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Rmd 81f440d kevinlkx 2022-08-25 unguided T cell res 
mkdir -p /project2/xinhe/kevinluo/GSFA/data

cp /project2/xinhe/yifan/Factor_analysis/Stimulated_T_Cells/processed_data/deviance_residual.all_T_cells_merged_top_6k.batch_uncorrected.rds \
  /project2/xinhe/kevinluo/GSFA/unguided_GSFA/Stimulated_T_Cells/processed_data/deviance_residual.all_T_cells_merged_top_6k.batch_uncorrected.rds

cp /project2/xinhe/yifan/Factor_analysis/Stimulated_T_Cells/processed_data/metadata.all_T_cells_merged.rds \
  /project2/xinhe/kevinluo/GSFA/unguided_GSFA/Stimulated_T_Cells/processed_data/metadata.all_T_cells_merged.rds

Analysis scripts

  • R script: /home/kaixuan/projects/GSFA_analysis/code/run_unguided_gsfa_Tcells.R
  • sbatch script: /home/kaixuan/projects/GSFA_analysis/code/run_unguided_gsfa_Tcells.sbatch
mkdir -p /project2/xinhe/kevinluo/GSFA/unguided_GSFA/log
cd /project2/xinhe/kevinluo/GSFA/unguided_GSFA/log

sbatch ~/projects/GSFA_analysis/code/run_unguided_gsfa_Tcells.sbatch

Load packages

suppressPackageStartupMessages(library(data.table))
# suppressPackageStartupMessages(library(Seurat))
suppressPackageStartupMessages(library(ComplexHeatmap))
suppressPackageStartupMessages(library(ggplot2))
require(reshape2)
require(dplyr)
require(ComplexHeatmap)
theme_set(theme_bw() + theme(plot.title = element_text(size = 14, hjust = 0.5),
                             axis.title = element_text(size = 14),
                             axis.text = element_text(size = 13),
                             legend.title = element_text(size = 13),
                             legend.text = element_text(size = 12),
                             panel.grid.minor = element_blank())
)
suppressPackageStartupMessages(library(gridExtra))
source("code/plotting_functions.R")

Set directories

res_dir <- "/project2/xinhe/kevinluo/GSFA/unguided_GSFA/Stimulated_T_Cells/"
dir.create(res_dir, recursive = TRUE, showWarnings = FALSE)

Load unguided GSFA result

fit <- readRDS("/project2/xinhe/kevinluo/GSFA/unguided_GSFA/Stimulated_T_Cells/unguided_gsfa_output/All.gibbs_obj_k20.svd.light.rds")

Load the cell by perturbation matrix.

data_folder <- "/project2/xinhe/kevinluo/GSFA/unguided_GSFA/Stimulated_T_Cells/"
metadata <- readRDS(paste0(data_folder, "processed_data/metadata.all_T_cells_merged.rds"))
sample_group <- endsWith(metadata$orig.ident, "S") * 1 # 0: unstimulated, 1: stimulated
metadata <- metadata[which(sample_group == 1), ]

# Perturbation info:
G_mat <- metadata[, 4:24]
G_mat <- as.matrix(G_mat)
KO_names <- colnames(G_mat)
negctrl_index <- which(KO_names == "NonTarget")

Use linear regression to test for the association between perturbations and factors

Z_pm <- fit$posterior_means$Z_pm
if(!all.equal(rownames(G_mat), rownames(Z_pm))){
  stop("Rownames of G_mat do not match with Z_pm!")
}

perturb_matrix <- G_mat
factor_matrix <- Z_pm

summary_df <- expand.grid(colnames(perturb_matrix), colnames(factor_matrix))
colnames(summary_df) <- c("perturb", "factor")

summary_df <- cbind(summary_df, beta = NA, statistic = NA, pval = NA)

for(i in 1:nrow(summary_df)){
  df <- data.frame(perturb = perturb_matrix[,summary_df$perturb[i]], 
                   factor = factor_matrix[,summary_df$factor[i]])
  lm.res <- lm(factor ~ perturb, data=df)
  summary_df[i, ]$beta <- summary(lm.res)$coefficients["perturb",1]
  summary_df[i, ]$statistic <- summary(lm.res)$coefficients["perturb",3]
  summary_df[i, ]$pval <- summary(lm.res)$coefficients["perturb",4]
}

