Last updated: 2019-05-10

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Knit directory: dsc-log-fold-change/

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Rmd eafe173 Joyce Hsiao 2019-05-08 downstream effects of data transformation methods in null datasets: initial assessment

Summary

Applying data transformation methods to single-cell gene expression count and computing type I error rate in null datasets.


Data transformation methods compared:

\(X_{ig}\): gene expression count in cell \(i\) from gene \(g\)

\(X_{i+}\): gene expression count in cell \(i\), i.e., \(X_{i+} = \sum_g X_{ig}\)

\(S_i\): library size normalization factor for cell \(i\)

\(p\): a positive pseudo-count added to expression matrix; traditionally it is used to ensure log-transformation of the expression matrix is well-defined. For now we use pseudo-count of 1.

  1. log2_none_p1: \(log2(X_{ig} + p)\) where \(p=1\).

  2. log2_libsum_p1: \(log2(X_{ig}/X_{i+} + p)\) where \(p=1\)

  3. log2_libscale_TMM_p1: \(log2(X_{ig}/S_{i} + p)\) where \(p=1\) using edgeR TMM method to estimate \(S_i\).

  4. log2_libscale_RLE_p1: \(log2(X_{ig}/S_{i} + p)\) where \(p=1\) using DESeq2 RLE method to estimate \(S_i\).

  5. counts_pearsons: Pearson’s residuals of expression counts, derived using sctransform (Hafemeister and Satija, 2019).


Pipelines compared:


More about counts_pearson:

For a given gene \(g\), use the sum of all molecules assigned to a cell as a proxy for sequencing depth, and use this cell attribute in a regression model with negative binomial distribution and log link function. Thus, let \(X_g\) be the vector of UMI counts assigned to gene \(g\), and \(m\) be the vector of molecules assigned to the cells, i.e., \(m_i = \sum_i X_{ig}\). For a given \(g\), we have

\(log(E(X_g)) = \beta_0 + \beta_1 log10 m\)

Using the NB parametrization with mean \(\mu\) and variance \(\mu + \mu^2/\theta\),

Pearson’s residuals are defined as:

\(z_{ig} = (X_{ig}-\mu_{ig})/\sigma_{ig}\)

where

\(\mu_{ig} = exp(\beta_{0g} + \beta_{1g}log10 m_i)\),

\(\sigma_{ig} = \sqrt(\mu_{ig} + \mu^2_{ig}/\theta_{g})\)


Data simulation parameters:


Required packages and functions

library(tidyverse)
library(seqgendiff)
library(sctransform)
#source("dsc/modules/poisthin.R")
source("dsc/modules/filter_genes.R")
source("dsc/modules/transform_data.R")
source("dsc/modules/t_test.R")
source("dsc/modules/wilcoxon.R")
source("dsc/modules/limma_voom.R")
source("dsc/modules/edger.R")
source("dsc/modules/deseq2.R")
source("dsc/modules/sva.R")

run_methods <- function(data_obj, nsim, verbose=F) {
  
  Y <- t(data_obj$Y)
  X <- data_obj$X
  beta <- data_obj$beta
  keep_genes <- filter_genes(Y, min_cell_detected=5)
  Y <- Y[keep_genes,]
  beta <- beta[keep_genes]
  
