workflows/ingestion/cellranger_multi

Description

A pipeline for running Cell Ranger multi.

Input files

Name	Type & Properties	Description
--input	file multiple	The FASTQ files to be analyzed. FASTQ files should conform to the naming conventions of bcl2fastq and mkfastq: `[Sample Name]_S[Sample Index]_L00[Lane Number]_[Read Type]_001.fastq.gz`

Name

Type & Properties

--input

file

multiple

Feature type-specific input files

Name	Type & Properties	Description
--gex_input	file multiple	The FASTQ files to be analyzed for Gene Expression. FASTQ files should conform to the naming conventions of bcl2fastq and mkfastq: `[Sample Name]_S[Sample Index]_L00[Lane Number]_[Read Type]_001.fastq.gz`
--abc_input	file multiple	The FASTQ files to be analyzed for Antibody Capture. FASTQ files should conform to the naming conventions of bcl2fastq and mkfastq: `[Sample Name]_S[Sample Index]_L00[Lane Number]_[Read Type]_001.fastq.gz`
--cgc_input	file multiple	The FASTQ files to be analyzed for CRISPR Guide Capture. FASTQ files should conform to the naming conventions of bcl2fastq and mkfastq: `[Sample Name]_S[Sample Index]_L00[Lane Number]_[Read Type]_001.fastq.gz`
--mux_input	file multiple	The FASTQ files to be analyzed for Multiplexing Capture. FASTQ files should conform to the naming conventions of bcl2fastq and mkfastq: `[Sample Name]_S[Sample Index]_L00[Lane Number]_[Read Type]_001.fastq.gz`
--vdj_input	file multiple	The FASTQ files to be analyzed for VDJ. FASTQ files should conform to the naming conventions of bcl2fastq and mkfastq: `[Sample Name]_S[Sample Index]_L00[Lane Number]_[Read Type]_001.fastq.gz`
--vdj_t_input	file multiple	The FASTQ files to be analyzed for VDJ-T. FASTQ files should conform to the naming conventions of bcl2fastq and mkfastq: `[Sample Name]_S[Sample Index]_L00[Lane Number]_[Read Type]_001.fastq.gz`
--vdj_t_gd_input	file multiple	The FASTQ files to be analyzed for VDJ-T-GD. FASTQ files should conform to the naming conventions of bcl2fastq and mkfastq: `[Sample Name]_S[Sample Index]_L00[Lane Number]_[Read Type]_001.fastq.gz`
--vdj_b_input	file multiple	The FASTQ files to be analyzed for VDJ-B. FASTQ files should conform to the naming conventions of bcl2fastq and mkfastq: `[Sample Name]_S[Sample Index]_L00[Lane Number]_[Read Type]_001.fastq.gz`
--agc_input	file multiple	The FASTQ files to be analyzed for Antigen Capture. FASTQ files should conform to the naming conventions of bcl2fastq and mkfastq: `[Sample Name]_S[Sample Index]_L00[Lane Number]_[Read Type]_001.fastq.gz`

Name

Type & Properties

--gex_input

file

multiple

--abc_input

file

multiple

--cgc_input

file

multiple

--mux_input

file

multiple

--vdj_input

file

multiple

--vdj_t_input

file

multiple

--vdj_t_gd_input

file

multiple

--vdj_b_input

file

multiple

--agc_input

file

multiple

Library arguments

Name	Type & Properties	Description
--library_id	string multiple	The Illumina sample name to analyze. This must exactly match the 'Sample Name'part of the FASTQ files specified in the `--input` argument.
--library_type	string multiple	The underlying feature type of the library.
--library_subsample	string multiple	The rate at which reads from the provided FASTQ files are sampled. Must be strictly greater than 0 and less than or equal to 1.
--library_lanes	string multiple	Lanes associated with this sample. Defaults to using all lanes.
--library_chemistry	string	Only applicable to FRP. Library-specific assay configuration. By default, the assay configuration is detected automatically. Typically, users will not need to specify a chemistry.

Name

Type & Properties

--library_id

string

multiple

--library_type

string

multiple

--library_subsample

string

multiple

--library_lanes

string

multiple

--library_chemistry

string

Sample parameters

Name	Type & Properties	Description
--sample_ids --cell_multiplex_sample_id	string multiple	A name to identify a multiplexed sample. Must be alphanumeric with hyphens and/or underscores, and less than 64 characters. Required for Cell Multiplexing libraries.
--sample_description --cell_multiplex_description	string multiple	A description for the sample.
--sample_expect_cells	integer multiple	Expected number of recovered cells, used as input to cell calling algorithm.
--sample_force_cells	integer multiple	Force pipeline to use this number of cells, bypassing cell detection.