summary_df$fdr <- p.adjust(summary_df$pval, method = "BH")
summary_df$bonferroni_adj <- p.adjust(summary_df$pval, method = "bonferroni")

saveRDS(summary_df, file = file.path(res_dir, "Tcells_simultated_unguidedGSFA_guide_factor_lm_summary_df.rds"))

stat_mat <- reshape2::dcast(summary_df %>% dplyr::select(perturb, factor, statistic), perturb ~ factor, value.var = "statistic") 
rownames(stat_mat) <- stat_mat$perturb
stat_mat$perturb <- NULL
stat_mat <- as.matrix(stat_mat)

beta_mat <- reshape2::dcast(summary_df %>% dplyr::select(perturb, factor, beta), perturb ~ factor, value.var = "beta")
rownames(beta_mat) <- beta_mat$perturb
beta_mat$perturb <- NULL
beta_mat <- as.matrix(beta_mat)

fdr_mat <- reshape2::dcast(summary_df %>% dplyr::select(perturb, factor, fdr), perturb ~ factor, value.var = "fdr")
rownames(fdr_mat) <- fdr_mat$perturb
fdr_mat$perturb <- NULL
fdr_mat <- as.matrix(fdr_mat)

bonferroni_mat <- reshape2::dcast(summary_df %>% dplyr::select(perturb, factor, bonferroni_adj), 
                                  perturb ~ factor, value.var = "bonferroni_adj")
rownames(bonferroni_mat) <- bonferroni_mat$perturb
bonferroni_mat$perturb <- NULL
bonferroni_mat <- as.matrix(bonferroni_mat)
# pdf(file.path(res_dir, "stat-fdr-dotplot.pdf"), width = 9, height = 5.5)
KO_names <- rownames(fdr_mat)
dotplot_effectsize(t(beta_mat), t(fdr_mat),
                   reorder_markers = c(KO_names[KO_names!="NonTarget"], "NonTarget"),
                   color_lgd_title = "Estimated effect size",
                   size_lgd_title = "FDR",
                   max_score = 0.6,
                   min_score = -0.6,
                   by_score = 0.3) + coord_flip()

Version Author Date
43d8586 kevinlkx 2022-08-25 
# dev.off()

Plot perturbation ~ cluster associations (show Bonferroni adjusted p-values)

# pdf(file.path(res_dir, "stat-bonferroni-dotplot.pdf"), width = 9, height = 5.5)
KO_names <- rownames(bonferroni_mat)
dotplot_effectsize(t(beta_mat), t(bonferroni_mat),
                   reorder_markers = c(KO_names[KO_names!="NonTarget"], "NonTarget"),
                   color_lgd_title = "Estimated effect size",
                   size_lgd_title = "Bonferroni\nadjusted p-value",
                   max_score = 0.6,
                   min_score = -0.6,
                   by_score = 0.3) + coord_flip()

Version Author Date
43d8586 kevinlkx 2022-08-25 
# dev.off()

Find DE genes for each factor and assign DE genes to associated perturbations

First, find DE genes for each factor using F matrix (PIP>0.95).

Then, for each perturbation, find the associated factors, and pull the DE genes for those factors.

F_pm <- fit$posterior_means$F_pm
# dim(F_pm)
# feature.names <- data.frame(fread(file.path(data_dir, "LUHMES_GSM4219575_Run1_genes.tsv.gz"),
#                                   header = FALSE), stringsAsFactors = FALSE)

de.genes.factors <- vector("list", length = ncol(F_pm))
names(de.genes.factors) <- colnames(F_pm)
for( i in 1:length(de.genes.factors)){
  de_genes <- rownames(F_pm[F_pm[,i] > 0.95,])
  # de_genes <- feature.names$V2[match(de_genes, feature.names$V1)]
  de.genes.factors[[i]] <- de_genes
}

