rhapsodist

Rhapsodist is a Snakemake workflow to process BD Rhapsody WTA (enhanced beads) single-cell RNA-seq data. It pre-processes raw FASTQ reads through barcode standardisation, then runs alignment and UMI counting in parallel with STARsolo, kallisto/bustools, salmon/alevin, and/or the official BD Rhapsody CWL pipeline (locally). Each path produces a SingleCellExperiment object. Cell filtering can use each tool's native approach or DropletUtils emptyDrops. The workflow also handles sample tag demultiplexing and renders comparison reports across methods.

Workflow layout

flowchart TD
    reads[raw FASTQ\nR1 + R2] --> cutadapt[cutadapt\nbarcode standardisation]

    cutadapt --> starsolo[STARsolo\nalignment + UMI count]
    cutadapt --> kallisto[kallisto bus\nalignment]
    cutadapt --> alevin[salmon alevin\nalignment + UMI count]
    reads --> sbg[BD Rhapsody CWL\nvia cwl-runner]

    starsolo --> sce_star[SingleCellExperiment\nSTARsolo]
    kallisto --> bustools[bustools sort + count]
    bustools --> sce_kallisto[SingleCellExperiment\nkallisto]
    alevin --> sce_alevin[SingleCellExperiment\nalevin]
    sbg --> sce_sbg[SingleCellExperiment\nSBG]

    sce_star & sce_kallisto & sce_alevin & sce_sbg --> report[comparison report]

    starsolo --> sampletags[sampletag\ndemultiplexing]
    sampletags --> st_report[sampletag report]

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Quickstart

Install the CLI (optional):

pip install -e .

Run the simulation test:

rhapsodist --configfile configs/sim_config.yaml --cores 10

Run on real data (update the YAML first to point to your R1/R2 files):

rhapsodist --configfile configs/config.yaml --cores 10

Extra snakemake arguments can be appended directly:

rhapsodist --configfile configs/config.yaml --cores 10 --rerun-incomplete --nolock

Or call snakemake directly if preferred:

snakemake --use-conda --cores 10 --configfile configs/config.yaml

Repository layout

configs/          pipeline config yaml files (config.yaml, sim_config.yaml, real_config.yaml)
workflow/
  Snakefile       main snakemake workflow
  data/           reference data: barcode whitelists, sampletag sequences
  envs/           conda environment yaml files used by snakemake
  src/
    *.R           per-aligner SCE generation and report scripts
    *.Rmd         rmarkdown reports rendered by the pipeline
    *.py          python helpers and simulation scripts
    simulate.snmk snakemake rules for synthetic data generation
    reports/      standalone benchmark and overview documents
rhapsodist/       installable cli package
tests/            pytest unit tests

Configuration

Copy configs/config.yaml and fill in the fields before running on real data.

Resources:

• nthreads: number of CPU threads
• max_mem_mb: RAM limit in MB
• working_dir: path where outputs will be written (relative or absolute)

Configuration:

• gtf_origin: gencode or ensembl
• gtf: path to GTF annotation file (uncompressed)
• genome: path to genome FASTA (uncompressed)
• transcriptome: path to transcriptome FASTA (can be gzipped)
• sjdbOverhang: read length minus 1 (e.g. 70 for 71 bp reads)

Aligners/pipelines:

• aligner: list of aligners to run, any combination of starsolo, kallisto, alevin, sbg

Samples:

samples:
  - name: my_sample
    uses:
      cb_umi_fq: /path/to/R1.fastq.gz   # barcode + UMI read
      cdna_fq: /path/to/R2.fastq.gz     # cDNA read
      whitelist: 384x3                   # 384x3 for EnhV2 beads, 96x3 for Enh beads
      species: human                     # human or mouse

Cell filtering (cell_filtering key):

• native: each aligner uses its own method
• emptydrops: apply DropletUtils::emptyDrops uniformly across all aligners

BD Rhapsody official pipeline (optional): add sbg to the aligner list and set sbg_cwl:

aligner: [starsolo, kallisto, alevin, sbg]
sbg_cwl: third_party/cwl/v2.2.1/rhapsody_pipeline_2.2.1.cwl

The reference archive is built automatically from the STAR index and GTF. to use a pre-built BD archive:

sbg_reference_url: "http://bd-rhapsody-public.s3-website-us-east-1.amazonaws.com/..."
# or
sbg_reference_archive: /path/to/Rhapsody_reference.tar.gz

Contributors

• Izaskun Mallona
• Jiayi Wang
• Giulia Moro

Tools used: STAR, samtools, kallisto, bustools, salmon/alevin, cutadapt, pigz, R/Bioconductor.

Contact

izaskun.mallona at mls.uzh.ch, Mark D. Robinson lab https://www.mls.uzh.ch/en/research/robinson.html

History

started 30 July 2024, keeping history from https://github.com/imallona/rock_roi_method