Name

Type & Properties

--sample_ids

--cell_multiplex_sample_id

string

multiple

--sample_description

--cell_multiplex_description

string

multiple

--sample_expect_cells

integer

multiple

--sample_force_cells

integer

multiple

Feature Barcode library specific arguments

Name	Type & Properties	Description
--feature_reference	file	Path to the Feature reference CSV file, declaring Feature Barcode constructs and associated barcodes. Required only for Antibody Capture or CRISPR Guide Capture libraries. See https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/using/feature-bc-analysis#feature-ref for more information."
--feature_r1_length	integer	Limit the length of the input Read 1 sequence of V(D)J libraries to the first N bases, where N is the user-supplied value. Note that the length includes the Barcode and UMI sequences so do not set this below 26.
--feature_r2_length	integer	Limit the length of the input Read 2 sequence of V(D)J libraries to the first N bases, where N is a user-supplied value. Trimming occurs before sequencing metrics are computed and therefore, limiting the length of Read 2 may affect Q30 scores.
--min_crispr_umi	integer	Set the minimum number of CRISPR guide RNA UMIs required for protospacer detection. If a lower or higher sensitivity is desired for detection, this value can be customized according to specific experimental needs. Applicable only to datasets that include a CRISPR Guide Capture library.

Name

Type & Properties

--feature_reference

file

--feature_r1_length

integer

--feature_r2_length

integer

--min_crispr_umi

integer

Gene expression arguments

Name	Type & Properties	Description
--gex_reference	file required	Genome refence index built by Cell Ranger mkref.
--gex_secondary_analysis	boolean	Whether or not to run the secondary analysis e.g. clustering.
--gex_generate_bam	boolean	Whether to generate a BAM file.
--tenx_cloud_token_path	file	The 10x Cloud Analysis user token used to enable cell annotation.
--cell_annotation_model	string	"Cell annotation model to use. If auto, uses the default model for the species. If not given, does not run cell annotation."
--gex_expect_cells	integer	Expected number of recovered cells, used as input to cell calling algorithm.
--gex_force_cells	integer	Force pipeline to use this number of cells, bypassing cell detection.
--gex_include_introns	boolean	Whether or not to include intronic reads in counts. This option does not apply to Fixed RNA Profiling analysis.
--gex_r1_length	integer	Limit the length of the input Read 1 sequence of V(D)J libraries to the first N bases, where N is the user-supplied value. Note that the length includes the Barcode and UMI sequences so do not set this below 26.
--gex_r2_length	integer	Limit the length of the input Read 2 sequence of V(D)J libraries to the first N bases, where N is a user-supplied value. Trimming occurs before sequencing metrics are computed and therefore, limiting the length of Read 2 may affect Q30 scores.
--gex_chemistry	string	Assay configuration. Either specify a single value which will be applied to all libraries, or a number of values that is equal to the number of libararies. The latter is only applicable to only applicable to Fixed RNA Profiling. - auto: Chemistry autodetection (default) - threeprime: Single Cell 3' - SC3Pv1, SC3Pv2, SC3Pv3(-polyA), SC3Pv4(-polyA): Single Cell 3' v1, v2, v3, or v4 - SC3Pv3HT(-polyA): Single Cell 3' v3.1 HT - SC-FB: Single Cell Antibody-only 3' v2 or 5' - fiveprime: Single Cell 5' - SC5P-PE: Paired-end Single Cell 5' - SC5P-PE-v3: Paired-end Single Cell 5' v3 - SC5P-R2: R2-only Single Cell 5' - SC5P-R2-v3: R2-only Single Cell 5' v3 - SCP5-PE-v3: Single Cell 5' paired-end v3 (GEM-X) - SC5PHT : Single Cell 5' v2 HT - SFRP: Fixed RNA Profiling (Singleplex) - MFRP: Fixed RNA Profiling (Multiplex, Probe Barcode on R2) - MFRP-R1: Fixed RNA Profiling (Multiplex, Probe Barcode on R1) - MFRP-RNA: Fixed RNA Profiling (Multiplex, RNA, Probe Barcode on R2) - MFRP-Ab: Fixed RNA Profiling (Multiplex, Antibody, Probe Barcode at R2:69) - MFRP-Ab-R2pos50: Fixed RNA Profiling (Multiplex, Antibody, Probe Barcode at R2:50) - MFRP-RNA-R1: Fixed RNA Profiling (Multiplex, RNA, Probe Barcode on R1) - MFRP-Ab-R1: Fixed RNA Profiling (Multiplex, Antibody, Probe Barcode on R1) - ARC-v1 for analyzing the Gene Expression portion of Multiome data. If Cell Ranger auto-detects ARC-v1 chemistry, an error is triggered. See https://kb.10xgenomics.com/hc/en-us/articles/115003764132-How-does-Cell-Ranger-auto-detect-chemistry- for more information.

