Cell Ranger7.1, printed on 11/05/2024
Cell Ranger outputs certain files that are specific to the Antibody Capture analysis, besides the Gene Expression outputs.
Starting from Cell Ranger 3.0, all Feature Barcode counts, including Antibody Capture counts, simply become new features in addition to the standard per-gene features, and are output alongside gene counts in the feature-barcode matrix. For every row in the Feature Barcode Reference CSV file where feature_type is specified as Antibody Capture, there will be a corresponding row in the feature-barcode matrix. That row will get its title from the id field in the Feature Reference file for that feature, and the counts can be visualized via Loupe Browser by searching for the human-readable name from the name field of the Feature Reference file (for antibody applications, the id and name fields can typically be the same as long as the id is unique).
To visualize cells in 2-D space, secondary analysis dimensionality reduction outputs for Antibody Capture libraries are provided in the analysis/ directory. Log-transformed antibody counts are used to perform these analyses for Antibody Capture libraries. This is in contrast to the gene expression side of the feature-barcode matrix, where these projections are run on the PCA-reduced space from raw counts.
Below are some examples of the PCA, t-SNE, and UMAP output projection files.
Principal Components Analysis (PCA):
$ head -5 analysis/pca/antibody_capture_10_components/projection.csv Barcode,PC-1,PC-2,PC-3,PC-4,PC-5,PC-6,PC-7,PC-8,PC-9,PC-10 AAACAAGCACCATACT-1,-5.574515404720648,4.1250677853049735,0.3343758325171491,-0.9529782537962408,-1.8811942105099764,-0.4217695409442901,-1.9900329330389255,-1.2255017468251315,-1.3980947791205285,-1.1176859809904909 AAACAAGCACGTAATG-1,-6.983452898884609,-1.9379476767294177,-0.042479446422044376,-1.264967360758824,4.167549425417305,0.12065395835962933,-0.707084060668425,-2.9769215409849656,0.9053984182888417,-0.061563257127632665 AAACAAGCATGCAATG-1,-4.430486543723384,3.7442086078976002,-0.9447490398187632,-1.9902233589725338,-0.6258151384415838,-1.3582451690099415,-0.107256076231657,-1.6254493516586832,-0.43820589495677176,2.3253990939137505 AAACAAGCATTTGGGA-1,-4.945904594634436,4.017097394368968,-0.16688953081917113,-0.5729444140584459,-1.8303228981840096,-0.7755095535305054,-1.4069565944426259,-0.7969252558721216,-0.0011689859429466765,0.39202448730849027
The t-distributed Stochastic Neighbor Embedding (t-SNE):
$ head -5 analysis/tsne/antibody_capture_2_components/projection.csv Barcode,TSNE-1,TSNE-2 AAACCCAAGTGGTCAG-1,-29.97926190939189,-3.5125258285933603 AAAGGTATCAACTACG-1,20.762905594110116,-6.946344013493825 AAAGTCCAGCGTGTCC-1,11.156075443007484,-5.489821984514518 AACACACTCAAGAGTA-1,-26.08126312702518,-5.167458628104057
The Uniform Manifold Approximation and Projection (UMAP):
$ head -5 analysis/umap/antibody_capture_2_components/projection.csv Barcode,UMAP-1,UMAP-2 AAACGAAAGACCAGCA-1,-0.27067992,-6.089176 AAACGAAAGACGGAAA-1,-6.584536,4.711372 AAACGAAAGCGAAACC-1,-7.544384,5.097423 AAACGAATCAATCTCT-1,2.8123982,3.3911235
Cell Ranger 6.1 and later outputs detailed information about detected aggregate barcodes in a file named aggregate_barcodes.csv. If your data was analyzed using cellranger multi v7.1 and later, this output is found under the outs/per_sample_outs/<sample_name>/count/aggregate_barcodes.csv folder, and in outs/ for the cellranger count pipeline. In older versions of Cell Ranger, the aggregate_barcodes.csv is contained in a subfolder called /count/antibody_analysis/aggregate_barcodes.csv.
aggregate_barcodes.csv shows antibody (and antigen) barcodes that were detected as aggregates, the number of UMI-corrected reads, the fraction of reads corrected for a particular barcode, and the fraction of total reads on that barcode.
$ head -5 antibody_analysis/aggregate_barcodes.csv barcode,library_type,umis,umi_corrected_reads,frac_corrected_reads,frac_total_reads ATCGTAGCAGCGTATT-1,Antibody Capture,1976,1965,0.016,0.002 CTACGGGGTTCTCGTC-1,Antibody Capture,2147,1900,0.014,0.002 CTCCGATGTAGTAAGT-1,Antibody Capture,2999,2968,0.018,0.002 CTCCGATGTTGATGTC-1,Antibody Capture,3691,2878,0.02,0.003
The aggregate_barcodes.csv is only produced if antibody or antigen aggregates are detected.
Summary metrics for the antibody counts will be displayed in the Summary view of the count web summary under Antibody Sequencing and Antibody Application, and in the Cells and Library views of the multi web summary under the Antibody tabs.
Starting from Cell Ranger 6.1, on both count and multi web summaries, the histograms on UMI counts will be shown for features that were specified as Antibody Capture in the Feature Barcode reference file.
Any specific antibody analysis steps are described in the Antibody Algorithms section.