Contents

library(ggbio)
library(dplyr)
library(IHW)
library(fdrtool)
library(cowplot)
theme_set(theme_cowplot())
library(tidyr)
library(scales)
library(latex2exp)

Let us start by loading in the data:

file_loc <- system.file("extdata","real_data", "hqtl_chrom1_chrom2", package = "IHWpaper")

First the two tables with the p-values corresponding to the two chromosomes. Note that only p-values <= 1e-4 are stored in these.

chr1_df <- readRDS(file.path(file_loc, "chr1_subset.Rds"))
chr2_df <- readRDS(file.path(file_loc, "chr2_subset.Rds"))
pval_threshold <- 10^(-4)

Also recall each hypothesis corresponds to a peak (which we call gene below) and a SNP. Hence let us load files about each of the SNPs and peaks:

snp_chr1 <- readRDS(file.path(file_loc, "snppos_chr1.Rds"))
snp_chr2 <-  readRDS(file.path(file_loc, "snppos_chr2.Rds"))

all_peaks <- readRDS(file.path(file_loc, "peak_locations.Rds"))
peaks_chr1 <- dplyr::filter(all_peaks, chr=="chr1")
peaks_chr2 <- dplyr::filter(all_peaks, chr=="chr2")

We can use these both to infer how many hypotheses were conducted in total (or at a given distance), but also to calculate our covariates which are a function of SNP and peak (their distance).

Now let us attach the new column with the covariate (distance) to the data frames.

chr1_df <- left_join(chr1_df, select(snp_chr1, snp, pos), by=(c("SNP"="snp"))) %>%
       left_join(peaks_chr1, by=(c("gene"="id"))) %>%
       mutate( dist = pmin( abs(pos-start), abs(pos-end)))

chr2_df <- left_join(chr2_df, select(snp_chr2, snp, pos), by=(c("SNP"="snp"))) %>%
       left_join(peaks_chr2, by=(c("gene"="id"))) %>%
       mutate( dist = pmin( abs(pos-start), abs(pos-end)))

Now let us convert the distance to a categorical covariate by binning:

my_breaks <- c(-1, 
                 seq(from=10000,to=290000, by=10000) , 
                 seq(from=300000, to=0.9*10^6, by=100000),
                 seq(from=10^6, to=25.1*10^7, by=10^7))
myf1 <- cut(chr1_df$dist, my_breaks)
myf2 <- cut(chr2_df$dist, my_breaks)

To apply our method despite the fact that only small p-values are available, we will count how many hypotheses there are in each of the bins. The above code is not very efficient, so we have precomputed these and do not run the below chunk.

cnt = 0
ms <- rep(0, length(levels(myf1)))
pb = txtProgressBar(min = 0, max = nrow(peaks_chr1), initial = 0) 

for (i in 1:nrow(peaks_chr1)){
  setTxtProgressBar(pb,i)
  start_pos <- peaks_chr1$start[i]
  end_pos <- peaks_chr1$end[i]
  dist_vec <- pmin( abs(snp_chr1$pos - start_pos), abs(snp_chr1$pos - end_pos) )
  ms <- ms + table(cut(dist_vec, my_breaks))
}

saveRDS( ms, file = "m_groups_chr1.Rds" )


cnt = 0
ms_chr2 <- table(myf2)*0
pb = txtProgressBar(min = 0, max = nrow(peaks_chr2), initial = 0) 

for (i in 1:nrow(peaks_chr2)){
  setTxtProgressBar(pb,i)
  start_pos <- peaks_chr1$start[i]
  end_pos <- peaks_chr1$end[i]
  dist_vec <- pmin( abs(snp_chr2$pos - start_pos), abs(snp_chr2$pos - end_pos) )
  ms_chr2 <- ms_chr2 + table(cut(dist_vec, my_breaks))
}

saveRDS( ms_chr2, file = "m_groups_chr2.Rds" )

Let us load the result from the above execution:

ms_chr1 <- readRDS(file.path(file_loc, "m_groups_chr1.Rds"))
ms_chr2 <- readRDS(file.path(file_loc, "m_groups_chr2.Rds"))

