bd_rhapsody/bd_rhapsody_sequence_analysis

rna-seq

single-cell

multiomic

atac-seq

targeted

abseq

tcr

bcr

Description

BD Rhapsody Sequence Analysis CWL pipeline v2.2.

This pipeline performs analysis of single-cell multiomic sequence read (FASTQ) data. The supported
sequencing libraries are those generated by the BD Rhapsody™ assay kits, including: Whole Transcriptome
mRNA (WTA), Targeted mRNA, AbSeq Antibody-Oligonucleotides (ABC), Single-Cell Multiplexing (SMK),
TCR/BCR (VDJ), and ATAC-Seq.

Inputs

Name	Type & Properties	Description
--reads	file multiple	Reads (optional) - Path to your FASTQ.GZ formatted read files from libraries that may include: - WTA mRNA - Targeted mRNA - AbSeq - Sample Multiplexing - VDJ You may specify as many R1/R2 read pairs as you want.
--reads_atac	file multiple	Path to your FASTQ.GZ formatted read files from ATAC-Seq libraries. You may specify as many R1/R2/I2 files as you want.

Name

Type & Properties

--reads

file

multiple

--reads_atac

file

multiple

References

Name	Type & Properties	Description
--reference_archive	file	Path to Rhapsody WTA Reference in the tar.gz format. Structure of the reference archive: - `BD_Rhapsody_Reference_Files/`: top level folder - `star_index/`: sub-folder containing STAR index, that is files created with `STAR --runMode genomeGenerate` - GTF for gene-transcript-annotation e.g. "gencode.v43.primary_assembly.annotation.gtf"
--targeted_reference	file multiple	Path to the targeted reference file in FASTA format.
--abseq_reference	file multiple	Path to the AbSeq reference file in FASTA format. Only needed if BD AbSeq Ab-Oligos are used.
--supplemental_reference -s	file multiple	Path to the supplemental reference file in FASTA format. Only needed if there are additional transgene sequences to be aligned against in a WTA assay experiment.

Name

Type & Properties

--reference_archive

file

--targeted_reference

file

multiple

--abseq_reference

file

multiple

--supplemental_reference

-s

file

multiple

Outputs

Name	Type & Properties	Description
--output_dir -o	file required output	The unprocessed output directory containing all the outputs from the pipeline.
--output_seurat	file output	Single-cell analysis tool inputs. Seurat (.rds) input file containing RSEC molecules data table and all cell annotation metadata.
--output_mudata	file output	Single-cell analysis tool inputs. Scanpy / Muon input file containing RSEC molecules data table and all cell annotation metadata.
--metrics_summary	file output	Metrics Summary. Report containing sequencing, molecules, and cell metrics.
--pipeline_report	file output	Pipeline Report. Summary report containing the results from the sequencing analysis pipeline run.
--rsec_mols_per_cell	file output	Molecules per bioproduct per cell bassed on RSEC
--dbec_mols_per_cell	file output	Molecules per bioproduct per cell bassed on DBEC. DBEC data table is only output if the experiment includes targeted mRNA or AbSeq bioproducts.
--rsec_mols_per_cell_unfiltered	file output	Unfiltered tables containing all cell labels with ≥10 reads.
--bam	file output	Alignment file of R2 with associated R1 annotations for Bioproduct.
--bam_index	file output	Index file for the alignment file.
--bioproduct_stats	file output	Bioproduct Stats. Metrics from RSEC and DBEC Unique Molecular Identifier adjustment algorithms on a per-bioproduct basis.
--dimred_tsne	file output	t-SNE dimensionality reduction coordinates per cell index
--dimred_umap	file output	UMAP dimensionality reduction coordinates per cell index
--immune_cell_classification	file output	Immune Cell Classification. Cell type classification based on the expression of immune cell markers.

Name

Type & Properties

--output_dir

-o

file

required

output

--output_seurat

file

output

--output_mudata

file

output

--metrics_summary

file

output

--pipeline_report

file

output

--rsec_mols_per_cell

file

output

--dbec_mols_per_cell

file

output

--rsec_mols_per_cell_unfiltered

file

output

--bam

file

output

--bam_index

file

output

--bioproduct_stats

file

output

--dimred_tsne

file

output

--dimred_umap

file

output

--immune_cell_classification

file

output

Multiplex outputs

Name	Type & Properties	Description
--sample_tag_metrics	file output	Sample Tag Metrics. Metrics from the sample determination algorithm.
--sample_tag_calls	file output	Sample Tag Calls. Assigned Sample Tag for each putative cell
--sample_tag_counts	file multiple output	Sample Tag Counts. Separate data tables and metric summary for cells assigned to each sample tag. Note: For putative cells that could not be assigned a specific Sample Tag, a Multiplet_and_Undetermined.zip file is also output.
--sample_tag_counts_unassigned	file output	Sample Tag Counts Unassigned. Data table and metric summary for cells that could not be assigned a specific Sample Tag.

