methylclock 1.11.0
This manual describes how to estimate chronological and gestational DNA methylation (DNAm) age as well as biological age using different methylation clocks. The package includes the following estimators:
The biological DNAm clocks implemented in our package are:
The main aim of this package is to facilitate the interconnection with R and
Bioconductor’s infrastructure and, hence, avoiding submitting data to online
calculators. Additionally, methylclock
also provides an unified way of
computing DNAm age to help downstream analyses.
The package depends on some R packages that can be previously installed into your computer by:
install.packages(c("tidyverse", "impute", "Rcpp"))
Then methylclock
package is installed into your computer by executing:
if (!requireNamespace("BiocManager", quietly = TRUE))
install.packages("BiocManager")
BiocManager::install("methylclock")
The package is loaded into R as usual:
library(methylclockData)
library(methylclock)
These libraries are required to reproduce this document:
library(Biobase)
library(tibble)
library(impute)
library(ggplot2)
library(ggpmisc)
library(GEOquery)
The main function to estimate chronological and biological mDNA age is called
DNAmAge
while the gestational DNAm age is estimated using DNAmGA
function.
Both functions have similar input arguments. Next subsections detail some of
the important issues to be consider before computind DNAm clocks.
The methylation data is given in the argument x
. They can be either beta or
M values. The argument toBetas
should be set to TRUE when M values are
provided. The x
object can be:
A matrix with CpGs in rows and individuals in columns having the name of the CpGs in the rownames.
A data frame or a tibble with CpGs in rows and individuals in columns having the name of the CpGs in the first column (e.g. cg00000292, cg00002426, cg00003994, …) as required in the Horvath’s DNA Methylation Age Calculator website (https://dnamage.genetics.ucla.edu/home).
A GenomicRatioSet object, the default method to encapsulate methylation
data in minfi
Bioconductor package.
An ExpressionSet object as obtained, for instance, when downloading methylation data from GEO (https://www.ncbi.nlm.nih.gov/geo/).
In principle, data can be normalized by using any of the existing standard
methods such as QN, ASMN, PBC, SWAN, SQN, BMIQ (see a revision of those
methods in Wang et al. (2015)). DNAmAge
function includes the BMIQ method
proposed by Teschendorff et al. (2012) using Horvath’s robust implementation that
basically consists of an optimal R code implementation and optimization
procedures. This normalization is recommended by Horvath since it improves
the predictions for his clock. This normalization procedure is very
time-consuming. In order to overcome these difficulties, we have parallelize
this process using BiocParallel
library. This step is not mandatory, so that,
you can use your normalized data and set the argument normalize
equal to
FALSE (default).
All the implemented methods require complete cases. DNAmAge
function has an
imputation method based on KNN implemented in the function knn.impute
from
impute
Bioconductor package. This is performed when missing data is present
in the CpGs used in any of the computed clocks. There is also another option
based on a fast imputation method that imputes missing values by the median of
required CpGs as recommended in Bohlin et al. (2016). This is recommended when
analyzing 450K arrays since knn.impute
for large datasets may be very time
consuming. Fast imputation can be performed by setting fastImp=TRUE
which is
not the default value.
By default the package computes the different clocks when there are more than
80% of the required CpGs of each method. Nothing is required when having
missing CpGs since the main functions will return NA for those estimators
when this criteria is not meet. Let us use a test dataset (TestDataset
)
which is available within the package to illustrate the type of information
we are obtaining:
# Get TestDataset data
TestDataset <- get_TestDataset()
cpgs.missing <- checkClocks(TestDataset)
clock Cpgs_in_clock missing_CpGs percentage
1 Horvath 353 2 0.6
2 Hannum 71 64 90.1
3 Levine 513 3 0.6
4 SkinHorvath 391 283 72.4
5 PedBE 94 91 96.8
6 Wu 111 2 1.8
7 TL 140 137 97.9
8 BLUP 319607 300288 94.0
9 EN 514 476 92.6
cpgs.missing.GA <- checkClocksGA(TestDataset)
clock Cpgs_in_clock missing_CpGs percentage
1 Knight 148 0 0.0
2 Bohlin 87 87 100.0
3 Mayne 62 0 0.0
4 Lee 1125 1072 95.3
5 EPIC 176 170 96.6
The objects cpgs.missing
and cpgs.missing.GA
are lists having the missing
CpGs of each clock
names(cpgs.missing)
[1] "Horvath" "Hannum" "Levine" "skinHorvath" "PedBE"
[6] "Wu" "TL" "BLUP" "EN"
We can see which are those CpGs for a given clock (for example Hannum) with
the function commonClockCpgs
:
commonClockCpgs(cpgs.missing, "Hannum" )
[1] "cg20822990" "cg22512670" "cg25410668" "cg04400972"
[5] "cg16054275" "cg10501210" "ch.2.30415474F" "cg22158769"
[9] "cg02085953" "cg06639320" "cg22454769" "cg24079702"
[13] "cg23606718" "cg22016779" "cg03607117" "cg07553761"
[17] "cg00481951" "cg25478614" "cg25428494" "cg02650266"
[21] "cg08234504" "cg23500537" "cg20052760" "cg16867657"
[25] "cg06685111" "cg00486113" "cg13001142" "cg20426994"
[29] "cg14361627" "cg08097417" "cg07955995" "cg22285878"
[33] "cg03473532" "cg08540945" "cg07927379" "cg16419235"
[37] "cg07583137" "cg22796704" "cg19935065" "cg23091758"
[41] "cg23744638" "cg04940570" "cg11067179" "cg22213242"
[45] "cg06419846" "cg02046143" "cg00748589" "cg18473521"
[49] "cg01528542" "ch.13.39564907R" "cg03032497" "cg04875128"
[53] "cg09651136" "cg03399905" "cg04416734" "cg07082267"
[57] "cg14692377" "cg06874016" "cg21139312" "cg02867102"
[61] "cg19283806" "cg14556683" "cg07547549" "cg08415592"
commonClockCpgs(cpgs.missing.GA, "Bohlin" )
[1] "cg00153101" "cg00602416" "cg00711496" "cg01190109" "cg01635555"
[6] "cg01833485" "cg02324006" "cg02405476" "cg02567958" "cg02642822"
[11] "cg03108070" "cg03281561" "cg03337084" "cg03507326" "cg03710860"
[16] "cg03729251" "cg03773820" "cg03963689" "cg04347477" "cg04685228"
[21] "cg05053327" "cg05544807" "cg05877497" "cg06753281" "cg06897661"
[26] "cg07106169" "cg07676709" "cg07738730" "cg07749613" "cg07788865"
[31] "cg07835443" "cg08326019" "cg08620426" "cg08943494" "cg09447786"
[36] "cg10308785" "cg11124260" "cg11294761" "cg11864574" "cg12880227"
[41] "cg12999267" "cg13036381" "cg13066703" "cg13433246" "cg13641317"
[46] "cg13733403" "cg13959344" "cg13982823" "cg14276580" "cg14427590"
[51] "cg15035133" "cg15131146" "cg15165154" "cg15908709" "cg16187883"
[56] "cg16348385" "cg17022232" "cg18183624" "cg18217136" "cg18954401"
[61] "cg19057830" "cg19439123" "cg19875532" "cg20301308" "cg20303561"
[66] "cg20816447" "cg21081878" "cg21143441" "cg21155834" "cg21221899"
[71] "cg21707172" "cg21878650" "cg22761205" "cg22796593" "cg22797644"
[76] "cg23051248" "cg23346945" "cg23403099" "cg23457357" "cg24041556"
[81] "cg24087613" "cg24366564" "cg25150953" "cg25531857" "cg25639749"
[86] "cg26077811" "cg26092675"
In Section 4.1 we describe how to change this 80% threshold.
