.. GSSNNG documentation master file, created by sphinx-quickstart on Wed Apr 27 09:20:15 2022. You can adapt this file completely to your liking, but it should at least contain the root `toctree` directive. Genesets as .gmt files ====================== Gene Set Scoring on the Nearest Neighbor Graph (gssnng) for Single Cell RNA-seq (scRNA-seq). .. .. toctree:: :caption: Table of Contents :maxdepth: 1 `**Notebook using gmt files** `_ `**See the paper** `_ This package works with AnnData objects stored as h5ad files. Expression values are taken from adata.X. For creating groups, up to four categorical variables can be used, which are found in the adata.obs table. Gene sets can be provided by using .gmt files or through the OmniPath API (see below). Scoring functions work with ranked or unranked data (**"your mileage may vary"**): Method references (singscore, RBO) are below. Some methods have additional parameters, see below! Installation ------------ Install the package using the following commands:: python3 -m pip install gssnng # or to from github python3 -m pip install git+https://github.com/IlyaLab/gssnng Example script -------------- Copy the script out from the cloned repo and run, check the paths if you get an error. :: cp gssnng/gssnng/test/example_gmt_input.py . python3.10 example_gmt_input.py Usage ----- See gssnng/notebooks for examples on all methods. 1. Read in an AnnData object using scanpy (an h5ad file). 2. Get gene sets formatted as a .gmt file. (default is UP, also uses _UP, _DN, and split gene sets _UP+_DN), see below for more details. 3. Score cells, each gene set will show up as a column in adata.obs. :: from gssnng import score_cells q = sc.datasets.pbmc3k_processed() scores_cells.with_gene_sets(adata=q, # AnnData object gene_set_file='cibersort_lm22.gmt', # File path of gene sets groupby='louvain', # Will sample neighbors within this group, can take a list smooth_mode='connectivity', # Smooths matrix using distance weights from NN graph. recompute_neighbors=32, # Rebuild nearest neighbor graph with groups, 0 turns off function score_method='singscore', # Method of scoring method_params={'normalization':'theoretical'}, # Special parameters for some methods ranked=True, # Use ranked data, True or False cores=8) # Groups are scored in parallel. sc.pl.umap(q, color=['louvain','T.cells.CD8.up'], wspace=0.35) Scoring Functions ----------------- The list of scoring functions:: **geneset_overlap**: For each geneset, number (or fraction) of genes expressed past a given threshold. **singscore**: Normalised mean (median centered) ranks (requires ranked data) **ssGSEA**: Single sample GSEA based on ranked data. **rank_biased_overlap**: RBO, Weighted average of agreement between sorted ranks and gene set. **robust_std**: Med(x-med / mad), median of robust standardized values (recommend unranked). **mean_z**: Mean( (x - mean)/stddv ), average z score. (recommend unranked). **average_score**: Mean ranks or counts **median_score**: Median of counts or ranks **summed_up**: Sum up the ranks or counts. Parameters ---------- These parameters are used with the "scores_cells.with_gene_sets" function.:: adata: AnnData object from scanpy.read_* AnnData containing the cells to be scored gene_set_file: str[path] The gene set file with list of gene sets, gmt, one per line. See `this definition `_ . groupby: [str, list, dict] either a column label in adata.obs, and all categories taken, or a dict specifies one group. SEE DESCRIPTION BELOW smooth_mode: "adjacency", "connectivity", or "off" Dictates how to use the neighborhood graph. `adjacency` weights all neighbors equally, `connectivity` weights close neighbors more recompute_neighbors: int should neighbors be recomputed within each group, 0 for no, >0 for yes and specifies N score_method: str which scoring method to use method_params: dict python dict with XGBoost params. ranked: bool whether the gene expression counts should be rank ordered cores: int number of parallel processes to work through groupby groups Groupby ------- The specific neighborhood for each cell can be controlled by using the groupby parameter. In the example above, by setting groupby='louvain', only cells within a louvain cluster will be considered as being part of the neighborhood and will available for sampling. Groupby specifies a column name that's found in the AnnData.obs table, and it can also take a list of column names. In that case, cells will be grouped as the intersection of categories. For example, using groupby=['louvain','phenotype'] will take cells that are first in a given louvain cluster and then also in a given phenotype group. By also setting the recompute_neighbors, the nearest neighbor graph is recomputed within this subset of cells. Controlling the neighborhood leads to more controlled smoothing of the count matrix and is more suitable for downstream comparisons. Gene sets --------- We are following the MSigDB nomenclature, where gene sets default to up, but can have direction specified with the suffix "_UP" (example: CD8_signature_UP or CD8.signature.up). If the gene set name has suffix "_DN" (example: CD8_signature_DN or CD8.signature.dn), then low expressed genes will have large ranks and produce positive scores. In the use of singscore or Z scores, the undirected case is based on absolute values, so either direction, in the extreme, will result in a large score. Method parameters ----------------- Some methods have some additional options. They are passed as a dictionary, method_params={param_name, param_value}.:: singscore: {'normalization', 'theoretical'}, {'normalization', 'standard'} The singscore manuscript describes the theoretical method of standardization which involves determining the theoretical max and minimum ranks for the given gene set.:: rank_biased_overlap: {'rbo_depth', n} (n: int) Here, n is the depth that is decended down the ranks, where at each step, the overlap with the gene set is measured and added to the score.:: ssGSEA: {'omega': 0.75} The ssGSEA method uses this parameter as a exponent to the ranks. It has been strongly suggested to use 0.75. *The following methods do not have additional options.* robust_std mean_z average_score median_score summed_up References ---------- rank biased overlap: https://arxiv.org/pdf/1408.3587.pdf singscore: https://pubmed.ncbi.nlm.nih.gov/30400809/ anndata: https://anndata.readthedocs.io/en/latest/ MSigDB: https://www.gsea-msigdb.org/gsea/msigdb/ ssGSEA: https://gsea-msigdb.github.io/ssGSEA-gpmodule/v10/index.html decoupler: https://academic.oup.com/bioinformaticsadvances/article/2/1/vbac016/6544613 omnipath: https://omnipathdb.org/