  foo_m <- do.call(rbind, lapply(1:length(de_methods_list), function(j) {

  if (de_methods_list[j] == "edger") {
    if (verbose) message("edger")
      res <- edger(Y=Y, X=X)
      pvals <- res$pval
      return(data.frame(type1error_01=mean(pvals < .01, na.rm=TRUE),
                        type1error_001=mean(pvals < .001, na.rm=TRUE),
                        mse = mean((res$est - beta)^2, na.rm=T),
                        transform_method = de_methods_list[j],
                        de_method = de_methods_list[j],
                        nsim = nsim))
  } 
  if (de_methods_list[j] == "deseq2") {
      if (verbose) message("deseq2")
      res <- deseq2(Y=Y, X=X)
      pvals <- res$pval
      return(data.frame(type1error_01=mean(pvals < .01, na.rm=TRUE),
                        type1error_001=mean(pvals < .001, na.rm=TRUE),
                        mse = mean((res$est - beta)^2, na.rm=T),
                        transform_method = de_methods_list[j],
                        de_method = de_methods_list[j],
                        nsim = nsim))
  } 
  if (de_methods_list[j] == "limma") {
      if (verbose) message("limma")
      foo_l <- do.call(rbind, lapply(1:(length(transform_methods_list)-1), function(k) {
          if (transform_methods_list[k] == "log2_none_p1") {
          transformed_Y <- transform_data(Y, libscale_method = "none", 
                                          log="log2", pseudo_count=1)
          }
          if (transform_methods_list[k] == "log2_libsum_p1") {
          transformed_Y <- transform_data(Y, libscale_method = "sum", 
                                          log="log2", pseudo_count=1)
          }
          if (transform_methods_list[k] == "log2_libscale_TMM_p1") {
          transformed_Y <- transform_data(Y, libscale_method = "TMM", 
                                          log="log2", pseudo_count=1)
          }
          if (transform_methods_list[k] == "log2_libscale_RLE_p1") {
          transformed_Y <- transform_data(Y, libscale_method = "RLE", 
                                          log="log2", pseudo_count=1)
          }
          if (sum(is.na(transformed_Y))==nrow(transformed_Y)*ncol(transformed_Y)) {
          return(data.frame(type1error_01=NA,
                            type1error_001=NA,
                            mse=NA,
                            transform_method = transform_methods_list[k],
                            de_method = de_methods_list[j],
                            nsim = nsim))
          } else {
          fit <- lmFit(transformed_Y,design=X)
          fit.ebayes <- eBayes(fit)
          pvals <- fit.ebayes$p.value[,2]
          return(data.frame(type1error_01=mean(pvals < .01, na.rm=TRUE),
                            type1error_001=mean(pvals < .001, na.rm=TRUE),
                            mse=mean((fit.ebayes$coefficients[,2]-beta)^2,na.rm=T),
                            transform_method = transform_methods_list[k],
                            de_method = de_methods_list[j],
                            nsim = nsim))
          }
      }) )
      return(foo_l)      
    }
  if (de_methods_list[j] == "limma_voom") {
      if (verbose) message("limma_voom")
      res <- limma_voom(Y=Y, X=X)
      pvals <- res$pvalue
      return(data.frame(type1error_01=mean(pvals < .01, na.rm=TRUE),
                        type1error_001=mean(pvals < .001, na.rm=TRUE),
                        mse=mean((res$betahat-beta)^2,na.rm=T),
                        transform_method = de_methods_list[j],
                        de_method = de_methods_list[j],
                        nsim = nsim))
  } 
  if (de_methods_list[j] == "limma_voom_libscale_tmm") {
  if (verbose) message("limma_voom_libscale_tmm")
  libnorm_factors <- edgeR::calcNormFactors(Y, method="TMM")
  res <- limma_voom(Y, X, libnorm_factors=libnorm_factors)
  pvals <- res$pvalue
  return(data.frame(type1error_01=mean(pvals < .01, na.rm=TRUE),
                    type1error_001=mean(pvals < .001, na.rm=TRUE),
                    mse=mean((res$betahat-beta)^2,na.rm=T),
                    transform_method = de_methods_list[j],
                    de_method = de_methods_list[j],
                    nsim = nsim))
  } 
  if (de_methods_list[j] == "limma_voom_libscale_rle") {
    if (verbose) message("limma_voom_libscale_rle")
    libnorm_factors <- edgeR::calcNormFactors(Y, method="RLE")
    if (anyNA(libnorm_factors)) {
    return(data.frame(type1error_01=NA,
                      type1error_001=NA,
                      mse=NA,
                      transform_method = de_methods_list[j],
                      de_method = de_methods_list[j],
                      nsim = nsim))
    } else {
    res <- limma_voom(Y, X, libnorm_factors=libnorm_factors)
    pvals <- res$pvalue
    return(data.frame(type1error_01=mean(pvals < .01, na.rm=TRUE),
                      type1error_001=mean(pvals < .001, na.rm=TRUE),
                      mse=mean((res$betahat-beta)^2,na.rm=T),
                      transform_method = de_methods_list[j],
                      de_method = de_methods_list[j],
                      nsim = nsim))
    }
  } 
  if (de_methods_list[j] == "sva_limma_voom") {
      if (verbose) message("sva_limma_voom")
      output_sva <- sva(Y, X)
      res <- limma_voom(Y, X=output_sva$X.sv)
      pvals <- res$pval
      return(data.frame(type1error_01=mean(pvals < .01, na.rm=TRUE),
                        type1error_001=mean(pvals < .001, na.rm=TRUE),
                        mse=mean((res$betahat-beta)^2,na.rm=T),
                        transform_method = de_methods_list[j],
                        de_method = de_methods_list[j],
                        nsim = nsim))
  } 
  if (de_methods_list[j] == "t_test") {
      if (verbose) message("t_test")
      foo_t <- do.call(rbind, lapply(1:length(transform_methods_list), function(k) {
          if (transform_methods_list[k] == "log2_none_p1") {
          transformed_Y <- transform_data(Y, libscale_method = "none", 
                                          log="log2", pseudo_count=1)
          }
          if (transform_methods_list[k] == "log2_libsum_p1") {
          transformed_Y <- transform_data(Y, libscale_method = "sum", 
                                          log="log2", pseudo_count=1)
          }
          if (transform_methods_list[k] == "log2_libscale_TMM_p1") {
          transformed_Y <- transform_data(Y, libscale_method = "TMM", 
                                          log="log2", pseudo_count=1)
          }
          if (transform_methods_list[k] == "log2_libscale_RLE_p1") {
          transformed_Y <- transform_data(Y, libscale_method = "RLE", 
                                          log="log2", pseudo_count=1)
          }
          if (transform_methods_list[k] == "counts_pearsons") {
          transformed_Y <- transform_data(Y, libscale_method = "pearsons_residual", 
                                          log="none", pseudo_count=1)
          }
          res <- t_test(transformed_Y, X)
          pvals <- res[2,]
          return(data.frame(type1error_01=mean(pvals < .01, na.rm=TRUE),
                            type1error_001=mean(pvals < .001, na.rm=TRUE),
                            mse=mean((res[1,]-beta)^2,na.rm=T),
                            transform_method = transform_methods_list[k],
                            de_method = de_methods_list[j],
                            nsim = nsim))
      }) )
      return(foo_t)      
    }
  }) )
   return(foo_m)
}


