2.1.1 From TXT files

The function readExposome will create an ExposomeSet from the three txt files. The following lines are used to locate these three files, that were included in the package for demonstration purposes.

path <- file.path(path.package("rexposome"), "extdata")
description <- file.path(path, "description.csv")
phenotype <- file.path(path, "phenotypes.csv")
exposures <- file.path(path, "exposures.csv")

These files follows the rules described in Data Format section. They are csv files, meaning each values is split from the others by a comma (,). Function readExposome allows to load most any type of files containing exposome data:

args(readExposome)

## function (exposures, description, phenotype, sep = ",", na.strings = c("NA", 
##     "-", "?", " ", ""), exposures.samCol = "sample", description.expCol = "exposure", 
##     description.famCol = "family", phenotype.samCol = "sample", 
##     exposures.asFactor = 5, warnings = TRUE) 
## NULL

readExposome expects, by default, csv files. Changing the content of the argument sep will allow to load other files types. The missing values are set using the argument na.strings. This means that the character assigned to this argument will be interpreted as a missing value. By default, those characters are "NA", "-", "?", " " and "". Then, the columns with the exposures’ names and the individual’s names need to be indicated. Arguments exposures.samCol and phenotype.samCol indicates the column with the individuals’ names at exposures file and phenotypes file. The arguments description.expCol and description.famCol indicates the column containing the exposures’ names and the exposures’ family in the description file.

exp <- readExposome(exposures = exposures, description = description, phenotype = phenotype, 
    exposures.samCol = 1, description.expCol = 2, description.famCol = 1, phenotype.samCol = 1)

The result is an object of class ExposomeSet, that can show all the information of the loaded exposome:

exp

## Object of class 'ExposomeSet' (storageMode: environment)
##  . exposures description:
##     . categorical:  4 
##     . continuous:  84 
##  . exposures transformation:
##     . categorical: 0 
##     . transformed: 0 
##     . standardized: 0 
##     . imputed: 0 
##  . assayData: 88 exposures 109 individuals
##     . element names: exp 
##     . exposures: AbsPM25, ..., Zn 
##     . individuals: id001, ..., id108 
##  . phenoData: 109 individuals 9 phenotypes
##     . individuals: id001, ..., id108 
##     . phenotypes: whistling_chest, ..., cbmi 
##  . featureData: 88 exposures 7 explanations
##     . exposures: AbsPM25, ..., Zn 
##     . descriptions: Family, ..., .imp 
## experimentData: use 'experimentData(object)'
## Annotation:

Under the section exposures description the number of continuous (84) and categorical (4) exposures are shown. The assayData, phenoData and featureData shows the content of the files we loaded with readExposome.

2.1.2 From data.frame

The function loadExposome allows to create an ExposomeSet through three data.frames: one as description data, one as exposures data and one as phenotypes data. The arguments are similar to the ones from readExposome:

args(loadExposome)

## function (exposures, description, phenotype, description.famCol = "family", 
##     exposures.asFactor = 5, warnings = TRUE) 
## NULL

In order to illustrate how to use loadExposome, we are loading the previous csv files as data.frames:

dd <- read.csv(description, header = TRUE)
ee <- read.csv(exposures, header = TRUE)
pp <- read.csv(phenotype, header = TRUE)

Then we rearrange the data.frames to fulfil with the requirements of the exposome data. The data.frame corresponding to description data needs to have the exposure’s names as rownames.

rownames(dd) <- dd[, 2]
dd <- dd[, -2]

The data.frame corresponding to exposures data needs to have the individual’s identifiers as rownames:

rownames(ee) <- ee[, 1]
ee <- ee[, -1]

The data.frame corresponding to phenotypes data needs to have the individual’s identifiers as a rownames, as the previous data.frame:

rownames(pp) <- pp[, 1]
pp <- pp[, -1]