Let us put the data for the two chromosomes together:

chr1_chr2_df <- rbind(chr1_df, chr2_df)
chr1_chr2_groups <- as.factor(c(myf1,myf2))
folds_vec <- as.factor(c(rep(1, nrow(chr1_df)), rep(2, nrow(chr2_df))))
m_groups <- cbind(ms_chr1, ms_chr2)
m <- sum(m_groups) #total number of hypotheses
m
## [1] 15725016812

1 Histogram plots

Get our colors:

beyonce_colors <- c("#b72da0", "#7c5bd2", "#0097ed","#00c6c3",
                   "#9cd78a", "#f7f7a7", "#ebab5f", "#e24344",
                   "#04738d")#,"#d8cdc9")
beyonce_colors[6] <- c("#dbcb09") # thicker yellow
pretty_colors <- beyonce_colors[c(2,1,3:5)]
qs <- c(0.025, 0.05)
cutoffs <- c(0, quantile(chr1_chr2_df$dist,qs), Inf)
cov_scatter_gg <- ggplot(chr1_chr2_df, aes(x=rank(dist)/nrow(chr1_chr2_df),
                                           y=-log10(pvalue))) +
     geom_bin2d(bins=150, drop=TRUE) + #  geom_point(alpha=0.2, col=pretty_colors[1]) + 
     geom_vline(xintercept=qs, linetype="dashed") + 
     ylab(expression(paste(-log[10],"(p-value)"))) +
     xlab(expression(paste("Quantile of distance"))) + 
     scale_fill_gradientn(trans="log10", colors=alpha(pretty_colors[1], c(0.2,1)))
cov_scatter_gg

ggsave(cov_scatter_gg, filename="cov_scatter_gg.pdf", width=4,height=3)
chr1_chr2_df$cutoff_groups <- cut(chr1_chr2_df$dist, cutoffs)
table(chr1_chr2_df$cutoff_groups)
## 
##        (0,1.13e+05] (1.13e+05,2.34e+06]      (2.34e+06,Inf] 
##               60404               60404             2295337

First let us plot the marginal histogram:

gg_marginal_hist <- ggplot(chr1_chr2_df, aes(x=pvalue*10^4)) + 
         geom_histogram(aes(y=..density..), alpha=0.5, binwidth=0.05, boundary = 0, colour="black",fill=pretty_colors[1]) +
          scale_x_continuous(expand = c(0.02, 0), breaks=c(0,0.5,1)) + 
          scale_y_continuous(expand = c(0.02, 0), limits=c(0,2.5)) + 
          ylab(expression(paste("Density")))+
          xlab(TeX("p-value ($\\times 10^{-4}$)")) 
gg_marginal_hist

ggsave(gg_marginal_hist, filename="gg_marginal_hist.pdf", width=4,height=3)
gg_stratified_hist <- ggplot(chr1_chr2_df, aes(x=pvalue*10^4)) + 
         geom_histogram(aes(y=..density..), alpha=0.5, binwidth=0.05, boundary = 0, colour="black",fill=pretty_colors[1]) +
          scale_x_continuous(expand = c(0.02, 0), breaks=c(0,0.5,1)) + 
          scale_y_continuous(expand = c(0.02, 0), limits=c(0,11)) + 
          ylab("Density")+
          xlab(TeX("p-value ($\\times 10^{-4}$)")) +
          facet_grid(~cutoff_groups) + 
          theme(strip.background = element_blank(), strip.text.y = element_blank()) + 
          theme(panel.spacing = unit(2, "lines"))
gg_stratified_hist      

ggsave(gg_stratified_hist, filename="gg_stratified_hist.pdf", width=7,height=3)

2 Apply IHW-BY and BY

We want to apply the Benjamini-Yekutieli at alpha=0.1, thus we will apply Benjamini-Hochberg at the corrected level:

alpha <- .01/(log(m)+1)

Now let us run the IHW procedure:

ihw_chr1_chr2 <- ihw(chr1_chr2_df$pvalue, chr1_chr2_groups, alpha, folds=folds_vec,
                     m_groups=m_groups, lambdas=2000)

Rejections of BH:

sum(p.adjust(chr1_chr2_df$pvalue, n = m, method="BH") <= alpha)
## [1] 9110

Rejections of IHW-BY:

rejections(ihw_chr1_chr2)
## [1] 21903

So we see that we more than doubled discoveries!