The EEAA method requires to estimate cell counts. We use the package meffil
(Min et al. (2018)) that provides some functions to estimate cell counts using
predefined datasets. This is performed by setting cell.count=TRUE
(default
value). The reference panel is passed through the argument
cell.count.reference
. So far, the following options are available:
miffil
package. It includes CD14, Bcell,
CD4T, CD8T, NK, Gran.Next we illustrate how to estimate the chronological DNAm age using several datasets which aim to cover different data input formats.
IMPORTANT NOTE: On some systems we can find an error in the DNAmAge()
function when parameter cell.count = TRUE
. This error is related to
preprocessCore
package and can be fixed by disabling multi-threading
when installing the preprocessCore package using the command
BiocManager::install("preprocessCore",
configure.args = "--disable-threading",
force = TRUE)
csv
with CpGs in rows)Let us start by reproducing the results proposed in Horvath (2013). It uses
the format available in the file ’MethylationDataExample55.csv" from his
tutorial (available here). These data
are available at methylclock
package. Although these data can be loaded into
R by using standard functions such as read.csv
we hihgly recommend to use
functions from tidiverse
, in particular read_csv
from readr
package.
The main reason is that currently researchers are analyzing Illumina 450K
or EPIC arrays that contains a huge number of CpGs that can take a long time
to be loaded when using basic importing R function. These functions import
csv
data as tibble which is one of the possible formats of DNAmAge
function
library(tidyverse)
MethylationData <- get_MethylationDataExample()
MethylationData
# A tibble: 27,578 × 17
ProbeID GSM946048 GSM946049 GSM946052 GSM946054 GSM946055 GSM946056 GSM946059
<chr> <dbl> <dbl> <dbl> <dbl> <dbl> <dbl> <dbl>
1 cg0000… 0.706 0.730 0.705 0.751 0.715 0.634 0.682
2 cg0000… 0.272 0.274 0.311 0.279 0.178 0.269 0.330
3 cg0000… 0.0370 0.0147 0.0171 0.0290 0.0163 0.0243 0.0127
4 cg0000… 0.133 0.120 0.121 0.107 0.110 0.129 0.102
5 cg0000… 0.0309 0.0192 0.0217 0.0132 0.0181 0.0243 0.0199
6 cg0000… 0.0700 0.0715 0.0655 0.0719 0.0914 0.0508 0.0294
7 cg0000… 0.993 0.993 0.993 0.994 0.991 0.994 0.993
8 cg0000… 0.0215 0.0202 0.0187 0.0169 0.0162 0.0143 0.0172
9 cg0000… 0.0105 0.00518 0.00410 0.00671 0.00758 0.00518 0.00543
10 cg0001… 0.634 0.635 0.621 0.639 0.599 0.591 0.594
# ℹ 27,568 more rows
# ℹ 9 more variables: GSM946062 <dbl>, GSM946064 <dbl>, GSM946065 <dbl>,
# GSM946066 <dbl>, GSM946067 <dbl>, GSM946073 <dbl>, GSM946074 <dbl>,
# GSM946075 <dbl>, GSM946076 <dbl>
IMPORTANT NOTE: Be sure that the first column contains the CpG names.
Sometimes, your imported data look like this one (it can happen, for
instance, if the csv
file was created in R without indicating
row.names=FALSE
)
> mydata
# A tibble: 473,999 x 6
X1 Row.names BIB_15586_1X BIB_33043_1X EDP_5245_1X KAN_584_1X
<int> <chr> <dbl> <dbl> <dbl> <dbl>
1 1 cg000000~ 0.635 0.575 0.614 0.631
2 2 cg000001~ 0.954 0.948 0.933 0.950
3 3 cg000001~ 0.889 0.899 0.901 0.892
4 4 cg000001~ 0.115 0.124 0.107 0.123
5 5 cg000002~ 0.850 0.753 0.806 0.815
6 6 cg000002~ 0.676 0.771 0.729 0.665
7 7 cg000002~ 0.871 0.850 0.852 0.863
8 8 cg000003~ 0.238 0.174 0.316 0.206
If so, the first column must be removed before being used as the input
object in DNAmAge
funcion. It can be done using dplyr
function
> mydata2 <- select(mydata, -1)
# A tibble: 473,999 x 5
Row.names BIB_15586_1X BIB_33043_1X EDP_5245_1X KAN_584_1X
<chr> <dbl> <dbl> <dbl> <dbl>
1 cg000000~ 0.635 0.575 0.614 0.631
2 cg000001~ 0.954 0.948 0.933 0.950
3 cg000001~ 0.889 0.899 0.901 0.892
4 cg000001~ 0.115 0.124 0.107 0.123
5 cg000002~ 0.850 0.753 0.806 0.815
6 cg000002~ 0.676 0.771 0.729 0.665
7 cg000002~ 0.871 0.850 0.852 0.863
8 cg000003~ 0.238 0.174 0.316 0.206
In any case, if you use the object mydata
that contains the CpGs in the
second column, you will see this error message:
> DNAmAge(mydata)
Error in DNAmAge(mydata) : First column should contain CpG names
Once data is in the proper format, DNAmAge can be estimated by simply:
age.example55 <- DNAmAge(MethylationData)
Warning in predAge(cpgs.imp, coefHannum, intercept = FALSE, min.perc): The number of missing CpGs forHannumclock exceeds 80%.
