Cell Ranger7.1, printed on 02/21/2024
antigen capture, the Feature Reference CSV that specifies the list of antigens (and MHC alleles) included in the experiment, and all the antigen specific customizable parameters in a multi config CSV are described in detail. Example multi config CSV for TCR and BCR Antigen Capture libraries are also provided.
antigen_specificity_scores.csv (new file)
per_barcode.csv (new file)
aggregate_barcodes.csv (updated location and format)
These genes have counterparts with identical V, D, J, and C gene sequences, but differ in the length of their 5' UTRs. Removing duplicates improves clonotype assignment.
|These updates are explained further in this Knowledge Base article: What are the major updates in Cell Ranger v7.1 that impacts V(D)J data?
region column, which indicates whether a probe spans a splice junction by at least 10 bp (spliced) or not (unspliced).
metrics_summary.csv files will include genomic DNA metrics. The
region column information is used to calculate these metrics.
molecule_info.h5 files include the probe to which each molecule is mapped.
included column is set to
FALSE for all deprecated probes.
expect-cells upper range has been restricted to the lower range of the EmptyDrops method. Previously, the upper range was 262,000 cells. The new upper range is 45,000 for single cell gene expression analyses. The exception is super-loaded multiplex Fixed RNA Profiling analyses, where the range is calculated as:
max(45,000, number of probe barcodes × 22,500).
.tar.xz, is available on the Cell Ranger downloads page. The smaller file size enables faster download.
ARC-v1 chemistry may be used to analyze only the Gene Expression library portion of a Multiome ATAC + Gene Expression experiment.
aggregate_barcodes.csv output file for Antibody Capture analyses is no longer stored in a
antibody_analysis/ sub-directory. In the cellranger multi pipeline, it is found in
outs/per_sample_outs/<sample_name>/count/aggregate_barcodes.csv. In the cellranger count pipeline, it is found in
outs/aggregate_barcodes.csv. For both pipelines, the CSV file is only generated if antibody aggregates are detected.
cmo-set instead of those used in the experiment.
The updates are explained further in these Knowledge Base articles:
Undetermined/ because the orientation of i5 (Index2) could not be autodetected.
Support for gamma-delta libraries: The cellranger multi pipeline can process T cell receptor (TCR) libraries enriched for gamma (TRG) and delta (TRD) chains. 10x Genomics does not officially support TRG/D analysis with a reagent kit. Please note that, only CDR3 annotation is available for TRG/D, and the quality of annotations cannot be guaranteed. Users must specify
VDJ-T-GD as the
feature_type in the cellranger multi config CSV as TRG/D chains cannot be autodetected. A
web_summary alert is displayed to indicate the use of an unsupported workflow. No TRG/D analysis is available via the cellranger vdj pipeline.
V(D)J Reference updated: The recommended V(D)J reference packages for human and mouse have been updated from version 5.0 to 7.0. The changes to the V(D)J reference sequences are listed below:
V(D)J web summary: In the
web_summary.html file produced by cellranger vdj, the Analysis tab has been renamed to VDJ.
The default for the
fiveprime_multiplexing parameter in the
cellranger-7.0.0/lib/bin/parameters.toml file has changed to
probe-set, to specify the probe set CSV file. Output files are described in the Understanding Outputs section. The Fixed RNA Profiling algorithms section includes descriptions of the new methods that were developed for processing Fixed RNA Profiling data.
To maximize sensitivity for whole transcriptome 3’/5’ Single Cell Gene Expression and 3’ Cell Multiplexing experiments, introns will be included in the analysis by default for cellranger count and multi. There will be an informational alert in the count and multi web summaries to indicate that intronic reads were included in your analysis. While not recommended, users can exclude introns by setting
include-introns=false in Cell Ranger. This change does not apply to the 3’/5’ Targeted Gene Expression or Fixed RNA Profiling assays, as both target exonic sequences. Learn more here.
CRISPR Guide Capture libraries can be aggregated with cellranger aggr. This addition allows users to combine large CRISPR assays across multiple GEM wells. There are no changes to aggr inputs – the presence of CRISPR libraries in the
molecule_info.h5 input files enables CRISPR aggregation. Normalization is enabled by default for both Gene Expression and CRISPR libraries; changes to the normalization parameters affect both libraries. Protospacer calling is performed again on the combined data included in the cellranger aggr run. CRISPR aggregation generates the
crispr_analysis folder in the
outs/ directory. The structure of the
crispr_analysis folder is similar to the CRISPR outputs from count.
Users no longer need to specify
expect-cells for cellranger count and multi pipelines due to improvements in the gene expression cell calling algorithm. The expected number of cells can either be auto-estimated (recommended) or users can still provide a reasonable estimate to
check-library-compatibility option allows users to disable the default check for 10x Barcode overlap when multiple libraries are specified for cellranger count and multi (3' Gene Expression, 5' Immune Profiling).
For 3’ Cell Multiplexing analysis in cellranger multi, users can override Cell Ranger’s cell calling and tag calling algorithms with the custom cell assignment input file specified by the
barcode-sample-assignment option in the multi config CSV file.
Modifications to the 3’ Cell Multiplexing CMO tag calling algorithm enable users to recover viable singlet data from “blank” assignments.
The following per-sample output files from cellranger multi have been renamed:
|Cell Ranger 6.1.2 outputs
|Cell Ranger 7.0 outputs
6. Secondary analysis outputs will be named to reflect which library they are specific to (
multiplexing_capture_*). The secondary analysis clustering, PCA/t-SNE/UMAP, and differential gene expression outputs are supported for Gene Expression and Antibody Capture libraries, while PCA/t-SNE/UMAP outputs are supported for CRISPR Guide Capture and Cell Multiplexing libraries. For example:
└── analysis └── pca ├── antibody_capture_10_components/ └── gene_expression_10_components/
The cellranger count web summary “Analysis” tab has been renamed to “Gene Expression”. There is an “Antibody” tab for Antibody Capture analysis, which includes a t-SNE projection plot by clustering and a histogram of antibody counts.
The cellranger multi web summary (3' Gene Expression, 5' Immune Profiling) “Sample” view has been renamed to “Cells”. The “Antibody” tab includes a t-SNE projection plot by clustering. The mapping metrics, sequencing saturation plot, and median genes per cell plot are displayed on the “Library” view (previously appeared on “Sample” and “Library” view).
Cell Ranger can now ingest FASTQs with a quality score up to the full supported range (93 instead of 41).
Improved error messages and better handling of poorly formatted inputs in cellranger mkref. Enable users to generate references for analyses with large genomes containing chromosomes longer than 512 Mbp. cellranger count and multi pipelines will output a
.csi BAM index file instead of
.bai in these cases.
Fixed a bug that resulted in a segmentation fault error when mapping to references that contain small contigs, for example, the rabbit genome.
Inconsistent Throughput Detected alert in web summary when it should not appear.
Fixed a bug where VDJ pipeline failed for specific CentOS/RHEL 7 kernels.
Bundles the latest version of bamtofastq (v1.4.1) in Cell Ranger 7.0 tarball.
Fixed a bug where bamtofastq failed if the R1 read length was >26bp.