Then, the ExposomeSet is creating by giving the three data.frames to loadExposome:

exp <- loadExposome(exposures = ee, description = dd, phenotype = pp, description.famCol = "Family")

2.1.3 Accessing to Exposome Data

The class ExposomeSet has several accessors to get the data stored in it. There are four basic methods that returns the names of the individuals (sampleNames), the name of the exposures (exposureNames), the name of the families of exposures (familyNames) and the name of the phenotypes (phenotypeNames).

head(sampleNames(exp))

## [1] "id001" "id002" "id003" "id004" "id005" "id006"

head(exposureNames(exp))

## [1] "AbsPM25" "As"      "BDE100"  "BDE138"  "BDE153"  "BDE154"

familyNames(exp)

##  [1] "Air Pollutants"    "Metals"            "PBDEs"            
##  [4] "Bisphenol A"       "Water Pollutants"  "Built Environment"
##  [7] "Cotinine"          "Organochlorines"   "Home Environment" 
## [10] "Phthalates"        "Noise"             "PFOAs"            
## [13] "Temperature"

phenotypeNames(exp)

## [1] "whistling_chest" "flu"             "rhinitis"        "wheezing"       
## [5] "birthdate"       "sex"             "age"             "cbmi"           
## [9] "blood_pre"

fData will return the description of the exposures (including internal information to manage them).

head(fData(exp), n = 3)

##                 Family                                          Name .fct .trn
## AbsPM25 Air Pollutants Measurement of the blackness of PM2.5 filters          
## As              Metals                                        Asenic          
## BDE100           PBDEs            Polybrominated diphenyl ether -100          
##         .std .imp   .type
## AbsPM25           numeric
## As                numeric
## BDE100            numeric

pData will return the phenotypes information.

head(pData(exp), n = 3)

##       whistling_chest flu rhinitis wheezing  birthdate  sex age cbmi blood_pre
## id001           never  no       no       no 2004-12-29 male 4.2 16.3       120
## id002           never  no       no       no 2005-01-05 male 4.2 16.4       121
## id003        7-12 epi  no       no      yes 2005-01-05 male 4.2 19.0       120

Finally, the method expos allows to obtain the matrix of exposures as a data.frame:

expos(exp)[1:10, c("Cotinine", "PM10V", "PM25", "X5cxMEPP")]

##          Cotinine       PM10V     PM25 X5cxMEPP
## id001  0.03125173  0.10373078 1.176255       NA
## id002  1.59401990 -0.47768393 1.155122       NA
## id003  1.46251090          NA 1.215834 1.859045
## id004  0.89059991          NA 1.171610       NA
## id005          NA          NA 1.145765       NA
## id006  0.34818304          NA 1.145382       NA
## id007  1.53591130          NA 1.174642       NA
## id008  2.26864700          NA 1.165078 1.291871
## id009  1.24842660          NA 1.171406 1.650948
## id010 -0.36758339  0.01593277 1.179240 2.112357

2.2.1 Missing Data in Exposures and Phenotypes

The number of missing data on each exposure and on each phenotype can be found by using the function tableMissings. This function returns a vector with the amount of missing data in each exposure or phenotype. The argument set indicates if the number of missing values is counted on exposures of phenotypes. The argument output indicates if it is shown as counts (output="n") or as percentage (output="p").

The current exposome data has no missing in the exposures nor in the phenotypes:

tableMissings(exp, set = "exposures", output = "n")

