10x Genomics
Chromium Single Cell Gene Expression

Cell Ranger6.0, printed on 04/28/2024

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 VDJ (GEX + VDJ) data, and in Cell Ranger 6.0 for 3' Cell Multiplexing data.

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

The files in the multi folder are generic to the entire multiplexing experiment, while the files in the per_sample_outs directory have been demultiplexed to single samples.

└─ 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 folder contains files including:

• feature_reference.csv : feature reference used for this sample
• raw_cloupe.cloupe : a Loupe-readable file containing all samples in the experiment
• raw_feature_bc_matrix : raw matrix (including background GEMs) of UMI counts per (feature, barcode) pair, in MEX format
• raw_feature_bc_matrix.h5: raw matrix (including background GEMs) of UMI counts per (feature, barcode) pair, in H5 format
• raw_molecule_info.h5 : information about all molecules in the experiment
• unassigned_alignments.bam : alignments from barcodes not assigned to any sample
• unassigned_alignments.bam.bai : alignments from barcodes not assigned to any sample (index)

The multiplexing_analysis folder contains files including:

• assignment_confidence_table.csv : table that contains all information from the tag assignment algorithm for each cell-associated barcode, in CSV format
• cells_per_tag.json : lists the cell-associated barcodes that were assigned a given tag, for each tag, in JSON format
• tag_calls_per_cell.csv : table that summarizes, for each cell-associated barcode assigned a tag, the tags assigned and the UMI counts per tag, in CSV format
• tag_calls_summary.csv : table that summarizes the multiplexing results, including the number of cells assigned no tag, 1 tag, and more than 1 tag, in CSV format

These files are further described here.

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 used for assigning tags to barcodes on their own in a data-science environment like Python or R to enable further downstream analysis, if they so desire.

Row,CMO301,CMO302,Barcodes,Multiplet,Blanks,Assignment,Assignment_Probability
1,1.7492220542497986e-10,0.999994,AAACCCAAGATTCGAA-1,5.0927025002342424e-06,7.880223262070917e-07,CMO302,0.999994
2,0.9936742,1.95217130055954e-09,AAACCCAAGCAACAGC-1,0.0063257301365501695,7.021953108383088e-09,CMO301,0.9936742
13,2.3960595409731583e-08,0.0064328,AAACCCATCATTCGGA-1,0.993567110839031,3.554702514325053e-12,Multiplet,0.9935671
35,2.74875783854256e-12,0.00021014,AAACGCTGTCCAAGAG-1,1.3255406427923249e-17,0.9997898543154408,Blanks,0.999789
39,1.4235671575612072e-05,0.55488715,AAACGCTGTTCTTCAT-1,4.071660981663313e-07,0.44509820,Unassigned,0.55488715

Column descriptions:

• CMO301, CMO302, etc.: one column per tag used in the experiment, to indicate the probability that a given barcode belongs to each of those singlet states
• Barcodes: the cell-associated barcode
• Multiplet: the coarse-grained Multiplet state, which contains the probability that the given barcode is some-type of multiplet. Obtained by summing the probabilities across each possible multiplet state in the experiment
• Blanks: contains the probability that the barcode contains un-tagged cells i.e. is considered to have too few tag counts for every tag
• Assignment: the state assigned by the tag-assignment algorithm for the given barcode. Values: one of the singlet states, Multiplet, Blanks, or Unassigned (if every state has probability less than 90%, which is the default minimum confidence threshold.)
• Assignment_Probability: the probability of the most-likely state

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:

• num_cells: number of cells in the category
• pct_cells: percent of cells in the category
• median_umis: median UMI counts for the tag(s), amongst cells assigned those tag(s)
• stddev_umis: standard deviation of the UMI counts for the tag(s), amongst cells assigned those tag(s)

The tag_calls_per_cell.csv file contains tag calls per cell, one line for each barcode.

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:

• cell_barcode: cell-barcode
• num_features: number of tags assigned to that cell
• feature_call: names of tag(s) assigned, de-limited by "|"
• num_umis: number of molecules (UMIs) for each tag assigned, de-limited by "|"