This program classifies single cell types from RNA-seq data in mice. Our approach is to use supervised
machine learning algorithms on a single cell RNA-seq dataset with a marked training set and unmarked
testing set. We used 4 machine learning algorithms with the hopes of determining the best supervised
learning algorithm to classify unmarked cells.
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Support Vector Machine using Radial Basis Function Kernel
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Random Forests, Decision Trees
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KNN
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Multi-Layer Perceptron (Neural Network)
We found that the Neural Network gave the best performance, and the Support Vector Machine
gave the worst performance.
This project is written in Python using version 2.7. All machine learning algorithms are from
the scikit-learn library which uses SciPy, NumPy and matplotlib to implement data mining and
data analysis tools. The stable version of scikit-learn is available here:
- Scikit-learn: http://scikit-learn.org/stable/
*** IMPORTANT NOTE: The Multi-Layer Perceptron is not supported in the stable version of
scikit (as of 5/7/16). In order to use this classifier, the development version needs to
be used. As such, the import for the Neural Network file is commented out in main.py so
that the program compiles on the stable version of scikit-learn. In order to use the
Multi-Layer Perceptron, download and compile the development version of scikit-learn and
uncomment 'import neuralNetwork_RNASeq' on line 7 of main.py. Then, the neural network
classifier can be used.
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GSE60361C13005Expression.txt: this file holds the raw RNA seq data. It is organized by cells (columns) and their gene expression levels (rows). It is roughly 3000 cells x 20000 genes in size
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expressionmRNAAnnotations.txt: this file holds the annotations for the raw data including molecule count and cell type (1-9) classification
*** IMPORTANT NOTE: Github's file size limit is 100MB. Both the raw data and annotations files exceed these limits. As such, both files are compressed into a directory, 'RawData_Annotations_Compress.zip'. Upon cloning the source code, unpack this .zip and either supply the path into the directory to pass the files to main.py, or move the 2 files into the root directory of the project and just supply the file name.
- RNASeq_SingleCellClassification_Results: this directory holds the results from each of the classifiers. There are 4 run options in terms of preprocessing, and each of these options has a subdirectory in the results folder. The two options which have a total of 4 combinations are down sampling or not down sampling, and 10-fold cross validation or 1-fold cross validation. In order to converge on the 'true' results of the classifiers, each classifier is run 5 times on each of the 4 preprocessing run options. In addition, as the user has the ability to supply the number of neighbors to be used for KNN, there are 5 runs for each K = 1-8, on each of the 4 preprocessing run options.
The usage of the program is as follows:
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python main.py {raw_data} {data_annotations} {classifier 1-4} {down_sample - 0 or 1} {cross_validate - 0 or 1} {n_neighbors}
- To select a classifier, supply a number 1-4 corresponding to:
- 1 = SVM
- 2 = Neural Network
- 3 = KNN
- 4 = Random Forest
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To enable down sampling, supply 1. To disable, supply 0
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To enable cross validation, supply 1. To disable, supply 0
*** ONLY IF USING KNN:
- Supply a valid integer n_neighbors
Examples:
KNN with 5 neighbors, down sampling enabled, cross validation disabled:
- python main.py GSE60361C13005Expression.txt expressionmRNAAnnotations.txt 3 1 0 5
SVM with down sampling disabled, cross validation enabled
- python main.py GSE60361C13005Expression.txt expressionmRNAAnnotations.txt 1 0 1
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main.py: This file processes the command line arguments supplied and runs the program using the selected classifier. Based on user input, the program decides whether to down sample, cross validate, and which classifier to use. This file defines the four classifiers supplied to the user for classification: Support Vector Machine using Radial Basis Function Kernel, Mutli-Layer Perceptron (Neural Network), K-Nearest Neighbor, and Random Forest. After calling the preproccess code and running the classification, this class sends the results to an analysis file that performs evaluations and writes the results to file.
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RNASeqData.py This class object represents the RNA Seq Data. It holds the raw data, the annotations, and provides methods for partitioning the data. The partitions (for both down sampling and non down sampling and cross validation and no cross validation) randomly make partitions of the data for both training and testing, while simultaneously holding the annotations for the randomly selected testing data. The class also provides accessor methods for all data, annotations, training data, testing data, and training data target values to evaluate performance.
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preprocess.py This file does all of the preprocessing work before running classification. This file loads raw data and annotations into memory. Next, this file is used to down dample by both cluster size and molecule count.
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analysis.py After classification, this file is used to evaluate the performance of the classifier and write the results to an output file. First, this file creates a confusion matrix and then computes the accuracy, sensitivity, specificity, MCC, and F1 Score at both the class level and the global level across the supplied number of folds (10 folds for cross validation and 1 fold for non-cross validation). This class also uses a basic metric of merely counting the number of correct classifications that was used initially to check the performance of the classifiers. After evaluating, the results are written to a file.
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knn_RNASeq.py This file defines the K Nearest Neighbor classifier. It allows the user to specificy the number of neighbors used and then fits the training data and the samples to the classifier. Then, it takes training data and makes predictions, returning the results of the predictions.
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neuralNetwork_RNASeq.py This file defines the Multi-Layer Perceptron (Neural Network). It fits the training data and the samples to the classifier. Then, it takes training data and makes predictions, returning the results of the predictions.
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randomForest_RNASeq.py This file defines the Random Forest Classifier. It fits the training data and the samples to the classifier. Then, it takes training data and makes predictions, returning the results of the predictions.
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rbfSVC_RNASeq.py This file defines the Support Vector Machine using a Radial Basis Function Kernel. It fits the training data and the samples to the classifier. Then, it takes training data and makes predictions, returning the results of the predictions.
Project motivated by data from:
- Zeisel, A. et al. Cell types in the mouse cortex and hippocampus revealed by single-cell RNA-seq. Science 347, 1138-1142 (2015).