transform_methods_list <- c("log2_none_p1", "log2_libsum_p1", "log2_libscale_TMM_p1", 
                            "log2_libscale_RLE_p1", "counts_pearsons")
de_methods_list <- c("edger", "deseq2", "limma", "limma_voom", 
                     "limma_voom_libscale_tmm",
                     "limma_voom_libscale_rle",
                     "sva_limma_voom", "t_test")

No differences in library sizes

Simulations

counts <- readRDS("dsc/data/pbmc_counts.rds")
nsamp <- 100
ngene <- 1000
prop_null <- 0
libsize_factor <- 0
signal_fun <- function(n) rep(libsize_factor, n)
signal_params <- list()
#pvals_thres <- .001
nsim <- 50
for (i in 1:nsim) {
  set.seed(i)
  data_obj <- poisthin(t(counts), nsamp=nsamp, ngene=ngene, 
                       signal_params=signal_params, signal_fun=signal_fun, 
                       prop_null = prop_null)
  saveRDS(data_obj, file = paste0("output/transform_null.Rmd/data_obj_",i,".rds"))
}



out <- do.call(rbind, lapply(1:nsim, function(i) {
  print(i)
  data_obj <- readRDS(file = paste0("output/transform_null.Rmd/data_obj_",i,".rds"))

  res <- run_methods(data_obj, i, verbose=T)
  return(res)
  }) )

out$method <- as.character(out$de_method)
which_relabel <- which(as.character(out$de_method) != as.character(out$transform_method))
out$method[which_relabel] <- paste(as.character(out$de_method), as.character(out$transform_method), sep="_")[which_relabel]

out$method <- factor(out$method)

saveRDS(out, file = "output/transform_null.Rmd/libfactor_0.rds")

