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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: pip3 install --upgrade xlsxwriter
4, openpyxl >= 3.0.9: pip3 install --upgrade openpyxl
5, scipy: pip3 install --upgrade scipy

Install: pip3 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: python3 -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
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.

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.
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/

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"