Number of DE genes for each perturbation (FDR < 0.05)

perturb_names <- colnames(perturb_matrix)
perturb_names <- c("NonTarget", perturb_names[perturb_names!="NonTarget"])

de.genes.perturbs <- vector("list", length = length(perturb_names))
names(de.genes.perturbs) <- perturb_names

for(i in 1:length(de.genes.perturbs)){
  perturb_name <- names(de.genes.perturbs)[i]
  associated_factors <- colnames(fdr_mat)[which(fdr_mat[perturb_name, ] < 0.05)]
  if(length(associated_factors) > 0){
    de.genes.perturbs[[i]] <- unique(unlist(de.genes.factors[associated_factors]))
  }
}

num.de.genes.perturbs <- sapply(de.genes.perturbs, length)

unguided_GSFA_fdr0.05_genes <- de.genes.perturbs

dge_plot_df <- data.frame(Perturbation = names(num.de.genes.perturbs), Num_DEGs = num.de.genes.perturbs)
dge_plot_df$Perturbation <- factor(dge_plot_df$Perturbation, levels = names(num.de.genes.perturbs))

# pdf(file.path(res_dir, "count-de-genes.pdf"), width = 13, height = 5)
ggplot(data=dge_plot_df, aes(x = Perturbation, y = Num_DEGs+1)) +
  geom_bar(position = "dodge", stat = "identity") +
  geom_text(aes(label = Num_DEGs), position=position_dodge(width=0.9), vjust=-0.25) +
  scale_y_log10() +
  scale_fill_brewer(palette = "Set2") +
  labs(x = "Target gene",
       y = "Number of DEGs",
       title = "Number of DEGs detected by unguided GSFA") +
  theme(axis.text.x = element_text(angle = 45, hjust = 1, size = 12),
        legend.position = "bottom",
        legend.text = element_text(size = 13))

Version Author Date
43d8586 kevinlkx 2022-08-25 
# dev.off()

Number of DE genes for each perturbation (Bonferroni adjusted p-value < 0.05)

perturb_names <- colnames(perturb_matrix)
perturb_names <- c("NonTarget", perturb_names[perturb_names!="NonTarget"])

de.genes.perturbs <- vector("list", length = length(perturb_names))
names(de.genes.perturbs) <- perturb_names

for(i in 1:length(de.genes.perturbs)){
  perturb_name <- names(de.genes.perturbs)[i]
  associated_factors <- colnames(bonferroni_mat)[which(bonferroni_mat[perturb_name, ] < 0.05)]
  if(length(associated_factors) > 0){
    de.genes.perturbs[[i]] <- unique(unlist(de.genes.factors[associated_factors]))
  }
}

num.de.genes.perturbs <- sapply(de.genes.perturbs, length)
unguided_GSFA_bonferroni0.05_genes <- de.genes.perturbs

dge_plot_df <- data.frame(Perturbation = names(num.de.genes.perturbs), Num_DEGs = num.de.genes.perturbs)
dge_plot_df$Perturbation <- factor(dge_plot_df$Perturbation, levels = names(num.de.genes.perturbs))

ggplot(data=dge_plot_df, aes(x = Perturbation, y = Num_DEGs+1)) +
  geom_bar(position = "dodge", stat = "identity") +
  geom_text(aes(label = Num_DEGs), position=position_dodge(width=0.9), vjust=-0.25) +
  scale_y_log10() +
  scale_fill_brewer(palette = "Set2") +
  labs(x = "Target gene",
       y = "Number of DEGs",
       title = "Number of DEGs detected by unguided GSFA") +
  theme(axis.text.x = element_text(angle = 45, hjust = 1, size = 12),
        legend.position = "bottom",
        legend.text = element_text(size = 13))

Version Author Date
43d8586 kevinlkx 2022-08-25

Compare single-gene DE p-value distributions between GSFA and unguided GSFA

fdr_cutoff <- 0.05
lfsr_cutoff <- 0.05

Load the output of GSFA fit_gsfa_multivar() run.