Name

Type & Properties

--gex_reference

file

required

--gex_secondary_analysis

boolean

--gex_generate_bam

boolean

--tenx_cloud_token_path

file

--cell_annotation_model

string

--gex_expect_cells

integer

--gex_force_cells

integer

--gex_include_introns

boolean

--gex_r1_length

integer

--gex_r2_length

integer

--gex_chemistry

string

VDJ related parameters

Name	Type & Properties	Description
--vdj_reference	file	VDJ refence index built by Cell Ranger mkref.
--vdj_inner_enrichment_primers	file	V(D)J Immune Profiling libraries: if inner enrichment primers other than those provided in the 10x Genomics kits are used, they need to be specified here as a text file with one primer per line.
--vdj_r1_length	integer	Limit the length of the input Read 1 sequence of V(D)J libraries to the first N bases, where N is the user-supplied value. Note that the length includes the Barcode and UMI sequences so do not set this below 26.
--vdj_r2_length	integer	Limit the length of the input Read 2 sequence of V(D)J libraries to the first N bases, where N is a user-supplied value. Trimming occurs before sequencing metrics are computed and therefore, limiting the length of Read 2 may affect Q30 scores
--vdj_denovo	boolean	Run in reference-free mode (i.e., do not use annotations). This option is not supported for multiplexed experiments.

Name

Type & Properties

--vdj_reference

file

--vdj_inner_enrichment_primers

file

--vdj_r1_length

integer

--vdj_r2_length

integer

--vdj_denovo

boolean

3' Cell multiplexing parameters (CellPlex Multiplexing)

Name	Type & Properties	Description
--cell_multiplex_oligo_ids --cmo_ids	string multiple	The Cell Multiplexing oligo IDs used to multiplex this sample. If multiple CMOs were used for a sample, separate IDs with a pipe (e.g., CMO301\|CMO302). Required for Cell Multiplexing libraries.
--min_assignment_confidence	double	The minimum estimated likelihood to call a sample as tagged with a Cell Multiplexing Oligo (CMO) instead of "Unassigned". Users may wish to tolerate a higher rate of mis-assignment in order to obtain more singlets to include in their analysis, or a lower rate of mis-assignment at the cost of obtaining fewer singlets.
--cmo_set	file	Path to a custom CMO set CSV file, declaring CMO constructs and associated barcodes. If the default CMO reference IDs that are built into the Cell Ranger software are required, this option does not need to be used.
--barcode_sample_assignment	file	Path to a barcode-sample assignment CSV file that specifies the barcodes that belong to each sample.

Name

Type & Properties

--cell_multiplex_oligo_ids

--cmo_ids

string

multiple

--min_assignment_confidence

double

--cmo_set

file

--barcode_sample_assignment

file

Hashtag multiplexing parameters

Name	Type & Properties	Description
--hashtag_ids	string multiple	The hashtag IDs used to multiplex this sample. If multiple antibody hashtags were used for the same sample, you can separate IDs with a pipe.

Name

Type & Properties

--hashtag_ids

string

multiple

On-chip multiplexing parameters

Name	Type & Properties	Description
--ocm_barcode_ids	string multiple	The OCM barcode IDs used to multiplex this sample. Must be one of OB1, OB2, OB3, OB4. If multiple OCM Barcodes were used for the same sample, you can separate IDs with a pipe (e.g., OB1\|OB2).