3 Hypotheses shown in table

For our table we need one hypothesis in Chr1 that gets rejected both times (by BH and IHW):

 idx <- which(rejected_hypotheses(ihw_chr1_chr2) & (p.adjust(chr1_chr2_df$pvalue, n = m, method="BH") > alpha) &
                (covariates(ihw_chr1_chr2)==3) & (ihw_chr1_chr2@df$fold == 1))
idx_max <- which.max(pvalues(ihw_chr1_chr2)[idx])

ihw_chr1_chr2@df[idx[idx_max],]
##             pvalue   adj_pvalue weight weighted_pvalue group covariate fold
## 45723 9.738823e-07 0.0004046323 1731.2    5.625477e-10     3         3    1
chr1_df[idx[idx_max],]
## # A tibble: 1 x 11
##   SNP       gene   beta tstat     pvalue   FDR     pos chr    start    end  dist
##   <chr>    <int>  <dbl> <dbl>      <dbl> <dbl>   <int> <chr>  <int>  <int> <int>
## 1 rs61776… 13328 -0.907 -5.34    9.74e-7 0.168 1648027 chr1  1.62e6 1.62e6 23098

We need one in Chr1 that gets weight 0:

 idx <- which( !rejected_hypotheses(ihw_chr1_chr2) & (p.adjust(chr1_chr2_df$pvalue, n = m, method="BH") > alpha) &
                (covariates(ihw_chr1_chr2)==15) & (ihw_chr1_chr2@df$fold == 1))
idx_max <- which.max(pvalues(ihw_chr1_chr2)[idx])

ihw_chr1_chr2@df[idx[idx_max],]
##               pvalue adj_pvalue weight weighted_pvalue group covariate fold
## 1182156 9.999058e-05          1      0               1    15        15    1
ihw_chr1_chr2@df[idx[idx_max],]
##               pvalue adj_pvalue weight weighted_pvalue group covariate fold
## 1182156 9.999058e-05          1      0               1    15        15    1
chr1_df[idx[idx_max],]
## # A tibble: 1 x 11
##   SNP       gene  beta tstat   pvalue   FDR     pos chr     start     end   dist
##   <chr>    <int> <dbl> <dbl>    <dbl> <dbl>   <int> <chr>   <int>   <int>  <int>
## 1 rs12239… 16094 0.585  4.11 0.000100 0.666  1.83e8 chr1   1.83e8  1.83e8 141957

One which get rejected in both cases from Chr2 :

idx <- which( rejected_hypotheses(ihw_chr1_chr2) & (p.adjust(chr1_chr2_df$pvalue, n = m, method="BH") <= alpha) &
                (covariates(ihw_chr1_chr2)==3) & (ihw_chr1_chr2@df$fold == 2))
ihw_chr1_chr2@df[idx[9],]
##               pvalue   adj_pvalue   weight weighted_pvalue group covariate fold
## 1182483 1.237412e-19 2.792147e-15 1421.894     8.70256e-23     3         3    2
chr2_df[idx[9]-nrow(chr1_df),]
## # A tibble: 1 x 11
##   SNP      gene  beta tstat   pvalue      FDR     pos chr     start    end  dist
##   <chr>   <int> <dbl> <dbl>    <dbl>    <dbl>   <int> <chr>   <int>  <int> <int>
## 1 rs8014… 72797  2.03  12.3 1.24e-19 4.17e-12  2.29e8 chr2   2.29e8 2.29e8 29469

And another one that only gets rejected in one case

idx <- which( rejected_hypotheses(ihw_chr1_chr2) & (p.adjust(chr1_chr2_df$pvalue, n = m, method="BH") > alpha) &
                (covariates(ihw_chr1_chr2)==1) & (ihw_chr1_chr2@df$fold == 2))
idx_max <- which.max(pvalues(ihw_chr1_chr2)[idx])

ihw_chr1_chr2@df[idx[idx_max],]
##               pvalue   adj_pvalue   weight weighted_pvalue group covariate fold
## 1247287 1.710631e-06 0.0004075483 3015.383    5.673015e-10     1         1    2
chr2_df[idx[idx_max]-nrow(chr1_df),]
## # A tibble: 1 x 11
##   SNP        gene  beta tstat     pvalue   FDR     pos chr    start    end  dist
##   <chr>     <int> <dbl> <dbl>      <dbl> <dbl>   <int> <chr>  <int>  <int> <int>
## 1 rs2715879 78787 0.844  5.20 0.00000171 0.207 9515551 chr2  9.51e6 9.51e6  1542