---> This DNAm clock will be NA.
rows : 353 cols : 16
Warning in predAge(cpgs.imp, coefSkin, intercept = TRUE, min.perc): The number of missing CpGs forSkinclock exceeds 80%.
---> This DNAm clock will be NA.
Warning in predAge(cpgs.imp, coefPedBE, intercept = TRUE, min.perc): The number of missing CpGs forPedBEclock exceeds 80%.
---> This DNAm clock will be NA.
Warning in predAge(cpgs.imp, coefTL, intercept = TRUE, min.perc): The number of missing CpGs forTLclock exceeds 80%.
---> This DNAm clock will be NA.
Warning in predAge(cpgs.imp, coefBLUP, intercept = TRUE, min.perc): The number of missing CpGs forBLUPclock exceeds 80%.
---> This DNAm clock will be NA.
Warning in predAge(cpgs.imp, coefEN, intercept = TRUE, min.perc): The number of missing CpGs forENclock exceeds 80%.
---> This DNAm clock will be NA.
age.example55
# A tibble: 16 × 11
id Horvath Hannum Levine BNN skinHorvath PedBE Wu TL BLUP EN
<chr> <dbl> <lgl> <dbl> <dbl> <lgl> <lgl> <dbl> <lgl> <lgl> <lgl>
1 GSM946… 51.8 NA -30.3 56.4 NA NA 1.08 NA NA NA
2 GSM946… 39.8 NA -29.6 42.1 NA NA 0.808 NA NA NA
3 GSM946… 26.4 NA -33.3 25.6 NA NA 0.772 NA NA NA
4 GSM946… 34.0 NA -36.0 28.0 NA NA 0.941 NA NA NA
5 GSM946… 10.1 NA -52.8 13.4 NA NA 0.456 NA NA NA
6 GSM946… 20.4 NA -42.2 16.7 NA NA 0.621 NA NA NA
7 GSM946… 6.00 NA -44.8 7.54 NA NA 0.258 NA NA NA
8 GSM946… 34.6 NA -23.2 34.6 NA NA 0.624 NA NA NA
9 GSM946… 7.91 NA -49.8 12.0 NA NA 0.237 NA NA NA
10 GSM946… 4.72 NA -48.2 6.43 NA NA 0.396 NA NA NA
11 GSM946… 29.6 NA -39.9 28.5 NA NA 0.413 NA NA NA
12 GSM946… 1.38 NA -48.3 3.48 NA NA 0.122 NA NA NA
13 GSM946… 56.0 NA -26.7 47.3 NA NA 0.714 NA NA NA
14 GSM946… 24.0 NA -39.7 23.3 NA NA 0.676 NA NA NA
15 GSM946… 9.38 NA -45.4 11.9 NA NA 0.251 NA NA NA
16 GSM946… 38.8 NA -27.5 41.4 NA NA 0.599 NA NA NA
As mention in Section 3.4 some clocks returns NA when there are more than 80% of the required CpGs are missing as we can see when typing
missCpGs <- checkClocks(MethylationData)
clock Cpgs_in_clock missing_CpGs percentage
1 Horvath 353 0 0.0
2 Hannum 71 64 90.1
3 Levine 513 0 0.0
4 SkinHorvath 391 282 72.1
5 PedBE 94 91 96.8
6 Wu 111 0 0.0
7 TL 140 137 97.9
8 BLUP 319607 300192 93.9
9 EN 514 476 92.6
Here we can observe that 72.1% of the required CpGs for SkinHorvath clock are
missing. We could estimate DNAm age using this clock just changing the argument
min.perc
in DNAmAge
. For example, we can indicate that the minimum amount
of required CpGs for computing a given clock should be 25%.
age.example55.2 <- DNAmAge(MethylationData, min.perc = 0.25)
Warning in predAge(cpgs.imp, coefHannum, intercept = FALSE, min.perc): The number of missing CpGs forHannumclock exceeds 25%.
---> This DNAm clock will be NA.
rows : 353 cols : 16
Warning in predAge(cpgs.imp, coefPedBE, intercept = TRUE, min.perc): The number of missing CpGs forPedBEclock exceeds 25%.
---> This DNAm clock will be NA.
Warning in predAge(cpgs.imp, coefTL, intercept = TRUE, min.perc): The number of missing CpGs forTLclock exceeds 25%.
---> This DNAm clock will be NA.
Warning in predAge(cpgs.imp, coefBLUP, intercept = TRUE, min.perc): The number of missing CpGs forBLUPclock exceeds 25%.
---> This DNAm clock will be NA.
Warning in predAge(cpgs.imp, coefEN, intercept = TRUE, min.perc): The number of missing CpGs forENclock exceeds 25%.
