bai
│   ├── sample_filtered_barcodes.csv
│   ├── sample_filtered_feature_bc_matrix
│   ├── sample_filtered_feature_bc_matrix.h5
│   └── sample_molecule_info.h5
├── metrics_summary.csv
└── web_summary.html

For Cell Multiplexing experiments with CRISPR Guide Capture libraries, there will be additional outputs. These files are described in the CRISPR outputs overview. The output structure will include an additional crispr_analysis directory in the per_sample_outs/count directory:

├── count
│   ├── analysis
│   ├── sample_cloupe.cloupe
│   ├── crispr_analysis
│   │     ├── cells_per_protospacer.json
│   │     ├── feature_reference.csv
│   │     ├── perturbation_effects_by_feature
│   │     ├── perturbation_effects_by_target
│   │     ├── perturbation_efficiencies_by_feature.csv
│   │     ├── perturbation_efficiencies_by_target.csv
│   │     ├── protospacer_calls_per_cell.csv
│   │     ├── protospacer_calls_summary.csv
│   │     ├── protospacer_umi_thresholds.csv
│   │     └── protospacer_umi_thresholds.json
│   ├── feature_reference.csv
│   ├── sample_alignments.bam
│   ├── sample_alignments.bam.bai
│   ├── sample_filtered_barcodes.csv
│   ├── sample_filtered_feature_bc_matrix
│   ├── sample_filtered_feature_bc_matrix.h5
│   └── sample_molecule_info.h5
├── metrics_summary.csv
└── web_summary.html