Evaluating statistical learning methods for cell type classification and feature selection using RNA-seq data
BMC Bioinformatics volume 15, Article number: P26 (2014)
Single cell RNA-seq offers the opportunity to develop a systematic inventory of cell types in the brain.
Materials and methods
We evaluated several statistical learning methods for classifying RNA-seq data using samples obtained from different brain regions as surrogates for single cell data. These include the ventral tegmental area (VTA, n=36), the nucleus accumbens core (AcbC, n=34) and shell (AcbS, n=30) . We tested hierarchical clustering methods using different distance matrices (correlation, uncentered, abscor) and clustering algorithms. Multiscale bootstrap was used to evaluate the statistical significance of the clusters. We also tested the Bayesian Hierarchical Clustering method, which uses hypothesis testing to decide which cluster mergers increase the tree quality and calculates the optimum tree depth.
Both the Ward's algorithm and the Bayesian method correctly classified the majority of the samples. The reliability of the clusters were evaluated by repeated down sampling (without replacement). We found that sample sizes of 4-8/cluster were classified by the Ward's method with a very low error rate (0.02), while the error rate of BHC was slightly higher (0.11). We then compared Lasso and elastic net regularized logistic regression on the selection of genes that can be used as the molecular identity of each cluster. The Lasso method identified 15 genes (including Th, tyrosine hydroxylase) for the VTA cluster with cross validation error of 0.014. An elastic net regularized logistic regression model was also fitted for the VTA cluster. A two dimensional cross-validation procedure identified the parameters with the smallest cross validation error of 0.013. This model has 34 genes, among them are Th and Chrna4, two genes with high expression levels in the VTA. In addition, other models with slightly larger cross validation errors (~0.016) contain 35 -131 genes. We further fitted a multinomial elastic net model containing all three brain regions. The model with the smallest cross validation error (0.05) contained a set of 54 genes. Lastly, we tested the sparse hierarchical clustering method that combines clustering and feature selection.
We found that although this method classified all VTA samples correctly, it failed to separate the AcbS and AcbC samples, potentially because the variable selection step removed some key information differentiating these two similar populations. In summary, these results provided guidance on the selection of statistical learning methods for studying single cell transcriptome data, where the cell type is unknown.
Chen H, Luo R, Gong S, Matta SG, Sharp BM: Protection genes in nucleus accumbens shell affect vulnerability to nicotine self-administration across isogenic strains of adolescent rat. PLoS One. 2014, 9 (1): e86214-
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Chen, H. Evaluating statistical learning methods for cell type classification and feature selection using RNA-seq data. BMC Bioinformatics 15 (Suppl 10), P26 (2014). https://doi.org/10.1186/1471-2105-15-S10-P26