I have a question: What's the intended way to run the pipeline with 10x Flex data? I'm using the following nf-params, but it seems as if the probe set is not properly passed on to the multi config (frna_probeset = "".replace("./", work_dir) ), and it errors out. When I run it without cellranger_index, it also gives an error (although the index is not actually required as far as I can see it). What am I missing? 😄

{
  "input": "samplesheet.csv",
  "aligner": "cellrangermulti",
  "gex_frna_probe_set": "Chromium_Human_Transcriptome_Probe_Set_v2.0.0_GRCh38-2024-A.csv",
  "cellranger_index": "refdata-gex-GRCh38-2024-A",
  "skip_cellrangermulti_vdjref": true,
  "protocol": "auto",
  "skip_cellbender": true,
  "skip_fastqc": false,
  "save_align_intermeds": false
}

Per the thread above, this is what I used for an older probe set version

{
  "skip_cellbender": true,
  "skip_cellranger_renaming": true,
  "skip_cellrangermulti_vdjref": true,
  "skip_fastqc": false,
  "skip_multiqc": false,
  "save_align_intermeds": false,
  "save_reference": false,
  "igenomes_ignore": true,
  "igenomes_base": "s3://[bucket name]/igenomes/",
  "aligner": "cellrangermulti",
  "protocol": "auto",
  "input": "s3://[bucket name]/run_1234-samplesheet.csv",
  "cellranger_index": "s3://[bucket name]/10xgenomics/refdata-gex-GRCh38-2020-A/",
  "cellranger_multi_barcodes": "s3://[bucket name]/run_1234-multiplex.csv",
  "gex_frna_probe_set": "s3://[bucket name]/10xgenomics/Chromium_Human_Transcriptome_Probe_Set_v1.0.1_GRCh38-2020-A.csv"
}

We have the 10x reference data stored on s3, it appears you are using local directories? It may then just be a problem with staging

Thanks a lot! I dug down into the issue and I actually think the data is multiplexed without me knowing. Will close this post with a fix once I have figured it out 🙂 (or come back with more questions)