##         Dens         Temp         Conn      AbsPM25           NO          NO2 
##            0            0            1            2            2            2 
##          NOx         PM10       PM10Cu       PM10Fe        PM10K       PM10Ni 
##            2            2            2            2            2            2 
##        PM10S       PM10SI       PM10Zn         PM25       PM25CU       PM25FE 
##            2            2            2            2            2            2 
##        PM25K       PM25Ni        PM25S       PM25Sl       PM25Zn     PMcoarse 
##            2            2            2            2            2            2 
##      Benzene        PM25V          ETS G_pesticides          Gas         BTHM 
##            3            3            5            5            5            6 
##        CHCl3 H_pesticides      Noise_d      Noise_n          THM     Cotinine 
##            6            6            6            6            6            7 
##          DDE          DDT          HCB       PCB118       PCB138       PCB153 
##           13           13           13           13           13           13 
##       PCB180         bHCH          BPA           As           Cs           Mo 
##           13           13           21           24           24           24 
##           Ni           Tl           Zn           Hg           Cd           Sb 
##           24           24           24           27           28           30 
##        Green           Cu        PM10V           Se         MBzP        MEHHP 
##           31           40           41           45           46           46 
##         MEHP        MEOHP          MEP         MiBP         MnBP     X5cxMEPP 
##           46           46           46           46           46           46 
##           Co        PFHxS         PFNA         PFOA         PFOS    X7OHMMeOP 
##           47           48           48           48           48           49 
##           Pb     X2cxMMHP       BDE100       BDE138       BDE153       BDE154 
##           59           64           76           76           76           76 
##        BDE17       BDE183       BDE190       BDE209        BDE28        BDE47 
##           76           76           76           76           76           76 
##        BDE66        BDE71        BDE85        BDE99 
##           76           76           76           76

tableMissings(exp, set = "phenotypes", output = "n")

## whistling_chest             flu        rhinitis        wheezing             sex 
##               0               0               0               0               0 
##             age            cbmi       blood_pre       birthdate 
##               0               0               2               3

Alternatively to tableMissings, the function plotMissings draw a bar plot with the percentage of missing data in each exposure of phenotype.

plotMissings(exp, set = "exposures")

2.2.2 Exposures Normality

Most of the test done in exposome analysis requires that the exposures must follow a normal distribution. The function normalityTest performs a test on each exposure for normality behaviour. The result is a data.frame with the exposures’ names, a flag TRUE/FALSE for normality and the p-value obtained from the Shapiro-Wilk Normality Test (if the p-value is under the threshold, then the exposure is not normal).

nm <- normalityTest(exp)
table(nm$normality)

## 
## FALSE  TRUE 
##    55    29

So, the exposures that do not follow a normal distribution are:

nm$exposure[!nm$normality]

##  [1] "DDT"      "PM10SI"   "PM25K"    "PM25Sl"   "PCB118"   "Tl"      
##  [7] "PM10V"    "PM25Zn"   "PM25FE"   "PM10K"    "BDE17"    "PM25"    
## [13] "PMcoarse" "PM10"     "BPA"      "Green"    "NO2"      "Cs"      
## [19] "PFNA"     "PCB153"   "PM25CU"   "MEOHP"    "Cu"       "HCB"     
## [25] "MEHHP"    "DDE"      "BDE190"   "bHCH"     "PM10Zn"   "MnBP"    
## [31] "NO"       "NOx"      "PM10S"    "MEHP"     "PCB138"   "Zn"      
## [37] "X2cxMMHP" "PCB180"   "PFOA"     "Cotinine" "PM25S"    "Co"      
## [43] "Conn"     "PM25Ni"   "PFHxS"    "PM10Ni"   "Cd"       "Dens"    
## [49] "Se"       "X5cxMEPP" "BDE183"   "BDE28"    "Sb"       "BDE138"  
## [55] "PM25V"

Some of these exposures are categorical so they must not follow a normal distribution. This is the case, for example, of G_pesticides. If we plot the histogram of the values of the exposures it will make clear:

library(ggplot2)
plotHistogram(exp, select = "G_pesticides") + ggtitle("Garden Pesticides")

Some others exposures are continuous variables that do not overpass the normality test. A visual inspection is required in this case.

plotHistogram(exp, select = "BDE209") + ggtitle("BDE209 - Histogram")

If the exposures were following an anon normal distribution, the method plotHistogram has an argument show.trans that set to TRUE draws the histogram of the exposure plus three typical transformations:

plotHistogram(exp, select = "BDE209", show.trans = TRUE)

2.2.3 Exposures Imputation

The imputation process is out of rexposome scope. Nevertheless, rexposome incorporates a wrapper to run the imputation tools from the R packages and Hmisc. The imputation of the exposures in the ExposomeSet is done by using this code:

exp <- imputation(exp)

To use mice package instead of hmisc, see the vignette entitles Dealing with Multiple Imputations.