Name

Type & Properties

--ocm_barcode_ids

string

multiple

Flex multiplexing paramaters

Name	Type & Properties	Description
--probe_set	file	A probe set reference CSV file. It specifies the sequences used as a reference for probe alignment and the gene ID associated with each probe. It must include 4 columns (probe file format 1.0.0): gene_id,probe_seq,probe_id,included,region and an optional 5th column (probe file format 1.0.1). - gene_id: The Ensembl gene identifier targeted by the probe. - probe_seq: The nucleotide sequence of the probe, which is complementary to the transcript sequence. - probe_id: The probe identifier, whose format is described in Probe identifiers. - included: A TRUE or FALSE flag specifying whether the probe is included in the filtered counts matrix output or excluded by the probe filter. See filter-probes option of cellranger multi. All probes of a gene must be marked TRUE in the included column for that gene to be included. - region: Present only in v1.0.1 probe set reference CSV. The gene boundary targeted by the probe. Accepted values are spliced or unspliced. The file also contains a number of required metadata fields in the header in the format #key=value: - panel_name: The name of the probe set. - panel_type: Always predesigned for predesigned probe sets. - reference_genome: The reference genome build used for probe design. - reference_version: The version of the Cell Ranger reference transcriptome used for probe design. - probe_set_file_format: The version of the probe set file format specification that this file conforms to.
--filter_probes	boolean	If 'false', include all non-deprecated probes listed in the probe set reference CSV file. If 'true' or not set, probes that are predicted to have off-target activity to homologous genes are excluded from analysis. Not filtering will result in UMI counts from all non-deprecated probes, including those with predicted off-target activity, to be used in the analysis. Probes whose ID is prefixed with DEPRECATED are always excluded from the analysis.
--probe_barcode_ids	string multiple	The Fixed RNA Probe Barcode ID used for this sample, and for multiplex GEX + Antibody Capture libraries, the corresponding Antibody Multiplexing Barcode IDs. 10x recommends specifying both barcodes (e.g., BC001+AB001) when an Antibody Capture library is present. The barcode pair order is BC+AB and they are separated with a "+" (no spaces). Alternatively, you can specify the Probe Barcode ID alone and Cell Ranger's barcode pairing auto-detection algorithm will automatically match to the corresponding Antibody Multiplexing Barcode.
--emptydrops_minimum_umis	integer	For singleplex Flex experiments, use this option to adjust the UMI cutoff during the second step of cell calling. Cell Ranger will still perform the full cell calling process but will only evaluate barcodes with UMIs above the threshold you specify.

Name

Type & Properties

--probe_set

file

--filter_probes

boolean

--probe_barcode_ids

string

multiple

--emptydrops_minimum_umis

integer

Antigen Capture (BEAM) libary arguments

Name	Type & Properties	Description
--control_id	string multiple	A user-defined ID for any negative controls used in the T/BCR Antigen Capture assay. Must match id specified in the feature reference CSV. May only include ASCII characters and must not use whitespace, slash, quote, or comma characters. Each ID must be unique and must not collide with a gene identifier from the transcriptome.
--mhc_allele	string multiple	The MHC allele for TCR Antigen Capture libraries. Must match mhc_allele name specified in the Feature Reference CSV.

Name

Type & Properties

--control_id

string

multiple

--mhc_allele

string

multiple

General arguments

Name	Type & Properties	Description
--check_library_compatibility	boolean	Optional. This option allows users to disable the check that evaluates 10x Barcode overlap between ibraries when multiple libraries are specified (e.g., Gene Expression + Antibody Capture). Setting this option to false will disable the check across all library combinations. We recommend running this check (default), however if the pipeline errors out, users can bypass the check to generate outputs for troubleshooting.

Name

Type & Properties

--check_library_compatibility

boolean

Outputs

Name	Type & Properties	Description
--output_raw	file required output	The raw output folder.
--output_h5mu	file required output	Locations for the output files. Must contain a wildcard (*) character, which will be replaced with the sample name.
--uns_metrics	string	Name of the .uns slot under which to QC metrics (if any).

Name

Type & Properties

--output_raw

file

required

output

--output_h5mu

file

required

output

--uns_metrics

string

Run this component

Run the following command to execute this component with Nextflow:

cat > params.yaml <<'EOM'  
gex_secondary_analysis: [ false ]  
gex_generate_bam: [ false ]  
gex_include_introns: [ true ]  
gex_chemistry: [ "auto" ]  
check_library_compatibility: [ true ]  
output_raw: "$id.$key.output_raw.output_dir"  
output_h5mu: "$id.$key.output_h5mu.h5mu"  
uns_metrics: [ "metrics_cellranger" ]  
id: "run"  
publish_dir: "output/"  
EOM

nextflow run https://packages.viash-hub.com/vsh/openpipeline.git \  
  -revision v4.2.0 \  
  -main-script target/nextflow/workflows/ingestion/cellranger_multi/main.nf \  
  -params-file params.yaml

workflows/ingestion/cellranger_multi

Input files

Feature type-specific input files

Library arguments

Sample parameters

Feature Barcode library specific arguments

Gene expression arguments

VDJ related parameters

3' Cell multiplexing parameters (CellPlex Multiplexing)

Hashtag multiplexing parameters

On-chip multiplexing parameters

Flex multiplexing paramaters

Antigen Capture (BEAM) libary arguments

General arguments

Outputs

Run this component

Relationships

Used by

Uses