4 Set out to do the weight/threshold/lfdr plots

First get the threshold below which BY rejects:

t_bh <- get_bh_threshold(chr1_chr2_df$pvalue, alpha, mtests = m)
t_bh
## [1] 2.363825e-10

Next write a function to estimate the local fdr at a given threshold:

get_local_fdr <- function(fold, group){
  idx <- (chr1_chr2_groups == group) & (folds_vec == fold)
  pvals <- sort(chr1_chr2_df$pvalue[idx])
  m_true <-  m_groups[group,fold]
  gren <- IHW:::presorted_grenander(pvals, m_true)
  myt <- thresholds(ihw_chr1_chr2, levels_only=TRUE)[group,fold]
  id_ihw_myt <- which(myt < gren$x.knots)[1]
  local_fdr_ihw <- ifelse(myt == 0, 0, 1/gren$slope.knots[id_ihw_myt-1])
  
  
  id_bh_thresh <- which(t_bh < gren$x.knots)[1]
  local_fdr_bh <- 1/gren$slope.knots[id_bh_thresh-1]
  
  pi0 <- (m_true - length(pvals))/(1-10^(-4))/m_true
  data.frame(fold=fold, group=group, pi0=pi0, t_ihw=myt, local_fdr_ihw =  local_fdr_ihw, 
             local_fdr_bh = local_fdr_bh)
}
fold_groups <- expand.grid(1:62, 1:2)

Precompute the below too because it takes a while:

lfdrs <- bind_rows(mapply(get_local_fdr, fold_groups[[2]], fold_groups[[1]], SIMPLIFY = FALSE))
saveRDS(lfdrs,file="hqtl_estimated_lfdrs.Rds")
lfdrs <-  readRDS(file.path(file_loc, "hqtl_estimated_lfdrs.Rds"))
lfdrs <- mutate(lfdrs, Chromosome=paste0("chr", fold), stratum=group, t_bh=t_bh)

5 Start the plotting

breaks <-   my_breaks/10^3
breaks <- breaks[-1]
break_min <- 3000/10^3
breaks_left <- c(break_min,breaks[-length(breaks)])
stratum <- 1:62
step_df_weight <- data.frame(stratum=stratum, chr2=weights(ihw_chr1_chr2,levels_only=TRUE)[,1], 
                                       chr1=weights(ihw_chr1_chr2, levels_only=TRUE)[,2] ) %>%
           gather(Chromosome, weight , -stratum)

step_df_threshold <- data.frame(stratum=stratum,
                                chr2=thresholds(ihw_chr1_chr2,levels_only=TRUE)[,2], 
                                chr1=thresholds(ihw_chr1_chr2, levels_only=TRUE)[,1] ) %>%
           gather(Chromosome, threshold , -stratum)

step_df <- left_join(step_df_weight, step_df_threshold) %>% left_join(lfdrs)
## Joining, by = c("stratum", "Chromosome")
## Joining, by = c("stratum", "Chromosome")
step_df <- step_df %>% mutate(break_left = breaks_left[stratum],
                 break_right = breaks[stratum],
                 break_ratio = break_right/break_left , 
                 break_left =break_left * break_ratio^.2,
                 break_right = break_right *break_ratio^(-.2))

stratum_fun <- function(df, colname="weight"){
  stratum <- df$stratum
  weight <- df[[colname]]
  stratum_left <- stratum[stratum != length(stratum)]
  weight_left  <- weight[stratum_left]
  break_left <- df$break_right[stratum_left]
  stratum_right <- stratum[stratum != 1]
  weight_right <- weight[stratum_right]
  break_right <- df$break_left[stratum_right]
  data.frame(stratum_left= stratum_left, weight_left= weight_left, 
             stratum_right = stratum_right, weight_right = weight_right,
             break_left = break_left, break_right = break_right)
}




