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Add docs for flex libraries #494

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19e095d
include flex in processing documentation
allyhawkins Mar 18, 2026
db5ca7b
add flex metadata
allyhawkins Mar 18, 2026
5437932
don't forget about merged objects
allyhawkins Mar 18, 2026
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22 changes: 13 additions & 9 deletions docs/merged_objects.md
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Expand Up @@ -152,16 +152,20 @@ Each such list will contain the following fields:
| `sample_id` | Sample ID in the form `SCPCS000000` |
| `library_id` | Library ID in the form `SCPCL000000` |
| `project_id` | Project ID in the form `SCPCP000000` |
| `salmon_version` | Version of `salmon` used for initial mapping |
| `reference_index` | Transcriptome reference file used for mapping |
| `total_reads` | Total number of reads processed by `salmon` |
| `mapped_reads` | Number of reads successfully mapped |
| `mapping_tool` | Pipeline used for mapping and quantification (`alevin-fry` for all current data in ScPCA) |
| `alevinfry_version` | Version of `alevin-fry` used for mapping and quantification |
| `af_permit_type` | `alevin-fry generate-permit-list` method used for filtering cell barcodes |
| `af_resolution` | `alevin-fry quant` resolution mode used |
| `usa_mode` | Boolean indicating whether quantification was done using `alevin-fry` USA mode |
| `af_num_cells` | Number of cells reported by `alevin-fry` |
| `mapping_tool` | Pipeline used for mapping and quantification. `alevin-fry` for all tag-based experiments (e.g. 10Xv2, 10Xv3) and `cellranger-multi` for probe-based experiments (GEM-X Flex) |
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@sjspielman sjspielman Mar 19, 2026

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I can neither confirm nor deny that I just spent 3 minutes being really confused why we don't set this for spaceranger too, only to remember we do not make SPEs. That's the other thing I work on (training materials).

| `salmon_version` | Version of `salmon` used for initial mapping. Only present if the `mapping_tool` is `alevin-fry` |
| `alevinfry_version` | Version of `alevin-fry` used for mapping and quantification. Only present if the `mapping_tool` is `alevin-fry` |
| `af_permit_type` | `alevin-fry generate-permit-list` method used for filtering cell barcodes. Only present if the `mapping_tool` is `alevin-fry` |
| `af_resolution` | `alevin-fry quant` resolution mode used. Only present if the `mapping_tool` is `alevin-fry` |
| `usa_mode` | Boolean indicating whether quantification was done using `alevin-fry` USA mode. Only present if the `mapping_tool` is `alevin-fry` |
| `total_reads` | Total number of reads processed by `salmon` or `cellranger multi` |
| `mapped_reads` | Number of reads successfully mapped. Only present if the `mapping_tool` is `alevin-fry` |
| `af_num_cells` | Number of cells reported by `alevin-fry`. Only present if the `mapping_tool` is `alevin-fry` |
| `cellranger_version` | Total number of reads processed by `salmon` or `cellranger multi` |
| `reference_probeset` | Version of probe set from 10x Genomics used for mapping and quantification. Only present if the `mapping_tool` is `cellranger-multi` |
| `pct_mapped_reads` | Total percentage of reads mapped by `cellranger-multi`. Only present if the `mapping_tool` is `cellranger-multi` |
| `cellranger_num_cells` | Number of cells reported by `cellranger multi`. Only present if the `mapping_tool` is `cellranger-multi` |
| `tech_version` | A string indicating the technology and version used for the single-cell library, such as 10Xv2, 10Xv3, or 10Xv3.1 |
| `assay_ontology_term_id` | A string indicating the [Experimental Factor Ontology](https://www.ebi.ac.uk/ols/ontologies/efo) term ID associated with the `tech_version` |
| `seq_unit` | `cell` for single-cell samples or `nucleus` for single-nucleus samples |
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22 changes: 18 additions & 4 deletions docs/processing_information.md
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Expand Up @@ -5,7 +5,10 @@
### Mapping and quantification using alevin-fry

We used [`salmon`](https://salmon.readthedocs.io/en/latest) and [`alevin-fry`](https://alevin-fry.readthedocs.io/en/latest/) to generate gene by cell counts matrices for all single-cell and single-nuclei samples.
In brief, we utilized [selective alignment](#selective-alignment) to the [`splici` index](#reference-transcriptome-index) for all single-cell and single-nuclei samples.
In brief, we utilized [selective alignment](#selective-alignment) to the [`splici` index](#reference-transcriptome-index).

The only exception to this was single-nuclei samples generated using the probe-based GEM-X Flex platform from 10x Genomics.
See [Quantification for GEM-X Flex samples](#quantification-for-gem-x-flex-samples) for more information.
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Suggested change
See [Quantification for GEM-X Flex samples](#quantification-for-gem-x-flex-samples) for more information.
See [Quantification for GEM-X Flex samples](#mapping-and-quantification-for-gem-x-flex-samples) for more information.


#### Reference transcriptome index

Expand Down Expand Up @@ -36,12 +39,23 @@ In contrast to Cell Ranger, `cr-like-em` keeps multi-mapped reads and invokes an

3. With initial mapping to the `splici` index, `alevin-fry` quantification resulted in separate counts for spliced and unspliced transcripts, and an ambiguous count for reads compatible with either spliced or unspliced transcripts.

### Post alevin-fry processing

#### Combining counts from spliced cDNA and intronic regions

For single-cell and single-nuclei samples, the reads from spliced cDNA and intronic regions are combined by gene to produce a gene by cell counts matrix.
For single-cell and single-nuclei samples processed with `alevin-fry`, the reads from spliced cDNA and intronic regions are combined by gene to produce a gene by cell counts matrix.
After combining read counts, values are rounded to integer values.
The counts data from this step can be found in the `unfiltered` objects included with each library.

### Mapping and quantification for GEM-X Flex samples

Libraries that were generated using the GEM-X Flex technology from 10x Genomics were quantified with the [`cellranger multi` pipeline](https://www.10xgenomics.com/support/software/cell-ranger/latest/analysis/running-pipelines/cr-multi) within Cell Ranger.
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Comment that applies throughout PR - pick either cellranger multi or cellranger-multi

The `cellranger mkref` command was used to create a transcriptome reference index compatible with Cell Ranger.
The probe set associated with the specific GEM-X Flex version used for library preparation was provided as input alongside the transcriptome reference index and FASTQ files to `cellranger multi`.

If samples were multiplexed into a single library using GEM-X Flex, demultiplexing was performed as part of `cellranger multi` so that outputs were separated to have one gene by cell counts matrix for each sample.

The gene by cell counts matrix output for each sample in the `raw_feature_bc_matrix` folder was saved to the `unfiltered` objects included with each library.

### Post alignment processing

#### Filtering cells

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