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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 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:

cellranger web summary

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:

The multiplexing_analysis folder contains files including:

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:

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:

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: