10x Genomics Support/Cell Ranger 7.2/Analysis/

Cell Ranger Fixed RNA Profiling Outputs Overview

This page describes the output file structure from the cellranger multi subcommand specifically for Fixed RNA Profiling (FRP) data.

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


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 FRP experiment, while the files in the per_sample_outs/ directory have been demultiplexed to single samples.

Within the multi/ directory, there are count and multiplexing_analysis directories. The multiplexing_analysis sub-directory is only generated in the context of multiplex Fixed RNA Profiling inputs.

└─ multi ├── count │ ├── 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 │ ├── unassigned_alignments.bam │ └── unassigned_alignments.bam.bai └── multiplexing_analysis ├── cells_per_tag.json └── frp_gem_barcode_overlap.csv

The count directory contains raw files that include cell-associated and background data from all samples within an experiment:

Output FileDescription
raw_cloupe.cloupeA Loupe-readable file containing all cell-associated barcodes in the experiment.
raw_feature_bc_matrixA matrix of UMI counts per (feature, barcode) pair, in MEX format. This matrix contains every barcode from the full fixed list of known good barcode sequences that has at least one read. This includes background and cell-associated barcodes from all samples as well as valid barcodes that were not assigned to a sample in case of a multiplex Fixed RNA Profiling experiment. Distinct ligation events are counted for Fixed RNA Profiling rather than distinct transcripts.
raw_feature_bc_matrix.h5Same information as raw_feature_bc_matrix in HDF5 format.
raw_molecule_info.h5Information about all molecules in the experiment. This file includes background and cell-associated barcodes from all samples as well as valid barcodes that were not assigned to a sample. Starting in Cell Ranger v7.1, this file also includes UMI counts per probe. This file cannot be used as input for cellranger aggr pipeline.
raw_probe_bc_matrix.h5Contains UMI counts of each probe for all detected barcodes in HDF5 format.
unassigned_alignments.bamReads with either valid barcodes not assigned to a sample or invalid barcodes. Only generated if no-bam,false in config CSV.
unassigned_alignments.bam.baiIndexed file for unassigned_alignments.bam. Only generated if no-bam,false in config CSV.

The multiplexing_analysis directory contains:

Output FileDescription
cells_per_tag.jsonLists the cell-associated barcodes that were assigned a given Probe Barcode tag, for each tag, in JSON format.
frp_gem_barcode_overlap.csvContains 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. More details below.

The cells_per_tag.json file looks like this, for each Probe Barcode (e.g., BC001) the cell-associated barcodes (e.g., "AACAAGCTCCCTCAAAACTTTAGG-1", etc.) are listed below it:

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

The frp_gem_barcode_overlap.csv 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%. Please contact [email protected] if you have questions.

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 = Common GEMSmin(Barcode 1 GEMS, Barcode 2 GEMS)\frac{{\text{Common GEMS}}} {\text{min(Barcode 1 GEMS, Barcode 2 GEMS)}}

The per_sample_outs directory contains sample-level files with any data associated with a valid Probe Barcode that could be assigned to a sample, including both cells and background.

├── count │ ├── analysis │ │ ├── clustering │ │ ├── diffexp │ │ ├── pca │ │ ├── tsne │ │ └── umap │ ├── probe_set.csv │ ├── sample_cloupe.cloupe │ ├── 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_raw_feature_bc_matrix │ │ ├── barcodes.tsv.gz │ │ ├── features.tsv.gz │ │ └── matrix.mtx.gz │ ├── sample_raw_feature_bc_matrix.h5 │ ├── sample_molecule_info.h5 | └── sample_raw_probe_bc_matrix.h5 ├── metrics_summary.csv └── web_summary.html
Note that the sample_filtered_feature_bc_matrix directory and sample_filtered_feature_bc_matrix.h5 file are similar to the filtered_feature_bc_matrix and filtered_feature_bc_matrix.h5, respectively, generated by cellranger count. For more information about these files, see the Feature Barcode matrices section. These are the key files that contain expression levels, which can be used for downstream analysis and data interpretation.

The per_sample_outs directory contains:

Output FileDescription
count/Folder containing the results of any gene expression and Feature Barcode analysis, see table below.
metrics_summary.csvmetrics_summary.csv Run summary metrics file in CSV format. Metric definitions available in the web summary ? help text.
web_summary.htmlRun summary metrics and charts in HTML format, described in the multi web summary page.

