ANALYTICS

STACAS

Sample integration and cell clustering for scRNA-Seq

Input data

Files:

  • CellRanger output files from 10x Genomics (with one folder associated with each sample)
  • A design.csv file describing each sample (i.e. sample metadata), for example,
sample_id,is_treated
sample_1,yes
sample_2,no
sample_3,yes
sample_4,no

, where the sample identifiers must be in the first column.

Files are organized as follows:

my_project
|-- sample_1
|   |-- barcodes.tsv.gz
|   |-- features.tsv.gz
|   `-- matrix.mtx.gz
|-- sample_2
|   |-- barcodes.tsv
|   |-- genes.tsv
|   `-- matrix.mtx
|-- sample_3
|   |-- barcodes.tsv
|   |-- genes.tsv
|   `-- matrix.mtx
|-- sample_4
|   |-- barcodes.tsv
|   |-- genes.tsv
|   `-- matrix.mtx
|-- design.csv

Note: The folder names should match the sample identifiers in the design.csv file.

Output files

  • Clustering results (in metadata.tsv)
  • Normalized gene expression matrix (matrix.mtx)

For example,

output/
|-- barcodes.tsv
|-- cluster_marker.tsv
|-- file_description.json
|-- genes.tsv
|-- matrix.mtx
|-- metadata.tsv
|-- tsne_embedding.tsv

Parameter settings

Container:

  • g3bio/stacas

Options for cell filtering:

  • cutoff_MT — Mitochondrial reads cutoff (%), default: 25
  • max_n_features — Maximum number of features, default: 8000
  • min_n_features — Minimum number of features, default: 200
  • exclude_cycling — Exclude cells in cell cycle? default: “True”
  • organism — possible values: “human”, or “mouse”, default: “human”

Options for dimension reduction:

  • normalization_method — Normalization method, possible values: ‘LogNormalize’, ‘CLR’, ‘RC’, default: ‘LogNormalize’
  • number_of_features — Number of features selected for PCA, default: 2000
  • number_of_PCs — Number of principle components (PCs) selected, default: 40
  • resolution — Cluster resolution, default: 0.5
  • reduction_method — Nonlinear dimention reduction; possible values: ’tSNE’ or ‘UMAP’, default: tSNE

Options for cluster markers selection

  • log_fc_threshold — Difference between two groups of cells, default: 0.25
  • min_pct — Minimum percentage of cells expressed in any one group, default: 0.25
  • number_of_markers — Number of makers selected, default: 5

References

Please cite:

STACAS

Seurat