connecting_df_weights <- step_df %>% group_by(Chromosome) %>% 
                  do(stratum_fun(.)) %>%
                  mutate(dashed = factor(ifelse(abs(weight_left - weight_right) > 0.5 , TRUE, FALSE),
                         levels=c(FALSE,TRUE)))

weights_panel <- ggplot(step_df, aes(x=break_left, xend=break_right,y=weight, yend=weight, col=Chromosome)) +
                geom_segment(size=0.8)+                
                geom_segment(data= connecting_df_weights, aes(x=break_left, xend=break_right, 
                                                y=weight_left, yend=weight_right, 
                                                linetype=dashed),
                             size=0.8)+
                scale_x_log10(breaks=c(10^4, 10^5,10^6,10^7,10^8), 
                              labels = trans_format("log10", math_format(10^.x))) +
                xlab("Genomic distance (bp)")+
                ylab("Weight")+
                theme(legend.position=c(0.8,0.6)) +
                theme(plot.margin = unit(c(2, 1.5, 1, 2.5), "lines"))+
                theme(axis.title = element_text(face="bold" ))+
                scale_color_manual(values=pretty_colors)+
                guides(linetype=FALSE)

weights_panel

# Weights chromosome 1

weights_panel_1 <- ggplot(filter(step_df, Chromosome == "chr1"), aes(x=break_left, xend=break_right,y=weight, yend=weight, col=Chromosome)) +
                geom_segment(size=0.8,lineend="round")+                
                geom_segment(data= filter(connecting_df_weights, Chromosome=="chr1"), aes(x=break_left, xend=break_right, 
                                                y=weight_left, yend=weight_right, 
                                                linetype=dashed),
                             size=0.8,lineend="round")+
                scale_x_log10(breaks=c(10, 10^2,10^3,10^4), 
                              labels = trans_format("log10", math_format(10^.x))) +
                scale_y_continuous(breaks=c(0,1000,2000))+
                xlab(expression(paste("Distance (kbp)")))+
                ylab(expression(paste("Weight")))+
                theme(legend.position="none") +
                theme(plot.margin = unit(c(2, 1.5, 1, 2.5), "lines"))+
                theme(axis.title = element_text(face="bold" ))+
                scale_color_manual(values=pretty_colors)+
                guides(linetype=FALSE)

weights_panel_1

ggsave(weights_panel_1, filename="chr1_weights.pdf", width=3.5,height=2.5)
weights_panel_2 <- ggplot(filter(step_df, Chromosome == "chr2"), aes(x=break_left, xend=break_right,y=weight, yend=weight, col=Chromosome)) +
                geom_segment(size=0.8, lineend="round")+                
                geom_segment(data= filter(connecting_df_weights, Chromosome=="chr2"), aes(x=break_left, xend=break_right, 
                                                y=weight_left, yend=weight_right, 
                                                linetype=dashed),
                             size=0.8, lineend="round")+
                scale_x_log10(breaks=c(10, 10^2,10^3,10^4), 
                              labels = trans_format("log10", math_format(10^.x))) +
                scale_y_continuous(breaks=c(0,1000,2000))+
                xlab(expression(paste("Distance (kbp)")))+
                ylab(expression(paste("Weight")))+
                theme(legend.position="none") +
                theme(plot.margin = unit(c(2, 1.5, 1, 2.5), "lines"))+
                theme(axis.title = element_text(face="bold" ))+
                scale_color_manual(values=pretty_colors)+
                guides(linetype=FALSE)

weights_panel_2

ggsave(weights_panel_2, filename="chr2_weights.pdf", width=3.5,height=2.5)
connecting_df_thresholds_ihw <- step_df %>% group_by(Chromosome) %>% 
                  do(stratum_fun(., colname="t_ihw")) %>%
                  mutate(dashed = FALSE)#factor(ifelse(abs(weight_left - weight_right) > 10^{-7} , TRUE, FALSE),
                        # levels=c(FALSE,TRUE)))