The count directory contains:

Output FileDescription
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
probe_set.csvA duplicate of the input probe_set.csv file.
sample_cloupe.cloupeA Loupe Browser visualization and analysis file with cell-associated barcodes for the specific sample.
sample_filtered_feature_bc_matrix/Contains only detected cell-associated barcodes and only genes in the filtered probe set 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. Distinct ligation events are counted for Fixed RNA Profiling rather than distinct transcripts. This file can be input into third-party packages and allows users to wrangle the barcode-feature matrix (e.g. to filter outlier cells, run dimensionality reduction, normalize gene expression).
sample_filtered_feature_bc_matrix.h5Same information as sample_filtered_feature_bc_matrix in HDF5 format.
sample_molecule_info.h5Contains per-molecule information for all molecules counted in the sample_raw_feature_bc_matrix for this sample that contain a valid barcode, valid UMI, and were assigned with high confidence to a gene or Feature Barcode. Starting in Cell Ranger v7.1, this file also includes UMI counts per probe. This file is a required input to run cellranger aggr. Learn more
sample_alignments.bamIndexed BAM file containing position-sorted reads aligned to the genome and transcriptome, as well as unaligned reads, annotated with barcode information. Learn more. Only generated if no-bam,false in config CSV.
sample_alignments.bam.baiIndex file for the sample_alignments.bam. Only generated if no-bam,false in config CSV.
sample_filtered_barcodes.csvFile containing a list of only cell-associated barcodes.
sample_raw_feature_bc_matrix/Contains all barcodes assigned to this sample, including cell-associated and background barcodes in MEX format. Genes in this matrix include ones with DEPRECATED probes and probes with predicted off-target activities, as well as the ones in the filtered probe set. 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. Distinct ligation events are counted for Fixed RNA Profiling rather than distinct transcripts. This file is only generated in the context of multiplex Fixed RNA Profiling configuration.
sample_raw_feature_bc_matrix_h5Same information as sample_raw_feature_bc_matrix in HDF5 format. This file is only generated in the context of multiplex Fixed RNA Profiling configuration.
sample_raw_probe_bc_matrix.h5Contains columns that indicate the probes in the filtered probe reference, the probes that passed gDNA filtering, and the probe barcodes that are in cells. It is similar to the feature-barcode matrix, but is organized at the probe level rather than the gene level.

Probes are flagged as not passing the gDNA filter if their aggregate count across all cells have fewer UMI counts than the metric "Estimated UMIs from genomic DNA per unspliced probe".

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

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

Within the multi/ directory, there is a count/ directory. This directory contains the same files as described above for the multiplex Fixed RNA Profiling count/ directory. There is no multiplexing_analysis/ directory for singleplex experiments.

└─ multi └── count ├── 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 ├── unassigned_alignments.bam └── unassigned_alignments.bam.bai

The per_sample_outs directory contains data associated with all valid barcodes in the singleplex Fixed RNA Profiling library. This directory contains the same files as described above for the multiplex Fixed RNA Profiling per_sample_outs directory, except for the sample_raw_feature_bc_matrix/ and sample_raw_feature_bc_matrix.h5 files. The web_summary.html is described on the multi web summary page.

├── count │ ├── analysis │ │ ├── clustering │ │ ├── diffexp │ │ ├── pca │ │ ├── tsne │ │ └── umap │ ├── probe_set.csv │ ├── sample_cloupe.cloupe │ ├── 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 │ └── sample_raw_probe_bc_matrix.h5 ├── metrics_summary.csv └── web_summary.html

For Fixed RNA Profiling singleplex or multiplex analyses with an Antibody Capture library, the per_sample_outs/count directory will also contain a feature_reference.csv, which is a duplicate of the input feature reference CSV file.

├── count │ ├── analysis │ │ ├── clustering │ │ ├── diffexp │ │ ├── pca │ │ ├── tsne │ │ └── umap │ ├── aggregate_barcodes.csv │ ├── feature_reference.csv │ ├── ...

The /multi/count directory also contains a copy of the feature_reference.csv file:

└─ multi ├── count │ ├── raw_cloupe.cloupe │ ├── raw_feature_bc_matrix │ │ ├── barcodes.tsv.gz │ │ ├── features.tsv.gz │ │ └── matrix.mtx.gz │ ├── feature_reference.csv │ ├── raw_feature_bc_matrix.h5 │ ├── raw_molecule_info.h5 │ ├── unassigned_alignments.bam │ └── unassigned_alignments.bam.bai

The web_summary.html is described on the multi web summary page.