2.2.4 Exposures Behaviour

We can get a snapshot of the behaviour of the full exposome using the method plotFamily or its wrapper plot. This function allows drawing a plot of a given family of exposures or a mosaic with all the exposures.

plotFamily(exp, family = "all")

This plotting method allows to group the exposure by a given phenotype using the argument group:

plotFamily(exp, family = "Phthalates", group = "sex")

The same method allows to include a second group using the argument group2:

plotFamily(exp, family = "Phthalates", group = "rhinitis", group2 = "rhinitis")

2.2.5 Exposures PCA

To properly perform a PCA analysis the exposures needs to be standardised. The standardisation is done using function standardize that allows using a normal and a robust approaches or use the interquartile range. The normal aproache scales the exposures using the mean as a centre and the standard variation used as dispersion. In robust aproach the median and the median absolute deviation are used. This transformation are only applied to continuous exposures. When interquartile range is used, the median is used as a center and the coeficient between the interquartile range of the exposure and the normal range between the percentile 75 and 25 as variance.

exp_std <- standardize(exp, method = "normal")
exp_std

## Object of class 'ExposomeSet' (storageMode: environment)
##  . exposures description:
##     . categorical:  4 
##     . continuous:  84 
##  . exposures transformation:
##     . categorical: 0 
##     . transformed: 0 
##     . standardized: 84 
##     . imputed: 88 
##  . assayData: 88 exposures 109 individuals
##     . element names: exp 
##     . exposures: AbsPM25, ..., Zn 
##     . individuals: id001, ..., id108 
##  . phenoData: 109 individuals 9 phenotypes
##     . individuals: id001, ..., id108 
##     . phenotypes: whistling_chest, ..., cbmi 
##  . featureData: 88 exposures 7 explanations
##     . exposures: AbsPM25, ..., Zn 
##     . descriptions: Family, ..., .imp 
## experimentData: use 'experimentData(object)'
## Annotation:

Once the exposures are standardized we can run a PCA on the ExposomeSet using the method pca.

exp_pca <- pca(exp_std)

The method pca returns an object of class ExposomePCA. This object encapsulates all the information generated by the principal component analysis. The method plotPCA can be used in several ways. The first way is setting the argument set to "all" to create a mosaic of plots.

plotPCA(exp_pca, set = "all")

The plots in the first row correspond to the exposures and samples space. The first plot shows all the exposures on the axis for the first and the second principal components. The second plot shows all the individuals on the axis for the first and second principal components.

The plots on the second row are a summary of the variability explained by each component. The first plot is a bar plot with the variability explained by each component highlighting the components that are being drawn in the two first plots. The second plot is a line plot indicating the cumulative variability explained until each principal component. The vertical dashed line indicates the last principal component that is drawn in the first two plots. The horizontal dashed line indicates the amount of explained variability.

A second way of using plotPCA is changing the content of the argument set to "samples" to see the samples’ space. When the set argument is filled with samples, the argument phenotype can be used to colour each sample with its phenotype value.

plotPCA(exp_pca, set = "samples", phenotype = "sex")

This plot shows the sample space of the first and the second principal component. Each dot is a sample and it is coloured depending on its value in sex. We can see that no cluster is done in terms of sex.