Results

#alpha <- .001
out <- readRDS(file = "output/transform_null.Rmd/libfactor_0.rds")
levels(out$method)
 [1] "deseq2"                      "edger"                      
 [3] "limma_log2_libscale_RLE_p1"  "limma_log2_libscale_TMM_p1" 
 [5] "limma_log2_libsum_p1"        "limma_log2_none_p1"         
 [7] "limma_voom"                  "limma_voom_libscale_rle"    
 [9] "limma_voom_libscale_tmm"     "sva_limma_voom"             
[11] "t_test_counts_pearsons"      "t_test_log2_libscale_RLE_p1"
[13] "t_test_log2_libscale_TMM_p1" "t_test_log2_libsum_p1"      
[15] "t_test_log2_none_p1"        
out %>% #filter(n1==50) %>% 
    group_by(method) %>%
    ggplot(., aes(x=method, y=type1error_001, col=method)) +
#        facet_wrap(~de_method) +
        geom_boxplot() + geom_point() + xlab("Type 1 error at alpha < .001") +
        geom_hline(yintercept = .001, col="gray50") +
        ylab("Type I error") +
      scale_x_discrete(position = "top") +
       theme(axis.text.x=element_text(angle = 20, vjust = -.3, hjust=.1)) +
       stat_summary(fun.y=mean, geom="point", shape=4, size=4, col="black")

out %>% #filter(n1==50) %>% 
    group_by(method) %>%
    ggplot(., aes(x=method, y=type1error_01, col=method)) +
#        facet_wrap(~de_method) +
        geom_boxplot() + geom_point() + xlab("Type 1 error at alpha < .01") +
        geom_hline(yintercept = .01, col="gray50") +
        ylab("Type I error") +
      scale_x_discrete(position = "top") +
       theme(axis.text.x=element_text(angle = 20, vjust = -.3, hjust=.1)) +
       stat_summary(fun.y=mean, geom="point", shape=4, size=4, col="black")

out %>% #filter(n1==50) %>% 
    group_by(nsim, method) %>%
    summarise(mse_mn =mean(mse, na.rm=T)) %>%
    ggplot(., aes(x=method, y=mse_mn, col=method)) +
#        facet_wrap(~de_method) +
        geom_boxplot() + geom_point() + xlab("Mean squared error") +
        ylab("Mean squared error") +
      scale_x_discrete(position = "top") +
       theme(axis.text.x=element_text(angle = 20, vjust = -.3, hjust=.1)) +
       stat_summary(fun.y=mean, geom="point", shape=4, size=4, col="black")


library size factor 1

Simulation

counts <- readRDS("dsc/data/pbmc_counts.rds")
nsamp <- 100
ngene <- 1000
prop_null <- 0
libsize_factor <- 1
signal_fun <- function(n) rep(libsize_factor, n)
signal_params <- list()
#pvals_thres <- .001
nsim <- 50
for (i in 1:nsim) {
  set.seed(i)
  data_obj <- poisthin(t(counts), nsamp=nsamp, ngene=ngene, 
                       signal_params=signal_params, signal_fun=signal_fun, 
                       prop_null = prop_null)
  saveRDS(data_obj, file = paste0("output/transform_null.Rmd/data_obj_libfactor_1_",i,".rds"))
}


nsim=50
out <- do.call(rbind, lapply(1:nsim, function(i) {

  data_obj <- readRDS(file = paste0("output/transform_null.Rmd/data_obj_libfactor_1_",
                                    i,".rds"))

  res <- run_methods(data_obj, i)
  return(res)
  }) )

out$method <- as.character(out$de_method)
which_relabel <- which(as.character(out$de_method) != as.character(out$transform_method))
out$method[which_relabel] <- paste(as.character(out$de_method), as.character(out$transform_method), sep="_")[which_relabel]

out$method <- factor(out$method)
saveRDS(out, file = "output/transform_null.Rmd/libfactor_1.rds")