data_folder <- "/project2/xinhe/yifan/Factor_analysis/Stimulated_T_Cells/"
fit <- readRDS(paste0(data_folder,
                      "gsfa_output_detect_01/all_uncorrected_by_group.use_negctrl/All.gibbs_obj_k20.svd_negctrl.restart.light.rds"))
gibbs_PM <- fit$posterior_means
lfsr_mat1 <- fit$lfsr1[, -ncol(fit$lfsr1)]
lfsr_mat0 <- fit$lfsr0[, -ncol(fit$lfsr0)]
total_effect1 <- fit$total_effect1[, -ncol(fit$total_effect1)]
total_effect0 <- fit$total_effect0[, -ncol(fit$total_effect0)]
KO_names <- colnames(lfsr_mat1)

DEGs detected by GSFA

   ARID1A      BTLA  C10orf54      CBLB      CD3D       CD5    CDKN1B      DGKA 
      393       107        66       631         0       645       468        32 
     DGKZ    HAVCR2      LAG3      LCP2     MEF2D NonTarget     PDCD1     RASA2 
      113        35         1       589        15         0         0       277 
    SOCS1     STAT6     TCEB2   TMEM222   TNFRSF9 
      356         1       300         4        14

Load MAST single-gene DE result

guides <- KO_names[KO_names!="NonTarget"]

mast_list <- list()
for (m in guides){
  fname <- paste0(data_folder, "processed_data/MAST/all_by_stim_negctrl/gRNA_", 
                  m, ".dev_res_top6k.vs_negctrl.rds")
  tmp_df <- readRDS(fname)
  tmp_df$geneID <- rownames(tmp_df)
  tmp_df <- tmp_df %>% dplyr::rename(FDR = fdr, PValue = pval)
  mast_list[[m]] <- tmp_df
}
mast_signif_counts <- sapply(mast_list, function(x){filter(x, FDR < fdr_cutoff) %>% nrow()})

QQ-plots of MAST DE p-values for the GSFA genes vs unguided GSFA genes.

qqplots <- list()
for(i in 1:length(guides)){
  guide <- guides[i]
  mast_res <- mast_list[[guide]]
  unguided_gsfa_de_genes <- unguided_GSFA_fdr0.05_genes[[guide]]
  gsfa_de_genes <- gsfa_sig_genes[[guide]]
  unguided_gsfa_de_genes <- intersect(unguided_gsfa_de_genes, rownames(mast_res))
  gsfa_de_genes <- intersect(gsfa_de_genes, rownames(mast_res))
  
  if(length(unguided_gsfa_de_genes)>0 && length(gsfa_de_genes)>0){
    # cat("plot", guide, "\n")
    mast_res$unguided_gsfa_gene <- 0
    mast_res[unguided_gsfa_de_genes, ]$unguided_gsfa_gene <- 1
    mast_res$gsfa_gene <- 0
    mast_res[gsfa_de_genes, ]$gsfa_gene <- 1
    pvalue_list <- list('Unguided GSFA'=dplyr::filter(mast_res,unguided_gsfa_gene==1)$PValue,
                        'GSFA'=dplyr::filter(mast_res,gsfa_gene==1)$PValue,
                        'all genes'=mast_res$PValue)
    qqplots[[guide]] <- qqplot.pvalue(pvalue_list, pointSize = 1, legendSize = 4) + 
      ggtitle(guide) + theme(plot.title = element_text(hjust = 0.5)) +
      scale_colour_discrete(name="Method")
  }
}

grid.arrange(grobs = qqplots, nrow = 5, ncol = 3)

Version Author Date
d712d7e kevinlkx 2022-09-03
7337ae0 kevinlkx 2022-08-31
43d8586 kevinlkx 2022-08-25