thresholds_ihw_panel <- ggplot(step_df, aes(x=break_left, xend=break_right,y=t_ihw*10^6, yend=t_ihw*10^6, col=Chromosome)) +
                geom_segment(size=0.8, lineend="round")+                
                geom_segment(data= connecting_df_thresholds_ihw, aes(x=break_left, xend=break_right, 
                                                y=weight_left*10^6, yend=weight_right*10^6, 
                                                linetype=dashed),
                             size=0.8, lineend="round")+
                scale_x_log10(breaks=c(10, 10^2,10^3,10^4), 
                              labels = trans_format("log10", math_format(10^.x))) +
                scale_y_continuous(limits=c(0,1.8), breaks=c(0,1))+
                xlab(expression(paste("Distance (kbp)")))+
                ylab(expression(paste("IHW t(x) (",10^-6,")")))+
                theme(legend.position=c(0.6,0.7), legend.title = element_blank()) +
                theme(plot.margin = unit(c(2, 1.5, 1, 2.5), "lines"))+
                theme(axis.title = element_text(face="bold" ))+
                scale_color_manual(values=pretty_colors)+
                guides(linetype=FALSE)

thresholds_ihw_panel

ggsave(thresholds_ihw_panel, filename="ihw_by_threshold.pdf", width=3.5,height=2.5)
connecting_df_thresholds_bh <- step_df %>% group_by(Chromosome) %>% 
                  do(stratum_fun(., colname="t_bh")) %>%
                  mutate(dashed = FALSE)#factor(ifelse(abs(weight_left - weight_right) > 10^{-11} , TRUE, TRUE),
                         #levels=c(FALSE,TRUE)))

scientific_10 = function(x) {ifelse(x==0, "0", parse(text=gsub("[+]", "", gsub("e", " %*% 10^", scientific_format()(x)))))} 
thresholds_bh_panel <- ggplot(step_df, aes(x=break_left, xend=break_right,y=10^10*t_bh, yend=10^10*t_bh, col=Chromosome)) +
                geom_segment(size=0.8)+                
                geom_segment(data= connecting_df_thresholds_bh, aes(x=break_left, xend=break_right, 
                                                y=weight_left*10^10, yend=weight_right*10^10, 
                                                linetype=dashed),
                             size=0.8)+
                scale_x_log10(breaks=c(10, 10^2,10^3,10^4), 
                              labels = trans_format("log10", math_format(10^.x))) +
                scale_y_continuous(limits=c(0,5), breaks=c(0,2,4))+
                xlab(expression(paste("Distance (kbp)")))+
                ylab(expression(paste("BY t(x) (",10^-10,")")))+
                theme(legend.position=c(0.6,0.7), legend.title = element_blank()) +
                theme(plot.margin = unit(c(2, 1.5, 1, 2.5), "lines"))+
                theme(axis.title = element_text(face="bold" ))+
                scale_color_manual(values=pretty_colors)+
                guides(linetype=FALSE)

thresholds_bh_panel

ggsave(thresholds_bh_panel, filename="by_threshold.pdf", width=3.5,height=2.5)
connecting_df_lfdr_ihw <- step_df %>% group_by(Chromosome) %>% 
                  do(stratum_fun(., colname="local_fdr_ihw")) %>%
                  mutate(dashed = FALSE)

lfdr_ihw_panel <- ggplot(step_df, aes(x=break_left, xend=break_right,y=10^1*local_fdr_ihw, yend=10^1*local_fdr_ihw, col=Chromosome)) +
                geom_segment(size=0.8, lineend="round")+                
                geom_segment(data= connecting_df_lfdr_ihw, aes(x=break_left, xend=break_right, 
                                                y=10^1*weight_left, yend=10^1*weight_right, 
                                                linetype=dashed),
                             size=0.8,lineend="round")+
                scale_x_log10(breaks=c(10, 10^2,10^3,10^4), 
                              labels = trans_format("log10", math_format(10^.x))) +
                xlab(expression(paste("Distance (kbp)")))+
                ylab(expression(paste("IHW fdr(t(x) | x)")))+
                theme(legend.position=c(0.6,0.7), legend.title = element_blank()) +
                theme(plot.margin = unit(c(2, 1.5, 1, 2.5), "lines"))+
                theme(axis.title = element_text(face="bold" ))+
                scale_color_manual(values=pretty_colors)+
                guides(linetype=FALSE)

lfdr_ihw_panel