---> This DNAm clock will be NA.
age.example55.2
# A tibble: 16 × 11
id Horvath Hannum Levine BNN skinHorvath PedBE Wu TL BLUP EN
<chr> <dbl> <lgl> <dbl> <dbl> <dbl> <lgl> <dbl> <lgl> <lgl> <lgl>
1 GSM946… 51.8 NA -30.3 56.4 7.15 NA 1.08 NA NA NA
2 GSM946… 39.8 NA -29.6 42.1 7.09 NA 0.808 NA NA NA
3 GSM946… 26.4 NA -33.3 25.6 5.93 NA 0.772 NA NA NA
4 GSM946… 34.0 NA -36.0 28.0 6.34 NA 0.941 NA NA NA
5 GSM946… 10.1 NA -52.8 13.4 5.76 NA 0.456 NA NA NA
6 GSM946… 20.4 NA -42.2 16.7 5.79 NA 0.621 NA NA NA
7 GSM946… 6.00 NA -44.8 7.54 5.64 NA 0.258 NA NA NA
8 GSM946… 34.6 NA -23.2 34.6 5.55 NA 0.624 NA NA NA
9 GSM946… 7.91 NA -49.8 12.0 5.06 NA 0.237 NA NA NA
10 GSM946… 4.72 NA -48.2 6.43 5.48 NA 0.396 NA NA NA
11 GSM946… 29.6 NA -39.9 28.5 6.19 NA 0.413 NA NA NA
12 GSM946… 1.38 NA -48.3 3.48 4.91 NA 0.122 NA NA NA
13 GSM946… 56.0 NA -26.7 47.3 7.07 NA 0.714 NA NA NA
14 GSM946… 24.0 NA -39.7 23.3 6.23 NA 0.676 NA NA NA
15 GSM946… 9.38 NA -45.4 11.9 5.57 NA 0.251 NA NA NA
16 GSM946… 38.8 NA -27.5 41.4 6.69 NA 0.599 NA NA NA
In that case, we see as SkinHorvath clock is estimated (though it can be observed that the estimation is not very accurate - this is why we considered at least having 80% of the required CpGs).
By default all available clocks (Hovarth, Hannum, Levine, BNN, skinHorvath,…)
are estimated. One may select a set of clocks by using the argument clocks
as follows:
age.example55.sel <- DNAmAge(MethylationData, clocks=c("Horvath", "BNN"))
rows : 353 cols : 16
age.example55.sel
# A tibble: 16 × 3
id Horvath BNN
<chr> <dbl> <dbl>
1 GSM946048 51.8 56.4
2 GSM946049 39.8 42.1
3 GSM946052 26.4 25.6
4 GSM946054 34.0 28.0
5 GSM946055 10.1 13.4
6 GSM946056 20.4 16.7
7 GSM946059 6.00 7.54
8 GSM946062 34.6 34.6
9 GSM946064 7.91 12.0
10 GSM946065 4.72 6.43
11 GSM946066 29.6 28.5
12 GSM946067 1.38 3.48
13 GSM946073 56.0 47.3
14 GSM946074 24.0 23.3
15 GSM946075 9.38 11.9
16 GSM946076 38.8 41.4
However, in epidemiological studies one is interested in assessing whether age acceleration is associated with a given trait or condition. Three different measures can be computed:
All this estimates can be obtained for each clock when providing chronological
age through age
argument. This information is normally provided in a
different file including different covariates (metadata or sample annotation
data). In this example data are available at ‘SampleAnnotationExample55.csv’
file that is also available at methylclock
package:
library(tidyverse)
path <- system.file("extdata", package = "methylclock")
covariates <- read_csv(file.path(path, "SampleAnnotationExample55.csv"))
covariates
# A tibble: 16 × 14
OriginalOrder id title geo_accession TissueDetailed Tissue diseaseStatus
<dbl> <chr> <chr> <chr> <chr> <chr> <dbl>
1 3 GSM946… Auti… GSM946048 Fresh frozen … occip… 1
2 4 GSM946… Cont… GSM946049 Fresh frozen … occip… 0
3 7 GSM946… Auti… GSM946052 Fresh frozen … occip… 1
4 9 GSM946… Auti… GSM946054 Fresh frozen … occip… 1
5 10 GSM946… Auti… GSM946055 Fresh frozen … occip… 1
6 11 GSM946… Auti… GSM946056 Fresh frozen … occip… 1
7 14 GSM946… Cont… GSM946059 Fresh frozen … occip… 0
8 17 GSM946… Cont… GSM946062 Fresh frozen … occip… 0
9 19 GSM946… Auti… GSM946064 Fresh frozen … occip… 1
10 20 GSM946… Auti… GSM946065 Fresh frozen … occip… 1
11 21 GSM946… Auti… GSM946066 Fresh frozen … occip… 1
12 22 GSM946… Cont… GSM946067 Fresh frozen … occip… 0
13 28 GSM946… Cont… GSM946073 Fresh frozen … occip… 0
14 29 GSM946… Cont… GSM946074 Fresh frozen … occip… 0
15 30 GSM946… Cont… GSM946075 Fresh frozen … occip… 0
16 31 GSM946… Cont… GSM946076 Fresh frozen … occip… 0
# ℹ 7 more variables: Age <dbl>, PostMortemInterval <dbl>, CauseofDeath <chr>,
# individual <dbl>, Female <dbl>, Caucasian <lgl>, FemaleOriginal <lgl>
In this case, chronological age is available at Age
column:
age <- covariates$Age
head(age)
[1] 60 39 28 39 8 22
The different methylation clocks along with their age accelerated estimates can be simply computed by:
age.example55 <- DNAmAge(MethylationData, age=age, cell.count=TRUE)
Warning in predAge(cpgs.imp, coefHannum, intercept = FALSE, min.perc): The number of missing CpGs forHannumclock exceeds 80%.
---> This DNAm clock will be NA.
rows : 353 cols : 16
Warning in predAge(cpgs.imp, coefSkin, intercept = TRUE, min.perc): The number of missing CpGs forSkinclock exceeds 80%.
---> This DNAm clock will be NA.
Warning in predAge(cpgs.imp, coefPedBE, intercept = TRUE, min.perc): The number of missing CpGs forPedBEclock exceeds 80%.
---> This DNAm clock will be NA.
Warning in predAge(cpgs.imp, coefTL, intercept = TRUE, min.perc): The number of missing CpGs forTLclock exceeds 80%.
---> This DNAm clock will be NA.
Warning in predAge(cpgs.imp, coefBLUP, intercept = TRUE, min.perc): The number of missing CpGs forBLUPclock exceeds 80%.
---> This DNAm clock will be NA.
Warning in predAge(cpgs.imp, coefEN, intercept = TRUE, min.perc): The number of missing CpGs forENclock exceeds 80%.
