inst/tests/test.plot_genes_jitter.R

In monocle: Clustering, differential expression, and trajectory analysis for single- cell RNA-Seq

library(monocle)
library(HSMMSingleCell)
context("plot_genes_jitter functions properly")

pd <- new("AnnotatedDataFrame", data = HSMM_sample_sheet)
fd <- new("AnnotatedDataFrame", data = HSMM_gene_annotation)

# First create a CellDataSet from the relative expression levels
HSMM <- newCellDataSet(as.matrix(HSMM_expr_matrix),   
                       phenoData = pd, 
                       featureData = fd,
                       lowerDetectionLimit=0.1,
                       expressionFamily=tobit(Lower=0.1))

# Next, use it to estimate RNA counts
rpc_matrix <- relative2abs(HSMM, method = "num_genes")

# Now, make a new CellDataSet using the RNA counts
HSMM <- newCellDataSet(as(as.matrix(rpc_matrix), "sparseMatrix"),
                       phenoData = pd, 
                       featureData = fd,
                       lowerDetectionLimit=0.5,
                       expressionFamily=negbinomial.size())

HSMM <- estimateSizeFactors(HSMM)
HSMM <- estimateDispersions(HSMM)

HSMM <- detectGenes(HSMM, min_expr = 0.1)
expressed_genes <- row.names(subset(fData(HSMM), num_cells_expressed >= 10))


pData(HSMM)$Total_mRNAs <- Matrix::colSums(exprs(HSMM))


HSMM <- HSMM[,pData(HSMM)$Total_mRNAs < 1e6]
upper_bound <- 10^(mean(log10(pData(HSMM)$Total_mRNAs)) + 2*sd(log10(pData(HSMM)$Total_mRNAs)))
lower_bound <- 10^(mean(log10(pData(HSMM)$Total_mRNAs)) - 2*sd(log10(pData(HSMM)$Total_mRNAs)))
qplot(Total_mRNAs, data=pData(HSMM), color=Hours, geom="density") + 
  geom_vline(xintercept=lower_bound) + 
  geom_vline(xintercept=upper_bound)

HSMM <- HSMM[,pData(HSMM)$Total_mRNAs > lower_bound & 
               pData(HSMM)$Total_mRNAs < upper_bound]								  
HSMM <- detectGenes(HSMM, min_expr = 0.1)

L <- log(exprs(HSMM[expressed_genes,]))

# Standardize each gene, so that they are all on the same scale,
# Then melt the data with plyr so we can plot it easily"
melted_dens_df <- melt(Matrix::t(scale(Matrix::t(L))))

MYF5_id <- row.names(subset(fData(HSMM), gene_short_name == "MYF5"))
ANPEP_id <- row.names(subset(fData(HSMM), gene_short_name == "ANPEP"))

cth <- newCellTypeHierarchy()
cth <- addCellType(cth, "Myoblast", classify_func=function(x) {x[MYF5_id,] >= 1})
cth <- addCellType(cth, "Fibroblast", classify_func=function(x)
{x[MYF5_id,] < 1 & x[ANPEP_id,] > 1})

HSMM <- classifyCells(HSMM, cth, 0.1)

disp_table <- dispersionTable(HSMM)
unsup_clustering_genes <- subset(disp_table, mean_expression >= 0.1)
HSMM <- setOrderingFilter(HSMM, unsup_clustering_genes$gene_id)

HSMM <- reduceDimension(HSMM, max_components=2, num_dim = 6, 
                        reduction_method = 'tSNE', verbose = T) 
HSMM <- clusterCells(HSMM,
                     num_clusters=2)

marker_diff <- markerDiffTable(HSMM[expressed_genes,], 
                               cth, 
                               residualModelFormulaStr="~Media + num_genes_expressed",
                               cores=detectCores())

set.seed(0)

candidate_clustering_genes <- row.names(subset(marker_diff, qval < 0.01))
marker_spec <- calculateMarkerSpecificity(HSMM[candidate_clustering_genes,], cth)
head(selectTopMarkers(marker_spec, 3))

semisup_clustering_genes <- unique(selectTopMarkers(marker_spec, 500)$gene_id)
HSMM <- setOrderingFilter(HSMM, semisup_clustering_genes)

HSMM <- reduceDimension(HSMM, max_components=2, num_dim = 3, norm_method = 'log', reduction_method = 'tSNE', 
                        residualModelFormulaStr="~Media + num_genes_expressed", verbose = T) 
HSMM <- clusterCells(HSMM, num_clusters=2) 

HSMM_myo <- HSMM[,pData(HSMM)$CellType == "Myoblast"]	
HSMM_myo <- estimateDispersions(HSMM_myo)

HSMM_myo <- setOrderingFilter(HSMM_myo, ordering_genes)
HSMM_myo <- reduceDimension(HSMM_myo, max_components=2, method = 'DDRTree')
HSMM_myo <- orderCells(HSMM_myo)

GM_state <- function(cds){
  if (length(unique(pData(cds)$State)) > 1){
    T0_counts <- table(pData(cds)$State, pData(cds)$Hours)[,"0"]
    return(as.numeric(names(T0_counts)[which(T0_counts == max(T0_counts))]))
  }else { return (1) } 
}
HSMM_myo <- orderCells(HSMM_myo, root_state=GM_state(HSMM_myo))

blast_genes <- row.names(subset(fData(HSMM_myo), gene_short_name %in% c("CCNB2", "MYOD1", "MYOG")))

test_that("plot_genes_jitter functions as it appears in vignette", 
          expect_error(plot_genes_jitter(HSMM_myo[blast_genes,], grouping="State", min_expr=0.1), NA))

test_that("plot_genes_jitter works")