Name

Type & Properties

--sample_tag_metrics

file

output

--sample_tag_calls

file

output

--sample_tag_counts

file

multiple

output

--sample_tag_counts_unassigned

file

output

VDJ Outputs

Name	Type & Properties	Description
--vdj_metrics	file output	VDJ Metrics. Overall metrics from the VDJ analysis.
--vdj_per_cell	file output	VDJ Per Cell. Cell specific read and molecule counts, VDJ gene segments, CDR3 sequences, paired chains, and cell type.
--vdj_per_cell_uncorrected	file output	VDJ Per Cell Uncorrected. Cell specific read and molecule counts, VDJ gene segments, CDR3 sequences, paired chains, and cell type.
--vdj_dominant_contigs	file output	VDJ Dominant Contigs. Dominant contig for each cell label chain type combination (putative cells only).
--vdj_unfiltered_contigs	file output	VDJ Unfiltered Contigs. All contigs that were assembled and annotated successfully (all cells).

Name

Type & Properties

--vdj_metrics

file

output

--vdj_per_cell

file

output

--vdj_per_cell_uncorrected

file

output

--vdj_dominant_contigs

file

output

--vdj_unfiltered_contigs

file

output

ATAC-Seq outputs

Name	Type & Properties	Description
--atac_metrics	file output	ATAC Metrics. Overall metrics from the ATAC-Seq analysis.
--atac_metrics_json	file output	ATAC Metrics JSON. Overall metrics from the ATAC-Seq analysis in JSON format.
--atac_fragments	file output	ATAC Fragments. Chromosomal location, cell index, and read support for each fragment detected
--atac_fragments_index	file output	Index of ATAC Fragments.
--atac_transposase_sites	file output	ATAC Transposase Sites. Chromosomal location, cell index, and read support for each transposase site detected
--atac_transposase_sites_index	file output	Index of ATAC Transposase Sites.
--atac_peaks	file output	ATAC Peaks. Peak regions of transposase activity
--atac_peaks_index	file output	Index of ATAC Peaks.
--atac_peak_annotation	file output	ATAC Peak Annotation. Estimated annotation of peak-to-gene connections
--atac_cell_by_peak	file output	ATAC Cell by Peak. Peak regions of transposase activity per cell
--atac_cell_by_peak_unfiltered	file output	ATAC Cell by Peak Unfiltered. Unfiltered file containing all cell labels with >=1 transposase sites in peaks.
--atac_bam	file output	ATAC BAM. Alignment file for R1 and R2 with associated I2 annotations for ATAC-Seq. Only output if the BAM generation flag is set to true.
--atac_bam_index	file output	Index of ATAC BAM.

Name

Type & Properties

--atac_metrics

file

output

--atac_metrics_json

file

output

--atac_fragments

file

output

--atac_fragments_index

file

output

--atac_transposase_sites

file

output

--atac_transposase_sites_index

file

output

--atac_peaks

file

output

--atac_peaks_index

file

output

--atac_peak_annotation

file

output

--atac_cell_by_peak

file

output

--atac_cell_by_peak_unfiltered

file

output

--atac_bam

file

output

--atac_bam_index

file

output

AbSeq Cell Calling outputs

Name	Type & Properties	Description
--protein_aggregates_experimental	file output	Protein Aggregates Experimental

Name

Type & Properties

--protein_aggregates_experimental

file

output

Putative Cell Calling Settings

Name	Type & Properties	Description
--cell_calling_data	string	Specify the dataset to be used for putative cell calling: mRNA, AbSeq, ATAC, mRNA_and_ATAC For putative cell calling using an AbSeq dataset, please provide an AbSeq_Reference fasta file above. For putative cell calling using an ATAC dataset, please provide a WTA+ATAC-Seq Reference_Archive file above. The default data for putative cell calling, will be determined the following way: - If mRNA Reads and ATAC Reads exist: mRNA_and_ATAC - If only ATAC Reads exist: ATAC - Otherwise: mRNA
--cell_calling_bioproduct_algorithm	string	Specify the bioproduct algorithm to be used for putative cell calling: Basic or Refined By default, the Basic algorithm will be used for putative cell calling.
--cell_calling_atac_algorithm	string	Specify the ATAC-seq algorithm to be used for putative cell calling: Basic or Refined By default, the Basic algorithm will be used for putative cell calling.
--exact_cell_count	integer	Set a specific number (>=1) of cells as putative, based on those with the highest error-corrected read count
--expected_cell_count	integer	Guide the basic putative cell calling algorithm by providing an estimate of the number of cells expected. Usually this can be the number of cells loaded into the Rhapsody cartridge. If there are multiple inflection points on the second derivative cumulative curve, this will ensure the one selected is near the expected.