This view be recreated in a 3D space using the function plot3PCA:

plot3PCA(exp_pca, cmpX = 1, cmpY = 2, cmpZ = 3, phenotype = "sex")

2.2.6 Exposures Correlation

The correlation between exposures, in terms of intra-family and inter-family exposures, is interesting to take into account. The correlation of the exposome can be computed using correlation.

exp_cr <- correlation(exp, use = "pairwise.complete.obs", method.cor = "pearson")

The values of the correlation can be obtained using the method extract. This returns a data.frame.

extract(exp_cr)[1:4, 1:4]

##             AbsPM25         As     BDE100      BDE138
## AbsPM25  1.00000000 -0.1681446  0.1098427 -0.03196037
## As      -0.16814460  1.0000000 -0.3727296  0.13768481
## BDE100   0.10984268 -0.3727296  1.0000000  0.11482844
## BDE138  -0.03196037  0.1376848  0.1148284  1.00000000

The best option to see the inter-family correlations is the circos of correlations while the matrix of correlations is a better way for studying the intra-family correlations. Both of them are drawn using the method plotCorrelation.

plotCorrelation(exp_cr, type = "circos")

plotCorrelation(exp_cr, type = "matrix")

2.2.7 Individuals Clustering

Clustering analysis on exposures data results in exposure profile. The method clustering allows applying most of any clustering method to an ExposomeSet method.

The argument of the method clustering are:

args(clustering)

## function (object, method, cmethod, ..., warnings = TRUE) 
## NULL

The argument method is filled with the clustering function. This clustering function needs to accept an argument called data, that will be filled with the exposures-matrix. The object obtained from the clustering function needs to have an accessor called classification. Otherwise the argument cmethod needs to be filled with a function that takes the results of the clustering function and returns a vector with the classification of each individual.

In this analysis we apply the clustering method hclust. Hence we create a function to accept an argument called data.

hclust_data <- function(data, ...) {
    hclust(d = dist(x = data), ...)
}

The argument ... allows passing arguments from recposome’s clustering method to hclust.

Then, a function to obtain the classification of each sample is also required. This function will use the cutree function to obtain the labels.

hclust_k3 <- function(result) {
    cutree(result, k = 3)
}

The new function hclust_k3 is a function that takes the results of hclust_data and applies it the cutree function, requesting 3 groups of individuals.

Having both clustering function (hclust_data) and the classification function (hclust_k3) we use them in the clustering method:

exp_c <- clustering(exp, method = hclust_data, cmethod = hclust_k3)

## Warning in clustering(exp, method = hclust_data, cmethod = hclust_k3): Non
## continuous exposures will be discarded.

exp_c

## Object of class 'ExposomeClust' (storageMode: environment)
##  . Method: .... TRUE 
##  . assayData: 84 exposures 109 samples
##     . element names: exp 
##     . exposures: AbsPM25, ..., Zn 
##     . samples: id001, ..., id108 
##  . featureData: 84 exposures 7 explanations
##     . exposures: AbsPM25, ..., Zn 
##     . descriptions: Family, ..., .imp 
##     . #Cluster: 3

The profile for each group of individuals can be plotted with plotClassification method.

plotClassification(exp_c)

The classification of each individual can be obtained using the method classification. We can get a table with the number of samples per group with:

table(classification(exp_c))

## 
##  1  2  3 
## 80 24  5

As seen, the groups are given as numbers and the plotClassification transforms it to names (Group 1, Group 2 and Group 3).

2.3.1 Understanding Principal Component Analysis

From the results of the PCA on the exposome data, two measures can be obtained: the correlation of the exposures with the principal components and the association of the phenotypes with the principal components.

The method plotEXP draws a heat map with the correlation of each exposure to the principal components.

plotEXP(exp_pca) + theme(axis.text.y = element_text(size = 6.5)) + ylab("")

From the plot, some conclusions can be obtained:

• PC 1 is hight correlated with particle matter (PM) exposures.
• PC 2 is correlated with the Polybrominated Diphenyl Ethers (BDES) exposures.
• PC 5 is negative correlated with Perfluorooctanoic Acid (PFOAS) and Polychlorinated Biphenyl (PCBS).