---> This DNAm clock will be NA.
age.example55
# A tibble: 16 × 24
id Horvath ageAcc.Horvath ageAcc2.Horvath ageAcc3.Horvath Hannum Levine
<chr> <dbl> <dbl> <dbl> <dbl> <lgl> <dbl>
1 GSM9460… 51.8 -8.22 -4.45 -4.91 NA -30.3
2 GSM9460… 39.8 0.754 2.00 1.59 NA -29.6
3 GSM9460… 26.4 -1.59 -1.67 -1.86 NA -33.3
4 GSM9460… 34.0 -5.00 -3.76 -0.463 NA -36.0
5 GSM9460… 10.1 2.06 -0.428 2.82 NA -52.8
6 GSM9460… 20.4 -1.61 -2.42 -2.88 NA -42.2
7 GSM9460… 6.00 2.00 -0.971 -0.827 NA -44.8
8 GSM9460… 34.6 6.65 6.57 5.32 NA -23.2
9 GSM9460… 7.91 2.91 0.0589 -2.61 NA -49.8
10 GSM9460… 4.72 2.72 -0.489 1.46 NA -48.2
11 GSM9460… 29.6 -0.427 -0.268 -1.37 NA -39.9
12 GSM9460… 1.38 0.375 -2.95 -2.19 NA -48.3
13 GSM9460… 56.0 -4.01 -0.242 1.62 NA -26.7
14 GSM9460… 24.0 2.03 1.23 -0.669 NA -39.7
15 GSM9460… 9.38 1.38 -1.11 -0.885 NA -45.4
16 GSM9460… 38.8 8.76 8.92 5.85 NA -27.5
# ℹ 17 more variables: ageAcc.Levine <dbl>, ageAcc2.Levine <dbl>,
# ageAcc3.Levine <dbl>, BNN <dbl>, ageAcc.BNN <dbl>, ageAcc2.BNN <dbl>,
# ageAcc3.BNN <dbl>, skinHorvath <lgl>, PedBE <lgl>, Wu <dbl>,
# ageAcc.Wu <dbl>, ageAcc2.Wu <dbl>, ageAcc3.Wu <dbl>, TL <lgl>, BLUP <lgl>,
# EN <lgl>, age <dbl>
By default, the argument cell.count
is set equal to TRUE and, hence, can
be omitted. This implies that ageAcc3
will be computed for all clocks.
In some occassions this can be very time consuming. In such cases one can
simply estimate DNAmAge, accAge and accAge2 by setting cell.count=FALSE
.
NOTE: see section 3.5 to see the reference panels available to estimate
cell counts.
Then, we can investigate, for instance, whether the accelerated age is associated with Autism. In that example we will use a non-parametric test (NOTE: use t-test or linear regression for large sample sizes)
autism <- covariates$diseaseStatus
kruskal.test(age.example55$ageAcc.Horvath ~ autism)
Kruskal-Wallis rank sum test
data: age.example55$ageAcc.Horvath by autism
Kruskal-Wallis chi-squared = 1.3346, df = 1, p-value = 0.248
kruskal.test(age.example55$ageAcc2.Horvath ~ autism)
Kruskal-Wallis rank sum test
data: age.example55$ageAcc2.Horvath by autism
Kruskal-Wallis chi-squared = 3.1875, df = 1, p-value = 0.0742
kruskal.test(age.example55$ageAcc3.Horvath ~ autism)
Kruskal-Wallis rank sum test
data: age.example55$ageAcc3.Horvath by autism
Kruskal-Wallis chi-squared = 2.8235, df = 1, p-value = 0.09289
ExpressionSet
dataOne may be interested in assessing association between chronologial age and DNA
methylation age or evaluating how well chronological age is predicted by
DNAmAge. In order to illustrate this analysis we downloaded data from GEO
corresponding to a set of healthy individuals (GEO accession number GSE58045).
Data can be retrieved into R by using GEOquery
package as an ExpressionSet
object that can be the input of our main function.
# To avoid connection buffer size
Sys.setenv("VROOM_CONNECTION_SIZE" = 131072 * 10)
# Download data
dd <- GEOquery::getGEO("GSE58045")
gse58045 <- dd[[1]]
# Restore connection buffer size
Sys.setenv("VROOM_CONNECTION_SIZE" = 131072)
gse58045
ExpressionSet (storageMode: lockedEnvironment)
assayData: 27578 features, 172 samples
element names: exprs
protocolData: none
phenoData
sampleNames: GSM1399890 GSM1399891 ... GSM1400061 (172 total)
varLabels: title geo_accession ... twin:ch1 (43 total)
varMetadata: labelDescription
featureData
featureNames: cg00000292 cg00002426 ... cg27665659 (27578 total)
fvarLabels: ID Name ... ORF (38 total)
fvarMetadata: Column Description labelDescription
experimentData: use 'experimentData(object)'
pubMedIds: 22532803
Annotation: GPL8490
The chronological age is obtained by using pData
function from Biobase
package that is able to deal with ExpressionSet
objects:
pheno <- pData(gse58045)
age <- as.numeric(pheno$`age:ch1`)
And the different DNA methylation age estimates are obtained by using DNAmAge
function (NOTE: as there are missing values, the program automatically runs
impute.knn
function to get complete cases):
age.gse58045 <- DNAmAge(gse58045, age=age)
Imputing missing data of the entire matrix ....
Data imputed. Starting DNAm clock estimation ...
Warning in predAge(cpgs.imp, coefHannum, intercept = FALSE, min.perc): The number of missing CpGs forHannumclock exceeds 80%.
---> This DNAm clock will be NA.
rows : 353 cols : 172
Warning in predAge(cpgs.imp, coefSkin, intercept = TRUE, min.perc): The number of missing CpGs forSkinclock exceeds 80%.
---> This DNAm clock will be NA.
Warning in predAge(cpgs.imp, coefPedBE, intercept = TRUE, min.perc): The number of missing CpGs forPedBEclock exceeds 80%.
---> This DNAm clock will be NA.
Warning in predAge(cpgs.imp, coefTL, intercept = TRUE, min.perc): The number of missing CpGs forTLclock exceeds 80%.
---> This DNAm clock will be NA.
