CytoSig 0.0.3
Prediction model for cytokine signaling activity
Requires Python: >=3.6

CytoSig prediction model of cytokine signaling activity

**Prerequisite**:  
1, data_significance: https://github.com/data2intelligence/data_significance  
Please read its README.md and run test to make sure the successful installation.  

2, pandas >= 1.1.4: You may install anaconda (https://www.anaconda.com) to include all required python packages.  
3, xlsxwriter >= 1.3.7: pip install --upgrade xlsxwriter  
4, openpyxl >= 3.0.9: pip install --upgrade openpyxl  
5, scipy: pip install --upgrade scipy

**Install**:
pip install .

**Note**: if you fail to install pre-requisite packages and CytoSig on your local computer, welcome to try our Docker solution in Usage 3 below.  

**Test**:
python -m unittest tests.prediction

Please see **tests/prediction.py** for examples of two usages explained below.  

**Usage 1, through command line**:  

CytoSig_run.py -i input_profile -o output_prefix -r random_count -a penalty_alpha -e generate_excel -s expand_signature -c minimum_read_count -z max_dropout_ratio  

1, input_profile: input matrix of biological profiles. Three categories of formats are acceptable.  

-Format a, matrix file in excel (xls, xlsx, csv) or tab formats (plain or gzip formats). Each column is a biological condition, and each row should be a human gene symbol. Please see "tests/GSE147507.diff.gz" as an example.  
The expression values, from either RNASeq or MicroArray, should be transformed by log2(x+1). x could be FPKM, RPKM, or TPM for RNASeq. For single-cell RNASeq data, we used log2(TPM/10 + 1). We recommend input differential profiles between the two conditions. If data is from a sample collection without pairs, please mean-centralize the value of each gene across all samples.  

-Format b, python pickle formats: for matrix format in option a, you can save them as python pickle with names as "*.pickle.gz", "*.pkl.gz" or "*.pickle", "*.pkl".   

-Format c, cell ranger output: with file path and file name prefix separated by ",". This option will normalize and log-transform counts data and mean centralize the expression value across all single cells for each gene. Please see Example 2 below.  
**For Seurat users**: please save your Seurat object as cell ranger output with the following R commands, and then run with this option.  
> library(DropletUtils)  
> write10xCounts(x = your_object_name@assays$RNA@counts, path = "folder_path", version="3")  
  
2, output_prefix: prefix of output files. Each column is a biological condition, and each row is a cytokine name  
    output_prefix.Coef: regression coefficients  
    output_prefix.StdErr: standard error  
    output_prefix.Zscore: Coef/StdErr  
    output_prefix.Pvalue: two-sided test p-value of Zscore, from permutation test if random_count > 0 or student t-test if random_count = 0  
    output_prefix.xlsx: only exist if generate_excel = 1. A excel summary of results, with each input condition as one tab  

3, random_count: number of randomizations in the permutation test, with a default value 1000. If value is 0, the program will use student t-test.    

4, penalty_alpha: penalty weight in the ridge regression, with a default value 10000.  

5, generate_excel: whether generate excel output. The value could be 1 (Yes) or 0 (No) with a default value 0. This option is only effective when the input condition count is less than 50.

6, expand_signature: whether use an expanded signature of cytokine response. Our initial cytokine response signature included 43 cytokines with high confidence data (-s 0). However, we can also set a less stringent filter to include 51 cytokines (-s 1). We also included a beta version of the response signature as we are expanding the CytoSig datasets now (-s 2). However, the beta version is not fully validated yet. Please use it at your caution.  

Following options will only be effective if the input is cell ranger output.  
7, minimum_read_count: Minimal read counts required for each barcode. Default 1000  
8, max_dropout_ratio: Maximal zero drop out rates allowed for each gene. Default 0.95  

Example 1:
In the directory of README.md, please type: CytoSig_run.py -i tests/GSE147507.diff.gz -o tests/output_test -e 1  
Then, open "tests/output_test.xlsx" to view results  

Example 2:
Download some sample cellranger output files:  
> wget --no-check-certificate [https://hpc.nih.gov/~Jiang_Lab/Tres/GSE139829_sample.tar.gz](https://hpc.nih.gov/~Jiang_Lab/Tres/GSE139829_sample.tar.gz)  
> tar xvf GSE139829_sample.tar.gz  
  
Then, run CytoSig like this:  
CytoSig_run.py -i GSE139829\_sample/GSM4147093\_UMM059\_,GSM4147096\_UMM063\_,GSM4147099\_UMM066\_ -o output    
  
*Note*: H5 files or Seurat objects are also common formats for single-cell RNASeq data. Please see our further demonstrations on how to convert H5 or Seurat files at [https://github.com/data2intelligence/CytoSig_prediction](https://github.com/data2intelligence/CytoSig_prediction).  

**Usage 2, through Python function inside your customized code**:  
Input:  
Y: the expression matrix of your samples in pandas data frame. Each column name is a sample ID. Each row name is a human gene symbol.  
  
Output: four pandas data frames  
beta: regression coefficients  
std: standard errors of coefficients  
zscore: beta/std  
pvalue: statistical significance  

Then, use the following code snippet in your program:  

import os, sys, pandas, CytoSig  

signature = CytoSig.find_signature_path() # load cytokine response signature installed  
    
signature = pandas.read_csv(signature, sep='\t', index_col=0)  
beta, std, zscore, pvalue = CytoSig.ridge_significance_test(signature, Y, alpha=1E4, alternative="two-sided", nrand=1000, cnt_thres=10, flag_normalize=True, verbose = True)  

**More Usage Examples**:  
We prepared a package for reproducing major prediction results in our paper and demonstrating more usages: [https://github.com/data2intelligence/CytoSig_prediction](https://github.com/data2intelligence/CytoSig_prediction).  
In particular, this package demonstrated more single-cell analyses using H5 files or Seurat object.  

**Usage 3, through Docker**:  
We prepared a Docker image "data2intelligence/data2intelligence-suite" for ARM64 (e.g., Apple silicon) and AMD64 (x86_64, works on Intel chip) architectures. Users don't have to install CytoSig locally to use it.  
Please first install Docker here: [https://www.docker.com/](https://www.docker.com/)

Example 1:
At the folder where you have downloaded CytoSig package, enter the test path with example data by "cd CytoSig/tests". Then, run the following docker command:  
docker run -it -w /tests -v "$(pwd):/tests" data2intelligence/data2intelligence-suite  
After this step, you will enter a Docker container with CytoSig pre-installed and your local folder "tests" mounted to folder "tests" in the container. Then, you can run the following command on the example data.  
CytoSig_run.py -i GSE147507.diff.gz -o output -e 1  

Example 2:
If you want to run CytoSig in the Docker container with local command lines on the host, you can do the followings:  
First, please run the Docker container in a detached mode:  
docker run -dit -w /tests -v "$(pwd):/tests" data2intelligence/data2intelligence-suite  
After this, you should see the Docker container ID, and run the following command for each input file:  
docker exec -it your_docker_container_ID bash -i -c "CytoSig_run.py -i GSE147507.diff.gz -o output -e 1"