Name

Type & Properties

--cell_calling_data

string

--cell_calling_bioproduct_algorithm

string

--cell_calling_atac_algorithm

string

--exact_cell_count

integer

--expected_cell_count

integer

Intronic Reads Settings

Name	Type & Properties	Description
--exclude_intronic_reads	boolean	By default, the flag is false, and reads aligned to exons and introns are considered and represented in molecule counts. When the flag is set to true, intronic reads will be excluded. The value can be true or false.

Name

Type & Properties

--exclude_intronic_reads

boolean

Multiplex Settings

Name	Type & Properties	Description
--sample_tags_version	string	Specify the version of the Sample Tags used in the run: * If Sample Tag Multiplexing was done, specify the appropriate version: human, mouse, flex, nuclei_includes_mrna, nuclei_atac_only * If this is an SMK + Nuclei mRNA run or an SMK + Multiomic ATAC-Seq (WTA+ATAC-Seq) run (and not an SMK + ATAC-Seq only run), choose the "nuclei_includes_mrna" option. * If this is an SMK + ATAC-Seq only run (and not SMK + Multiomic ATAC-Seq (WTA+ATAC-Seq)), choose the "nuclei_atac_only" option.
--tag_names	string multiple	Specify the tag number followed by '-' and the desired sample name to appear in Sample_Tag_Metrics.csv Do not use the special characters: &, (), [], {}, <>, ?, \|

Name

Type & Properties

--sample_tags_version

string

--tag_names

string

multiple

VDJ arguments

Name	Type & Properties	Description
--vdj_version	string	If VDJ was done, specify the appropriate option: human, mouse, humanBCR, humanTCR, mouseBCR, mouseTCR

Name

Type & Properties

--vdj_version

string

ATAC options

Name	Type & Properties	Description
--predefined_atac_peaks	file	An optional BED file containing pre-established chromatin accessibility peak regions for generating the ATAC cell-by-peak matrix.

Name

Type & Properties

--predefined_atac_peaks

file

Additional options

Name	Type & Properties	Description
--run_name	string	Specify a run name to use as the output file base name. Use only letters, numbers, or hyphens. Do not use special characters or spaces.
--generate_bam	boolean	Specify whether to create the BAM file output
--long_reads	boolean	Use STARlong (default: undefined - i.e. autodetects based on read lengths) - Specify if the STARlong aligner should be used instead of STAR. Set to true if the reads are longer than 650bp.

Name

Type & Properties

--run_name

string

--generate_bam

boolean

--long_reads

boolean

Advanced options

Name	Type & Properties	Description
--custom_star_params	string	Modify STAR alignment parameters - Set this parameter to fully override default STAR mapping parameters used in the pipeline. For reference this is the default that is used: Short Reads: `--outFilterScoreMinOverLread 0 --outFilterMatchNminOverLread 0 --outFilterMultimapScoreRange 0 --clip3pAdapterSeq AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA --seedSearchStartLmax 50 --outFilterMatchNmin 25 --limitOutSJcollapsed 2000000` Long Reads: Same as Short Reads + `--seedPerReadNmax 10000` This applies to fastqs provided in the Reads user input Do NOT set any non-mapping related params like `--genomeDir`, `--outSAMtype`, `--outSAMunmapped`, `--readFilesIn`, `--runThreadN`, etc. We use STAR version 2.7.10b
--custom_bwa_mem2_params	string	Modify bwa-mem2 alignment parameters - Set this parameter to fully override bwa-mem2 mapping parameters used in the pipeline The pipeline does not specify any custom mapping params to bwa-mem2 so program default values are used This applies to fastqs provided in the Reads_ATAC user input Do NOT set any non-mapping related params like `-C`, `-t`, etc. We use bwa-mem2 version 2.2.1

Name

Type & Properties

--custom_star_params

string

--custom_bwa_mem2_params

string

CWL-runner arguments

Name	Type & Properties	Description
--parallel	boolean	Run jobs in parallel.
--timestamps	boolean_true	Add timestamps to the errors, warnings, and notifications.