Results

#alpha <- .001
out <- readRDS(file = "output/transform_null.Rmd/libfactor_1.rds")

levels(out$method)
 [1] "deseq2"                      "edger"                      
 [3] "limma_log2_libscale_RLE_p1"  "limma_log2_libscale_TMM_p1" 
 [5] "limma_log2_libsum_p1"        "limma_log2_none_p1"         
 [7] "limma_voom"                  "limma_voom_libscale_rle"    
 [9] "limma_voom_libscale_tmm"     "sva_limma_voom"             
[11] "t_test_counts_pearsons"      "t_test_log2_libscale_RLE_p1"
[13] "t_test_log2_libscale_TMM_p1" "t_test_log2_libsum_p1"      
[15] "t_test_log2_none_p1"        
out %>% #filter(n1==50) %>% 
    group_by(method) %>%
    ggplot(., aes(x=method, y=type1error_001, col=method)) +
#        facet_wrap(~de_method) +
        geom_boxplot() + geom_point() + xlab("Type 1 error at alpha < .001") +
        geom_hline(yintercept = .001, col="gray50") +
        ylab("Type I error") +
      scale_x_discrete(position = "top") +
       theme(axis.text.x=element_text(angle = 20, vjust = -.3, hjust=.1)) +
       stat_summary(fun.y=mean, geom="point", shape=4, size=4, col="black")

out %>% #filter(n1==50) %>% 
    group_by(method) %>%
    ggplot(., aes(x=method, y=type1error_01, col=method)) +
#        facet_wrap(~de_method) +
        geom_boxplot() + geom_point() + xlab("Type 1 error at alpha < .01") +
        geom_hline(yintercept = .01, col="gray50") +
        ylab("Type I error") +
      scale_x_discrete(position = "top") +
       theme(axis.text.x=element_text(angle = 20, vjust = -.3, hjust=.1)) +
       stat_summary(fun.y=mean, geom="point", shape=4, size=4, col="black")

out %>% #filter(n1==50) %>% 
    group_by(nsim, method) %>%
    summarise(mse_mn =mean(mse, na.rm=T)) %>%
    ggplot(., aes(x=method, y=mse_mn, col=method)) +
#        facet_wrap(~de_method) +
        geom_boxplot() + geom_point() + xlab("Mean squared error") +
        ylab("Mean squared error") +
      scale_x_discrete(position = "top") +
       theme(axis.text.x=element_text(angle = 20, vjust = -.3, hjust=.1)) +
       stat_summary(fun.y=mean, geom="point", shape=4, size=4, col="black")


library size factor 2

counts <- readRDS("dsc/data/pbmc_counts.rds")
nsamp <- 100
ngene <- 1000
prop_null <- 0
libsize_factor <- 2
signal_fun <- function(n) rep(libsize_factor, n)
signal_params <- list()
#pvals_thres <- .001
nsim <- 50
for (i in 1:nsim) {
  set.seed(i)
  data_obj <- poisthin(t(counts), nsamp=nsamp, ngene=ngene, 
                       signal_params=signal_params, signal_fun=signal_fun, 
                       prop_null = prop_null)
  saveRDS(data_obj, file = paste0("output/transform_null.Rmd/data_obj_libfactor_2_",i,".rds"))
}


nsim=50
out <- do.call(rbind, lapply(1:nsim, function(i) {
  print(i)
  data_obj <- readRDS(file = paste0("output/transform_null.Rmd/data_obj_libfactor_2_",
                                    i,".rds"))

  res <- run_methods(data_obj, i, verbose = T)
  return(res)
  }) )
out$method <- as.character(out$de_method)
which_relabel <- which(as.character(out$de_method) != as.character(out$transform_method))
out$method[which_relabel] <- paste(as.character(out$de_method), as.character(out$transform_method), sep="_")[which_relabel]
out$method <- factor(out$method)

saveRDS(out, file = "output/transform_null.Rmd/libfactor_2.rds")