Pooling p-values from all perturbations

combined_mast_res <- data.frame()
for(i in 1:length(guides)){
  guide <- guides[i]
  mast_res <- mast_list[[guide]]
  unguided_gsfa_de_genes <- unguided_GSFA_fdr0.05_genes[[guide]]
  gsfa_de_genes <- gsfa_sig_genes[[guide]]
  unguided_gsfa_de_genes <- intersect(unguided_gsfa_de_genes, rownames(mast_res))
  gsfa_de_genes <- intersect(gsfa_de_genes, rownames(mast_res))
  mast_res$unguided_gsfa_gene <- 0
  if(length(unguided_gsfa_de_genes) >0){
    mast_res[unguided_gsfa_de_genes, ]$unguided_gsfa_gene <- 1
  }
  mast_res$gsfa_gene <- 0
  if(length(gsfa_de_genes) >0){
    mast_res[gsfa_de_genes, ]$gsfa_gene <- 1
  }
  combined_mast_res <- rbind(combined_mast_res, mast_res)
}

pvalue_list <- list('Unguided GSFA'=dplyr::filter(combined_mast_res,unguided_gsfa_gene==1)$PValue,
                    'GSFA'=dplyr::filter(combined_mast_res,gsfa_gene==1)$PValue,
                    'all genes'=combined_mast_res$PValue)

# pdf(file.path(res_dir, "qqplot_all_combined.pdf"))
qqplot.pvalue(pvalue_list, pointSize = 1, legendSize = 4) + 
      ggtitle("") + theme(plot.title = element_text(hjust = 0.5)) +
      scale_colour_discrete(name="Method")

Version Author Date
d712d7e kevinlkx 2022-09-03
7337ae0 kevinlkx 2022-08-31 
# dev.off()

QQ-plots of MAST DE p-values for the GSFA only genes vs unguided GSFA only genes.

qqplots <- list()
for(i in 1:length(guides)){
  guide <- guides[i]
  mast_res <- mast_list[[guide]]
  unguided_gsfa_de_genes <- unguided_GSFA_fdr0.05_genes[[guide]]
  gsfa_de_genes <- gsfa_sig_genes[[guide]]
  unguided_gsfa_de_genes <- intersect(unguided_gsfa_de_genes, rownames(mast_res))
  gsfa_de_genes <- intersect(gsfa_de_genes, rownames(mast_res))
  
  unguided_gsfa_only_genes <- setdiff(unguided_gsfa_de_genes, gsfa_de_genes)
  gsfa_only_genes <- setdiff(gsfa_de_genes, unguided_gsfa_de_genes)

  if(length(unguided_gsfa_only_genes)>0 && length(gsfa_only_genes) >0){
    # cat("plot", guide, "\n")
    mast_res$unguided_gsfa_only_gene <- 0
    mast_res[unguided_gsfa_only_genes, ]$unguided_gsfa_only_gene <- 1
    mast_res$gsfa_only_gene <- 0
    mast_res[gsfa_only_genes, ]$gsfa_only_gene <- 1
    
    pvalue_list <- list('Unguided GSFA only'=dplyr::filter(mast_res,unguided_gsfa_only_gene==1)$PValue,
                        'GSFA only'=dplyr::filter(mast_res,gsfa_only_gene==1)$PValue,
                        'all genes'=mast_res$PValue)
    qqplots[[guide]] <- qqplot.pvalue(pvalue_list, pointSize = 1, legendSize = 4) + 
      ggtitle(guide) + theme(plot.title = element_text(hjust = 0.5)) +
      scale_colour_discrete(name="Method")
  }
}

grid.arrange(grobs = qqplots, nrow = 5, ncol = 3)

Version Author Date
d712d7e kevinlkx 2022-09-03
7337ae0 kevinlkx 2022-08-31
43d8586 kevinlkx 2022-08-25

Pooling p-values from all perturbations

combined_mast_res <- data.frame()
for(i in 1:length(guides)){
  guide <- guides[i]
  mast_res <- mast_list[[guide]]
  unguided_gsfa_de_genes <- unguided_GSFA_fdr0.05_genes[[guide]]
  gsfa_de_genes <- gsfa_sig_genes[[guide]]
  unguided_gsfa_de_genes <- intersect(unguided_gsfa_de_genes, rownames(mast_res))
  gsfa_de_genes <- intersect(gsfa_de_genes, rownames(mast_res))
  mast_res$unguided_gsfa_only_gene <- 0
  if(length(setdiff(unguided_gsfa_de_genes, gsfa_de_genes)) >0){
    mast_res[setdiff(unguided_gsfa_de_genes, gsfa_de_genes), ]$unguided_gsfa_only_gene <- 1
  }
  mast_res$gsfa_only_gene <- 0
  if(length(setdiff(gsfa_de_genes, unguided_gsfa_de_genes)) >0){
    mast_res[setdiff(gsfa_de_genes, unguided_gsfa_de_genes), ]$gsfa_only_gene <- 1
  }
  combined_mast_res <- rbind(combined_mast_res, mast_res)
}