Warning in predAge(cpgs.imp, coefBLUP, intercept = TRUE, min.perc): The number of missing CpGs forBLUPclock exceeds 80%.
---> This DNAm clock will be NA.
Warning in predAge(cpgs.imp, coefEN, intercept = TRUE, min.perc): The number of missing CpGs forENclock exceeds 80%.
---> This DNAm clock will be NA.
age.gse58045
# A tibble: 172 × 24
id Horvath ageAcc.Horvath ageAcc2.Horvath ageAcc3.Horvath Hannum Levine
<chr> <dbl> <dbl> <dbl> <dbl> <lgl> <dbl>
1 GSM1399… 65.6 1.07 4.58 5.46 NA 50.7
2 GSM1399… 66.3 0.197 4.06 5.06 NA 51.3
3 GSM1399… 53.9 -5.31 -2.98 -2.42 NA 40.5
4 GSM1399… 40.6 -5.23 -5.89 -6.14 NA 31.3
5 GSM1399… 50.1 0.982 1.06 1.28 NA 41.1
6 GSM1399… 63.7 -0.895 2.64 2.92 NA 48.1
7 GSM1399… 44.7 -0.875 -1.59 -1.76 NA 29.2
8 GSM1399… 59.7 -8.55 -4.20 -3.48 NA 41.0
9 GSM1399… 48.4 -5.84 -4.63 -2.50 NA 43.8
10 GSM1399… 59.3 -3.93 -0.719 -0.609 NA 46.1
# ℹ 162 more rows
# ℹ 17 more variables: ageAcc.Levine <dbl>, ageAcc2.Levine <dbl>,
# ageAcc3.Levine <dbl>, BNN <dbl>, ageAcc.BNN <dbl>, ageAcc2.BNN <dbl>,
# ageAcc3.BNN <dbl>, skinHorvath <lgl>, PedBE <lgl>, Wu <dbl>,
# ageAcc.Wu <dbl>, ageAcc2.Wu <dbl>, ageAcc3.Wu <dbl>, TL <lgl>, BLUP <lgl>,
# EN <lgl>, age <dbl>
Figure shows the correlation between DNAmAge obtained from Horvath’s method and the chronological age, while Figure depicts the correlation of a new method based on fitting a Bayesian Neural Network to predict DNAmAge based on Horvath’s CpGs.
plotDNAmAge(age.gse58045$Horvath, age)
plotDNAmAge(age.gse58045$BNN, age, tit="Bayesian Neural Network")
Let us illustrate how to use DNAmAge information in association studies (e.g case/control, smokers/non-smokers, responders/non-responders, …). GEO number GSE19711 contains transcriptomic and epigenomic data of a study in lung cancer. Data can be retrieved into R by
# To avoid connection buffer size
Sys.setenv("VROOM_CONNECTION_SIZE" = 131072 * 10)
# Download data
dd <- GEOquery::getGEO("GSE19711")
gse19711 <- dd[[1]]
# Restore connection buffer size
Sys.setenv("VROOM_CONNECTION_SIZE" = 131072)
The object gse19711
is an ExpressionSet
that can contains CpGs and
phenotypic (e.g clinical) information
gse19711
ExpressionSet (storageMode: lockedEnvironment)
assayData: 27578 features, 540 samples
element names: exprs
protocolData: none
phenoData
sampleNames: GSM491937 GSM491938 ... GSM492476 (540 total)
varLabels: title geo_accession ... stage:ch1 (58 total)
varMetadata: labelDescription
featureData
featureNames: cg00000292 cg00002426 ... cg27665659 (27578 total)
fvarLabels: ID Name ... ORF (38 total)
fvarMetadata: Column Description labelDescription
experimentData: use 'experimentData(object)'
pubMedIds: 20219944
Annotation: GPL8490
Let us imagine we are interested in comparing the accelerated age between cases and controls. Age and case/control status information can be obtained by:
pheno <- pData(gse19711)
age <- as.numeric(pheno$`ageatrecruitment:ch1`)
disease <- pheno$`sample type:ch1`
table(disease)
disease
bi-sulphite converted genomic whole blood DNA from Case
266
bi-sulphite converted genomic whole blood DNA from Control
274
disease[grep("Control", disease)] <- "Control"
disease[grep("Case", disease)] <- "Case"
disease <- factor(disease, levels=c("Control", "Case"))
table(disease)
disease
Control Case
274 266
The DNAmAge estimates of different methods is computed by
age.gse19711 <- DNAmAge(gse19711, age=age)
Imputing missing data of the entire matrix ....
Data imputed. Starting DNAm clock estimation ...
Warning in predAge(cpgs.imp, coefHannum, intercept = FALSE, min.perc): The number of missing CpGs forHannumclock exceeds 80%.
---> This DNAm clock will be NA.
rows : 353 cols : 540
Warning in predAge(cpgs.imp, coefSkin, intercept = TRUE, min.perc): The number of missing CpGs forSkinclock exceeds 80%.
---> This DNAm clock will be NA.
Warning in predAge(cpgs.imp, coefPedBE, intercept = TRUE, min.perc): The number of missing CpGs forPedBEclock exceeds 80%.
---> This DNAm clock will be NA.
Warning in predAge(cpgs.imp, coefTL, intercept = TRUE, min.perc): The number of missing CpGs forTLclock exceeds 80%.
---> This DNAm clock will be NA.
Warning in predAge(cpgs.imp, coefBLUP, intercept = TRUE, min.perc): The number of missing CpGs forBLUPclock exceeds 80%.
---> This DNAm clock will be NA.
Warning in predAge(cpgs.imp, coefEN, intercept = TRUE, min.perc): The number of missing CpGs forENclock exceeds 80%.
---> This DNAm clock will be NA.
