Mapping Many Gene Expression Traits

Last updated on 2024-11-12 | Edit this page

Estimated time: 60 minutes

Overview

Questions

  • How do I map many genes?

Objectives

  • To map several genes at the same time

Load Libraries

For this lesson, we need to install two more libraries and load them. You can do this by typing the following code:

R 

BiocManager::install(c("AnnotationHub","rtracklayer"))

Let’s install our libraries, and source two other R scripts.

R 

library(tidyverse)
library(knitr)
library(broom)
library(qtl2)
library(qtl2ggplot)
library(RColorBrewer)

#source("../code/gg_transcriptome_map.R")
#source("../code/qtl_heatmap.R")

Before we begin this lesson, we need to create another directory called results in our main directory. You can do this by clicking on the “Files” tab and navigate into the main directory. Then select “New Folder” and name it “results”.

Load Data

R 

# expression data
load("../data/attie_DO500_expr.datasets.RData")

# data from paper
load("../data/dataset.islet.rnaseq.RData")

# phenotypes
load("../data/attie_DO500_clinical.phenotypes.RData")

# mapping data
load("../data/attie_DO500_mapping.data.RData")

# genotype probabilities
probs = readRDS("../data/attie_DO500_genoprobs_v5.rds")

Data Selection

For this lesson, lets choose a random set of 50 gene expression phenotypes.

R 

genes = colnames(norm)

sams <- sample(length(genes), 50, replace = FALSE, prob = NULL)

ERROR 

Error in sample.int(length(x), size, replace, prob): cannot take a sample larger than the population when 'replace = FALSE'

R 

genes <- genes[sams]

ERROR 

Error: object 'sams' not found

R 

gene.info <- dataset.islet.rnaseq$annots[genes,]

ERROR 

Error: object 'dataset.islet.rnaseq' not found

R 

rownames(gene.info) = NULL

ERROR 

Error: object 'gene.info' not found

R 

kable(gene.info[1:10,])

ERROR 

Error: object 'gene.info' not found

Expression Data

Lets check the distribution for the first 20 gene expression phenotypes. If you would like to check the distribution of all 50 genes, change for(gene in genes[1:20]) in the code below to for(gene in genes).

R 

par(mfrow=c(3,4))
for(gene in genes[1:20]){
  hist(norm[,gene], main = gene)
  }

Check the distributions. Do they all have a normal distribution?

You will notice that the distribtion of some genes are skewed to the left. This means that that only a small amount of samples have data and therefore, will need to be removed. A suitable qc would be keeping expression data that have at least 5% of the samples with more than 10 reads.

R 

genes_qc <- which(as.numeric(colSums(counts[ , genes] > 10)) >= 0.05 * nrow(counts[,genes]))

ERROR 

Error: object 'counts' not found

R 

genes <- genes[genes_qc]

ERROR 

Error: object 'genes_qc' not found

The Marker Map

We are using the same marker map as in the previous lesson

Genotype probabilities

We have explored this earlier in th previous lesson. But, as a reminder, we have already calculated genotype probabilities which we loaded above called probs. This contains the 8 state g enotype probabilities using the 69k grid map of the same 500 DO mice that also have clinical phenotypes.

Kinship Matrix

We have explored the kinship matrix in the previous lesson. It has already been calculated and loaded in above.

Covariates

Now let’s add the necessary covariates. For these 50 gene expression data, we will correct for DOwave,sex and diet_days.

R 

# convert sex and DO wave (batch) to factors
pheno_clin$sex = factor(pheno_clin$sex)

ERROR 

Error: object 'pheno_clin' not found

R 

pheno_clin$DOwave = factor(pheno_clin$DOwave)

ERROR 

Error: object 'pheno_clin' not found

R 

pheno_clin$diet_days = factor(pheno_clin$DOwave)

ERROR 

Error: object 'pheno_clin' not found

R 

covar = model.matrix(~sex + DOwave + diet_days, data = pheno_clin)[,-1]

ERROR 

Error: object 'pheno_clin' not found

Performing a genome scan

Now lets perform the genome scan! We are also going to save our qtl results in an Rdata file to be used in further lessons. We will not perform permutations in this lesson as it will take too long. Instead we will use 6, which is the LOD score used in the paper to determine significance.

QTL Scans

R 

qtl.file = "../results/gene.norm_qtl_cis.trans.Rdata"

if(file.exists(qtl.file)) {
  load(qtl.file)
  } else {
    qtl = scan1(genoprobs = probs, 
                pheno = norm[,genes, drop = FALSE],
                kinship = K, 
                addcovar = covar, 
                cores = 2)
    save(qtl, file = qtl.file)
    }

QTL plots

Let’s plot the first 20 gene expression phenotypes. If you would like to plot all 50, change for(i in 1:20) in the code below to for(i in 1:ncol(qtl)).

R 

par(mfrow=c(3,4))
for(i in 1:20) {
  plot_scan1(x = qtl, 
             map = map, 
             lodcolumn = i, 
             main = colnames(qtl)[i])
  abline(h = 6, col = 2, lwd = 2)
  }

ERROR 

Error: object 'qtl' not found

QTL Peaks

We are also going to save our peak results so we can use these again else where.
First, lets get out peaks with a LOD score greater than 6.

R 

lod_threshold = 6
peaks = find_peaks(scan1_output = qtl, 
                   map = map, 
                   threshold = lod_threshold, 
                   peakdrop = 4, 
                   prob = 0.95)

ERROR 

Error: object 'qtl' not found

We will save these peaks into a csv file.

R 

kable(peaks[1:10,] %>% 
        dplyr::select(-lodindex) %>% 
        arrange(chr, pos), caption = "Expression QTL (eQTL) Peaks with LOD >= 6")

# write_csv(peaks, "../results/gene.norm_qtl_peaks_cis.trans.csv")

ERROR 

Error: object 'peaks' not found

QTL Peaks Figure

R 

qtl_heatmap(qtl = qtl, map = map, low.thr = 3.5)

ERROR 

Error in qtl_heatmap(qtl = qtl, map = map, low.thr = 3.5): could not find function "qtl_heatmap"

Challenge 1:

What do the qtl scans for all gene exression traits look like? Note: Don’t worry, we’ve done the qtl scans for you!!! You can read in this file, ../data/gene.norm_qtl_all.genes.Rdata, which are the scan1 results for all gene expression traits.

R 

load("../data/gene.norm_qtl_all.genes.Rdata")

lod_threshold = 6
peaks = find_peaks(scan1_output = qtl.all, 
                map = map, 
                threshold = lod_threshold, 
                peakdrop = 4, 
                prob = 0.95)
write_csv(peaks, "../results/gene.norm_qtl_all.genes_peaks.csv")

## Heat Map
qtl_heatmap(qtl = qtl, map = map, low.thr = 3.5)

Key Points

  • Use .md files for episodes when you want static content
  • Use .Rmd files for episodes when you need to generate output
  • Run sandpaper::check_lesson() to identify any issues with your lesson
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