These conclusions are useful to give a meaning to the Principal Components in terms of exposures.

The method plotPHE test the association between the phenotypes and the principal components and draws a heat map with the score of the association.

plotPHE(exp_pca)

The conclusions that can be taken from the heat map are:

• age is associated to PC 10.
• sex is associated to PC 6 and PC 10.
• flu is associated to PC 5.

2.3.2 Exposome-Wide Association Studies (ExWAS)

Method exwas performs univariate test of the association between exposures and health outcomes. This method requests a formula to test and the family of the distribution of the health outcome (dependent variable).

The following line performs an ExWAS on flu and wheezing adjusted by sex and age. Since the content of flu and others in the ExposomeSet is dichotomous, the family is set to binomial (for more information see ?glm).

fl_ew <- exwas(exp, formula = blood_pre ~ sex + age, family = "gaussian")
fl_ew

## An object of class 'ExWAS'
## 
##        blood_pre ~ sex+age 
## 
## Tested exposures:  88 
## Threshold for effective tests (TEF):  1.28e-03 
##  . Tests < TEF: 7

we_ew <- exwas(exp, formula = wheezing ~ sex + age, family = "binomial")
we_ew

## An object of class 'ExWAS'
## 
##        wheezing ~ sex+age 
## 
## Tested exposures:  88 
## Threshold for effective tests (TEF):  1.28e-03 
##  . Tests < TEF: 0

The method exwas calculates the effective number of tests in base of the correlation between the exposures. This is transformed into a threshold for the p-values of the association. This threshold can be obtained using the method tef.

A table with the associations between the exposures and flu is obtained with method extract:

head(extract(fl_ew))

## DataFrame with 6 rows and 4 columns
##                       pvalue           effect             X2.5            X97.5
##                    <numeric>        <numeric>        <numeric>        <numeric>
## NOx     8.47991160367928e-06 13.3616339264834 7.77551854451183  18.947749308455
## NO      1.11507113009775e-05 10.9207305588316 6.28755096595306 15.5539101517102
## NO2     1.16035859730813e-05 15.4008797431827 8.85281995815165 21.9489395282138
## AbsPM25  1.6643636784392e-05 20.2280203055961 11.4542843772182  29.001756233974
## PM25    3.71919769956175e-05 37.7805209158959 20.6067815811759 54.9542602506159
## PM25CU  0.000135028997381526  11.517055715762 5.82591839662578 17.2081930348982

A Manhattan-like plot with the p-values of the association between each exposure and asthma, coloured by families of exposures, is draw by method plotExwas.

clr <- rainbow(length(familyNames(exp)))
names(clr) <- familyNames(exp)
plotExwas(fl_ew, we_ew, color = clr) + ggtitle("Exposome Association Study - Univariate Approach")

Then a plot for the effects of a given model can be obtained with plotEffect:

plotEffect(fl_ew)

2.3.3 Multivariate Exposome Analysis

The last approach is a multivariate analysis in order to find the group of exposures related to the health outcome. This can be done using methods like Elastic Net. The method mexwas applies elastic net to the exposures given a health outcome of interest.

bl_mew <- mexwas(exp_std, phenotype = "blood_pre", family = "gaussian")
we_mew <- mexwas(exp_std, phenotype = "wheezing", family = "binomial")

The coefficient of each exposure is plotted with plotExwas. The method draws a heat map with two columns and the exposures as rows. The heat map is coloured with the coefficient of each exposure in relation with the health outcome, so the ones in white are not interesting. The two columns of the heat map correspond to the minimum lambda (Min) and to the lambda which gives the most regularised model such that error is within one standard error of the minimum (1SE).

plotExwas(bl_mew, we_mew) + ylab("") + ggtitle("Exposome Association Study - Multivariate Approach")