Results

#alpha <- .001
out <- readRDS(file = "output/transform_null.Rmd/libfactor_2.rds")

levels(out$method)
 [1] "deseq2"                      "edger"                      
 [3] "limma_log2_libscale_RLE_p1"  "limma_log2_libscale_TMM_p1" 
 [5] "limma_log2_libsum_p1"        "limma_log2_none_p1"         
 [7] "limma_voom"                  "limma_voom_libscale_rle"    
 [9] "limma_voom_libscale_tmm"     "sva_limma_voom"             
[11] "t_test_counts_pearsons"      "t_test_log2_libscale_RLE_p1"
[13] "t_test_log2_libscale_TMM_p1" "t_test_log2_libsum_p1"      
[15] "t_test_log2_none_p1"        
out %>% #filter(n1==50) %>% 
    group_by(method) %>%
    ggplot(., aes(x=method, y=type1error_001, col=method)) +
#        facet_wrap(~de_method) +
        geom_boxplot() + geom_point() + xlab("Type 1 error at alpha < .001") +
        geom_hline(yintercept = .001, col="gray50") +
        ylab("Type I error") +
      scale_x_discrete(position = "top") +
       theme(axis.text.x=element_text(angle = 20, vjust = -.3, hjust=.1)) +
       stat_summary(fun.y=mean, geom="point", shape=4, size=4, col="black")
Warning: Removed 3 rows containing non-finite values (stat_boxplot).
Warning: Removed 3 rows containing non-finite values (stat_summary).
Warning: Removed 3 rows containing missing values (geom_point).

out %>% #filter(n1==50) %>% 
    group_by(method) %>%
    ggplot(., aes(x=method, y=type1error_01, col=method)) +
#        facet_wrap(~de_method) +
        geom_boxplot() + geom_point() + xlab("Type 1 error at alpha < .01") +
        geom_hline(yintercept = .01, col="gray50") +
        ylab("Type I error") +
      scale_x_discrete(position = "top") +
       theme(axis.text.x=element_text(angle = 20, vjust = -.3, hjust=.1)) +
       stat_summary(fun.y=mean, geom="point", shape=4, size=4, col="black")
Warning: Removed 3 rows containing non-finite values (stat_boxplot).
Warning: Removed 3 rows containing non-finite values (stat_summary).
Warning: Removed 3 rows containing missing values (geom_point).

out %>% #filter(n1==50) %>% 
    group_by(nsim, method) %>%
    summarise(mse_mn =mean(mse, na.rm=T)) %>%
    ggplot(., aes(x=method, y=mse_mn, col=method)) +
#        facet_wrap(~de_method) +
        geom_boxplot() + geom_point() + xlab("Mean squared error") +
        ylab("Mean squared error") +
      scale_x_discrete(position = "top") +
       theme(axis.text.x=element_text(angle = 20, vjust = -.3, hjust=.1)) +
       stat_summary(fun.y=mean, geom="point", shape=4, size=4, col="black")
Warning: Removed 3 rows containing non-finite values (stat_boxplot).
Warning: Removed 3 rows containing non-finite values (stat_summary).
Warning: Removed 3 rows containing missing values (geom_point).