We can observe there are missing data. The funcion automatically impute those
using impute.knn
function from impute
package since complete cases are
required to compute the different methylation clocks. The estimates are:
age.gse19711
# A tibble: 540 × 24
id Horvath ageAcc.Horvath ageAcc2.Horvath ageAcc3.Horvath Hannum Levine
<chr> <dbl> <dbl> <dbl> <dbl> <lgl> <dbl>
1 GSM4919… 62.9 -5.14 -0.351 -1.10 NA 61.1
2 GSM4919… 68.8 -12.2 -2.85 -2.13 NA 57.0
3 GSM4919… 60.0 3.96 4.54 4.37 NA 43.0
4 GSM4919… 57.9 -4.13 -1.45 -1.38 NA 40.9
5 GSM4919… 59.0 -13.0 -6.79 -6.98 NA 57.0
6 GSM4919… 57.0 -4.00 -1.66 -1.09 NA 44.7
7 GSM4919… 61.9 -3.08 0.657 0.183 NA 47.9
8 GSM4919… 59.1 -11.9 -6.07 -5.53 NA 50.0
9 GSM4919… 60.7 -16.3 -8.33 -9.33 NA 47.7
10 GSM4919… 51.1 -7.93 -6.30 -6.33 NA 52.5
# ℹ 530 more rows
# ℹ 17 more variables: ageAcc.Levine <dbl>, ageAcc2.Levine <dbl>,
# ageAcc3.Levine <dbl>, BNN <dbl>, ageAcc.BNN <dbl>, ageAcc2.BNN <dbl>,
# ageAcc3.BNN <dbl>, skinHorvath <lgl>, PedBE <lgl>, Wu <dbl>,
# ageAcc.Wu <dbl>, ageAcc2.Wu <dbl>, ageAcc3.Wu <dbl>, TL <lgl>, BLUP <lgl>,
# EN <lgl>, age <dbl>
The association between disease status and DNAmAge estimated using Horvath’s method can be computed by
mod.horvath1 <- glm(disease ~ ageAcc.Horvath ,
data=age.gse19711,
family="binomial")
summary(mod.horvath1)
Call:
glm(formula = disease ~ ageAcc.Horvath, family = "binomial",
data = age.gse19711)
Coefficients:
Estimate Std. Error z value Pr(>|z|)
(Intercept) -0.10995 0.09771 -1.125 0.2605
ageAcc.Horvath -0.02023 0.01154 -1.753 0.0795 .
---
Signif. codes: 0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1
(Dispersion parameter for binomial family taken to be 1)
Null deviance: 748.48 on 539 degrees of freedom
Residual deviance: 745.25 on 538 degrees of freedom
AIC: 749.25
Number of Fisher Scoring iterations: 4
mod.skinHorvath <- glm(disease ~ ageAcc2.Horvath ,
data=age.gse19711,
family="binomial")
summary(mod.skinHorvath)
Call:
glm(formula = disease ~ ageAcc2.Horvath, family = "binomial",
data = age.gse19711)
Coefficients:
Estimate Std. Error z value Pr(>|z|)
(Intercept) -0.02970 0.08617 -0.345 0.730
ageAcc2.Horvath -0.01315 0.01209 -1.087 0.277
(Dispersion parameter for binomial family taken to be 1)
Null deviance: 748.48 on 539 degrees of freedom
Residual deviance: 747.27 on 538 degrees of freedom
AIC: 751.27
Number of Fisher Scoring iterations: 3
mod.horvath3 <- glm(disease ~ ageAcc3.Horvath ,
data=age.gse19711,
family="binomial")
summary(mod.horvath3)
Call:
glm(formula = disease ~ ageAcc3.Horvath, family = "binomial",
data = age.gse19711)
Coefficients:
Estimate Std. Error z value Pr(>|z|)
(Intercept) -0.02993 0.08626 -0.347 0.729
ageAcc3.Horvath -0.01927 0.01283 -1.502 0.133
(Dispersion parameter for binomial family taken to be 1)
Null deviance: 748.48 on 539 degrees of freedom
Residual deviance: 746.13 on 538 degrees of freedom
AIC: 750.13
Number of Fisher Scoring iterations: 4
We do not observe statistical significant association between age acceleration estimated using Horvath method and the risk of developing lung cancer. It is worth to notice that Horvath’s clock was created to predict chronological age and the impact of age acceleration of this clock on disease may be limited. On the other hand, Levine’s clock aimed to distinguish risk between same-aged individuals. Let us evaluate whether this age acceleration usin Levine’s clock is associated with lung cancer
mod.levine1 <- glm(disease ~ ageAcc.Levine , data=age.gse19711,
family="binomial")
summary(mod.levine1)
Call:
glm(formula = disease ~ ageAcc.Levine, family = "binomial", data = age.gse19711)
Coefficients:
Estimate Std. Error z value Pr(>|z|)
(Intercept) 0.40956 0.17894 2.289 0.02209 *
ageAcc.Levine 0.03178 0.01133 2.806 0.00502 **
---
Signif. codes: 0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1
(Dispersion parameter for binomial family taken to be 1)
Null deviance: 748.48 on 539 degrees of freedom
Residual deviance: 740.17 on 538 degrees of freedom
AIC: 744.17
Number of Fisher Scoring iterations: 4
mod.levine2 <- glm(disease ~ ageAcc2.Levine , data=age.gse19711,
family="binomial")
summary(mod.levine2)
Call:
glm(formula = disease ~ ageAcc2.Levine, family = "binomial",
data = age.gse19711)
Coefficients:
Estimate Std. Error z value Pr(>|z|)
(Intercept) -0.02925 0.08718 -0.336 0.737225
ageAcc2.Levine 0.04430 0.01234 3.589 0.000332 ***
---
Signif. codes: 0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1
(Dispersion parameter for binomial family taken to be 1)
Null deviance: 748.48 on 539 degrees of freedom
Residual deviance: 734.49 on 538 degrees of freedom
AIC: 738.49
Number of Fisher Scoring iterations: 4
mod.levine3 <- glm(disease ~ ageAcc3.Levine , data=age.gse19711,
family="binomial")
summary(mod.levine3)
Call:
glm(formula = disease ~ ageAcc3.Levine, family = "binomial",
data = age.gse19711)
Coefficients:
Estimate Std. Error z value Pr(>|z|)
(Intercept) -0.02962 0.08622 -0.344 0.731
ageAcc3.Levine 0.01679 0.01244 1.350 0.177
(Dispersion parameter for binomial family taken to be 1)
Null deviance: 748.48 on 539 degrees of freedom
Residual deviance: 746.62 on 538 degrees of freedom
AIC: 750.62
Number of Fisher Scoring iterations: 3
Here we observe as the risk of developing lung cancer increases
3.23 percent per each unit in the
age accelerated variable (ageAcc
). Similar conclusion is obtained when using
ageAcc2
and ageAcc3
variables.