pvalue_list <- list('Unguided GSFA only'=dplyr::filter(combined_mast_res,unguided_gsfa_only_gene==1)$PValue,
                    'GSFA only'=dplyr::filter(combined_mast_res,gsfa_only_gene==1)$PValue,
                    'all genes'=combined_mast_res$PValue)

# pdf(file.path(res_dir, "qqplot_only_combined.pdf"))
qqplot.pvalue(pvalue_list, pointSize = 1, legendSize = 4) + 
      ggtitle("") + theme(plot.title = element_text(hjust = 0.5)) +
      scale_colour_discrete(name="Method") + 
      coord_cartesian(ylim = c(0, 30))

Version Author Date
d712d7e kevinlkx 2022-09-03
7337ae0 kevinlkx 2022-08-31 
# dev.off()

sessionInfo()
R version 4.2.0 (2022-04-22)
Platform: x86_64-pc-linux-gnu (64-bit)
Running under: CentOS Linux 7 (Core)

Matrix products: default
BLAS/LAPACK: /software/openblas-0.3.13-el7-x86_64/lib/libopenblas_haswellp-r0.3.13.so

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

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

other attached packages:
[1] lattice_0.20-45       gridExtra_2.3         dplyr_1.0.9          
[4] reshape2_1.4.4        ggplot2_3.3.6         ComplexHeatmap_2.12.0
[7] data.table_1.14.2     workflowr_1.7.0      

loaded via a namespace (and not attached):
 [1] Rcpp_1.0.8.3        circlize_0.4.15     getPass_0.2-2      
 [4] png_0.1-7           ps_1.7.0            assertthat_0.2.1   
 [7] rprojroot_2.0.3     digest_0.6.29       foreach_1.5.2      
[10] utf8_1.2.2          plyr_1.8.7          R6_2.5.1           
[13] stats4_4.2.0        evaluate_0.15       highr_0.9          
[16] httr_1.4.3          pillar_1.7.0        GlobalOptions_0.1.2
[19] rlang_1.0.2         rstudioapi_0.13     whisker_0.4        
[22] callr_3.7.0         jquerylib_0.1.4     S4Vectors_0.34.0   
[25] GetoptLong_1.0.5    rmarkdown_2.14      labeling_0.4.2     
[28] stringr_1.4.0       munsell_0.5.0       compiler_4.2.0     
[31] httpuv_1.6.5        xfun_0.30           pkgconfig_2.0.3    
[34] BiocGenerics_0.42.0 shape_1.4.6         htmltools_0.5.2    
[37] tidyselect_1.1.2    tibble_3.1.7        IRanges_2.30.0     
[40] codetools_0.2-18    matrixStats_0.62.0  fansi_1.0.3        
[43] withr_2.5.0         crayon_1.5.1        later_1.3.0        
[46] DBI_1.1.3           jsonlite_1.8.0      gtable_0.3.0       
[49] lifecycle_1.0.1     git2r_0.30.1        magrittr_2.0.3     
[52] scales_1.2.0        cli_3.3.0           stringi_1.7.6      
[55] farver_2.1.0        fs_1.5.2            promises_1.2.0.1   
[58] doParallel_1.0.17   bslib_0.3.1         ellipsis_0.3.2     
[61] vctrs_0.4.1         generics_0.1.2      rjson_0.2.21       
[64] RColorBrewer_1.1-3  iterators_1.0.14    tools_4.2.0        
[67] glue_1.6.2          purrr_0.3.4         processx_3.5.3     
[70] parallel_4.2.0      fastmap_1.1.0       yaml_2.3.5         
[73] clue_0.3-61         colorspace_2.0-3    cluster_2.1.3      
[76] knitr_1.39          sass_0.4.1