TBD

res_libfactor_null <- readRDS(file = "output/transform_null.Rmd/type1error.rds")
res_libfactor_null$libfactor <- 2^0
res_libfactor_1 <- readRDS(file = "output/transform_null.Rmd/type1error_libfactor_1.rds")
res_libfactor_1$libfactor <- 2^1
res_libfactor_2 <- readRDS(file = "output/transform_null.Rmd/type1error_libfactor_2.rds")
res_libfactor_2$libfactor <- 2^2

res <- rbind(res_libfactor_null,
             res_libfactor_1)
res$libfactor <- factor(res$libfactor)


# res %>% #filter(n1==50) %>% 
#     group_by(libfactor, transform_method) %>%
#     ggplot(., aes(x=transform_method, y=type1error_001, col=transform_method)) +
# #        facet_wrap(~libfactor) +
#         geom_boxplot() + geom_point() + xlab("Type 1 error at alpha < .001") +
#         geom_hline(yintercept = .001, col="gray50") +
#         ylab("Type I error") +
#       scale_x_discrete(position = "top",
#                        labels=labels_methods) +
#        theme(axis.text.x=element_text(angle = 20, vjust = -.3, hjust=.1)) +
#        stat_summary(fun.y=mean, geom="point", shape=4, size=4, col="black")

sessionInfo()
R version 3.5.1 (2018-07-02)
Platform: x86_64-pc-linux-gnu (64-bit)
Running under: Scientific Linux 7.4 (Nitrogen)

Matrix products: default
BLAS/LAPACK: /software/openblas-0.2.19-el7-x86_64/lib/libopenblas_haswellp-r0.2.19.so

locale:
 [1] LC_CTYPE=en_US.UTF-8       LC_NUMERIC=C              
 [3] LC_TIME=en_US.UTF-8        LC_COLLATE=en_US.UTF-8    
 [5] LC_MONETARY=en_US.UTF-8    LC_MESSAGES=en_US.UTF-8   
 [7] LC_PAPER=en_US.UTF-8       LC_NAME=C                 
 [9] LC_ADDRESS=C               LC_TELEPHONE=C            
[11] LC_MEASUREMENT=en_US.UTF-8 LC_IDENTIFICATION=C       

attached base packages:
[1] stats     graphics  grDevices utils     datasets  methods   base     

other attached packages:
 [1] sctransform_0.2.0 seqgendiff_0.1.0  forcats_0.3.0    
 [4] stringr_1.3.1     dplyr_0.8.0.1     purrr_0.3.2      
 [7] readr_1.3.1       tidyr_0.8.3       tibble_2.1.1     
[10] ggplot2_3.1.0     tidyverse_1.2.1  

loaded via a namespace (and not attached):
 [1] tidyselect_0.2.5   listenv_0.7.0      reshape2_1.4.3    
 [4] haven_1.1.2        lattice_0.20-38    colorspace_1.3-2  
 [7] generics_0.0.2     htmltools_0.3.6    yaml_2.2.0        
[10] rlang_0.3.4        pillar_1.3.1       glue_1.3.0        
[13] withr_2.1.2        modelr_0.1.2       readxl_1.1.0      
[16] plyr_1.8.4         munsell_0.5.0      gtable_0.2.0      
[19] workflowr_1.3.0    cellranger_1.1.0   rvest_0.3.2       
[22] future_1.12.0      codetools_0.2-15   evaluate_0.12     
[25] labeling_0.3       knitr_1.20         parallel_3.5.1    
[28] broom_0.5.1        Rcpp_1.0.1         scales_1.0.0      
[31] backports_1.1.2    jsonlite_1.6       fs_1.2.6          
[34] gridExtra_2.3      hms_0.4.2          digest_0.6.18     
[37] stringi_1.2.4      grid_3.5.1         rprojroot_1.3-2   
[40] cli_1.0.1          tools_3.5.1        magrittr_1.5      
[43] lazyeval_0.2.1     crayon_1.3.4       whisker_0.3-2     
[46] future.apply_1.2.0 pkgconfig_2.0.2    MASS_7.3-51.1     
[49] Matrix_1.2-17      xml2_1.2.0         lubridate_1.7.4   
[52] assertthat_0.2.0   rmarkdown_1.10     httr_1.3.1        
[55] rstudioapi_0.10    globals_0.12.4     R6_2.4.0          
[58] nlme_3.1-137       git2r_0.23.0       compiler_3.5.1