In some occasions cell composition should be used to assess association. This
information is calculated in DNAmAge
function and it can be incorporated in
the model by:
cell <- attr(age.gse19711, "cell_proportion")
mod.cell <- glm(disease ~ ageAcc.Levine + cell, data=age.gse19711,
family="binomial")
summary(mod.cell)
Call:
glm(formula = disease ~ ageAcc.Levine + cell, family = "binomial",
data = age.gse19711)
Coefficients:
Estimate Std. Error z value Pr(>|z|)
(Intercept) -9.768206 4.380382 -2.230 0.025748 *
ageAcc.Levine 0.003959 0.012208 0.324 0.745746
cellCD4T -3.339693 3.833531 -0.871 0.383656
cellMono 10.165096 4.594096 2.213 0.026922 *
cellNeu 16.319534 4.584745 3.560 0.000372 ***
cellNK -0.882134 4.296498 -0.205 0.837326
---
Signif. codes: 0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1
(Dispersion parameter for binomial family taken to be 1)
Null deviance: 748.48 on 539 degrees of freedom
Residual deviance: 686.56 on 534 degrees of freedom
AIC: 698.56
Number of Fisher Scoring iterations: 4
Here we observe as the positive association disapears after adjusting for cell counts.
dd <- GEOquery::getGEO("GSE109446")
gse109446 <- dd[[1]]
controls <- pData(gse109446)$`diagnosis:ch1`=="control"
gse <- gse109446[,controls]
age <- as.numeric(pData(gse)$`age:ch1`)
age.gse <- DNAmAge(gse, age=age)
rows : 353 cols : 29
plotCorClocks(age.gse)
Let us start by reproducing the example provided in Knight et al. (2016) as
a test data set (file ‘TestDataset.csv’). It consists on 3 individuals whose
methylation data are available as supplementary data of their paper. The data
is also available at methylclock
package as a data frame.
TestDataset[1:5,]
CpGName Sample1 Sample2 Sample3
1 cg00000292 0.72546496 0.72350947 0.69023377
2 cg00002426 0.85091763 0.80077888 0.80385777
3 cg00003994 0.05125853 0.05943935 0.05559333
4 cg00005847 0.08775420 0.11722333 0.10845113
5 cg00006414 0.03982478 0.06146891 0.03491992
The Gestational Age (in months) is simply computed by
ga.test <- DNAmGA(TestDataset)
Warning in DNAmGA(TestDataset): CpGs in all Gestational Age clocks are not present in your
data. Try 'checkClocksGA' function to find the missing CpGs of
each method.
Warning in predAge(cpgs.imp, coefBohlin, intercept = TRUE, min.perc): The number of missing CpGs forBohlinclock exceeds 80%.
---> This DNAm clock will be NA.
Warning in predAge(cpgs.imp, coefEPIC, intercept = TRUE, min.perc): The number of missing CpGs forEPICclock exceeds 80%.
---> This DNAm clock will be NA.
Warning in DNAmGA(TestDataset): The number of missing CpGs for Lee clocks exceeds 80%.
---> This DNAm clock will be NA.
ga.test
# A tibble: 3 × 6
id Knight Bohlin Mayne EPIC Lee
<chr> <dbl> <dbl> <dbl> <lgl> <lgl>
1 Sample1 38.2 NA 35.8 NA NA
2 Sample2 38.8 NA 36.5 NA NA
3 Sample3 40.0 NA 36.6 NA NA
like in DNAmAge we can use the parameter min.perc
to set the minimum missing
percentage.
The results are the same as those described in the additional file 7 of Knight et al. (2016) (link [here] (https://static-content.springer.com/esm/art%3A10.1186%2Fs13059-016-1068-z/MediaObjects/13059_2016_1068_MOESM7_ESM.docx))
Let us continue by illustrating how to compute GA of real examples.
The PROGRESS cohort data is available in the additional file 8 of
Knight et al. (2016). It is available at methylclock
as a tibble
:
data(progress_data)
This file also contains different variables that are available in this
tibble
.
data(progress_vars)
The Clinical Variables including clinical assesment of gestational age
(EGA) are available at this tibble
.
The Gestational Age (in months) is simply computed by
ga.progress <- DNAmGA(progress_data)
Warning in DNAmGA(progress_data): CpGs in all Gestational Age clocks are not present in your
data. Try 'checkClocksGA' function to find the missing CpGs of
each method.
Warning in predAge(cpgs.imp, coefBohlin, intercept = TRUE, min.perc): The number of missing CpGs forBohlinclock exceeds 80%.
---> This DNAm clock will be NA.
Warning in predAge(cpgs.imp, coefMayneGA, intercept = TRUE, min.perc): The number of missing CpGs forMayneclock exceeds 80%.
---> This DNAm clock will be NA.
Warning in predAge(cpgs.imp, coefEPIC, intercept = TRUE, min.perc): The number of missing CpGs forEPICclock exceeds 80%.
---> This DNAm clock will be NA.
Warning in DNAmGA(progress_data): The number of missing CpGs for Lee clocks exceeds 80%.
---> This DNAm clock will be NA.
ga.progress
# A tibble: 150 × 6
id Knight Bohlin Mayne EPIC Lee
<chr> <dbl> <dbl> <lgl> <lgl> <lgl>
1 784 38.8 NA NA NA NA
2 1052 37.2 NA NA NA NA
3 1048 40.3 NA NA NA NA
4 1017 39.2 NA NA NA NA
5 956 38.9 NA NA NA NA
6 1038 39.2 NA NA NA NA
7 989 37.2 NA NA NA NA
8 946 35.4 NA NA NA NA
9 941 33.5 NA NA NA NA
10 1024 37.4 NA NA NA NA
# ℹ 140 more rows
We can compare these results with the clinical GA available in the variable EGA
plotDNAmAge(ga.progress$Knight, progress_vars$EGA,
tit="GA Knight's method",
clock="GA")