Name

Type & Properties

--parallel

boolean

--timestamps

boolean_true

Undocumented arguments

Name	Type & Properties	Description
--abseq_umi	integer
--target_analysis	boolean
--vdj_jgene_evalue	double	e-value threshold for J gene. The e-value threshold for J gene call by IgBlast/PyIR, default is set as 0.001
--vdj_vgene_evalue	double	e-value threshold for V gene. The e-value threshold for V gene call by IgBlast/PyIR, default is set as 0.001
--write_filtered_reads	boolean

Name

Type & Properties

--abseq_umi

integer

--target_analysis

boolean

--vdj_jgene_evalue

double

--vdj_vgene_evalue

double

--write_filtered_reads

boolean

Run this component

Run the following command to execute this component with Nextflow:

cat > params.yaml <<'EOM'  
output_dir: "$id.$key.output_dir.output_dir"  
output_seurat: "$id.$key.output_seurat.rds"  
output_mudata: "$id.$key.output_mudata.h5mu"  
metrics_summary: "$id.$key.metrics_summary.csv"  
pipeline_report: "$id.$key.pipeline_report.html"  
rsec_mols_per_cell: "$id.$key.rsec_mols_per_cell.zip"  
dbec_mols_per_cell: "$id.$key.dbec_mols_per_cell.zip"  
rsec_mols_per_cell_unfiltered: "$id.$key.rsec_mols_per_cell_unfiltered.zip"  
bam: "$id.$key.bam.bam"  
bam_index: "$id.$key.bam_index.bai"  
bioproduct_stats: "$id.$key.bioproduct_stats.csv"  
dimred_tsne: "$id.$key.dimred_tsne.csv"  
dimred_umap: "$id.$key.dimred_umap.csv"  
immune_cell_classification: "$id.$key.immune_cell_classification.csv"  
sample_tag_metrics: "$id.$key.sample_tag_metrics.csv"  
sample_tag_calls: "$id.$key.sample_tag_calls.csv"  
sample_tag_counts: "$id.$key.sample_tag_counts._*.zip"  
sample_tag_counts_unassigned: "$id.$key.sample_tag_counts_unassigned.zip"  
vdj_metrics: "$id.$key.vdj_metrics.csv"  
vdj_per_cell: "$id.$key.vdj_per_cell.csv"  
vdj_per_cell_uncorrected: "$id.$key.vdj_per_cell_uncorrected.csv"  
vdj_dominant_contigs: "$id.$key.vdj_dominant_contigs.csv"  
vdj_unfiltered_contigs: "$id.$key.vdj_unfiltered_contigs.csv"  
atac_metrics: "$id.$key.atac_metrics.csv"  
atac_metrics_json: "$id.$key.atac_metrics_json.json"  
atac_fragments: "$id.$key.atac_fragments.gz"  
atac_fragments_index: "$id.$key.atac_fragments_index.tbi"  
atac_transposase_sites: "$id.$key.atac_transposase_sites.gz"  
atac_transposase_sites_index: "$id.$key.atac_transposase_sites_index.tbi"  
atac_peaks: "$id.$key.atac_peaks.gz"  
atac_peaks_index: "$id.$key.atac_peaks_index.tbi"  
atac_peak_annotation: "$id.$key.atac_peak_annotation.gz"  
atac_cell_by_peak: "$id.$key.atac_cell_by_peak.zip"  
atac_cell_by_peak_unfiltered: "$id.$key.atac_cell_by_peak_unfiltered.zip"  
atac_bam: "$id.$key.atac_bam.bam"  
atac_bam_index: "$id.$key.atac_bam_index.bai"  
protein_aggregates_experimental: "$id.$key.protein_aggregates_experimental.csv"  
run_name: [ "sample" ]  
generate_bam: [ false ]  
parallel: [ true ]  
id: "run"  
publish_dir: "output/"  
EOM

nextflow run https://packages.viash-hub.com/vsh/biobox.git \  
  -revision v0.4.0 \  
  -main-script target/nextflow/bd_rhapsody/bd_rhapsody_sequence_analysis/main.nf \  
  -params-file params.yaml

bd_rhapsody/bd_rhapsody_sequence_analysis

Inputs

References

Outputs

Multiplex outputs

VDJ Outputs

ATAC-Seq outputs

AbSeq Cell Calling outputs

Putative Cell Calling Settings

Intronic Reads Settings

Multiplex Settings

VDJ arguments

ATAC options

Additional options

Advanced options

CWL-runner arguments

Undocumented arguments

Run this component

Relationships

Used by

Uses