\documentclass{article}
\usepackage{url}
\usepackage{hyperref}
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%\VignetteIndexEntry{DMR calling from EPICv2 arrays}
%\VignetteEngine{knitr::knitr}
\begin{document}
\title{\texttt{DMRcate} for EPICv2}

\author{Peters TJ}
\maketitle


\renewcommand{\abstractname}{Summary}
\begin{abstract}
Worked example to find DMRs from EPICv2 arrays between B cell ALL and T cell ALL samples.
\end{abstract}

<<bioconductor, message=FALSE, warning=FALSE, eval=FALSE>>=
if (!require("BiocManager"))
	install.packages("BiocManager")
BiocManager::install("DMRcate")
@


The Illumina Infinium HumanMethylationEPIC v2.0 BeadChip (EPICv2) extends genomic coverage to more than
920,000 CpG sites. One of the new characteristics of this array compared to previous versions is that
it contains instances of multiple probes (or ``replicates'') that map to the same CpG site. This is
potentially useful for performance testing of probes, but for DMRcate we need a 1-to-1 mapping of probe 
to CpG site, otherwise the kernel will be biased towards those sites with more replicates. This vignette
will show how to pare down an EPICv2 dataset to a suitable format for DMR calling. We use the AnnotationHub package EPICv2manifest\cite{Peters2024} as a backend for this purpose.

<<libr, message=FALSE, warning=FALSE>>=
library(DMRcate)
@

We use a subset of samples kindly taken from Noguera-Castells \emph{et al.} (2023)\cite{Noguera}, for finding DMRs between T cell acute lymphoblastic leukaemia (TALL) and B cell acute lymphoblastic leukaemia (BALL). We load the beta 
matrix from ExperimentHub:

<<libreh, message=FALSE, warning=FALSE>>=
library(ExperimentHub)
eh <- ExperimentHub()
ALLbetas <- eh[["EH9451"]]
head(ALLbetas)
@


There are 5 TALL samples and 5 BALL samples, which, at a glance, may have distinct methylation profiles. 

<<density>>=
plot(density(ALLbetas[,1]), col="forestgreen", xlab="Beta value", ylim=c(0, 6), 
     main="Noguera-Castells et al. 2023 ALL samples", lwd=2)
invisible(sapply(2:5, function (x) lines(density(ALLbetas[,x]), col="forestgreen", lwd=2)))
invisible(sapply(6:10, function (x) lines(density(ALLbetas[,x]), col="orange", lwd=2)))
legend("topleft", c("B cell ALL", "T cell ALL"), text.col=c("forestgreen", "orange"))
@


Now let's \textrm{logit}2 transform the betas into \textit{M}-values, which approximate normality for linear modelling.

<<Ms>>=
ALLMs <- minfi::logit2(ALLbetas)
@


Just like with the previous EPIC array, there are subsets of probes whose target is near a SNP, and also subsets for which there is evidence of cross-reactivity with other parts of the genome\cite{Peters2024}, both which can be filtered out with with \texttt{rmSNPandCH()}. We will filter out the SNP-proximal probes, but for this vignette, we will retain the EPICv2 probes for which there is evidence for preferential binding to off-targets, using \texttt{rmcrosshyb=FALSE}, since these off-targets have also been mapped\cite{Peters2024}.


<<rmSNPandCH>>=
nrow(ALLMs)
ALLMs.noSNPs <- rmSNPandCH(ALLMs, rmcrosshyb = FALSE)
nrow(ALLMs.noSNPs)
@


EPICv2 data contains groups of probes we call ``replicates'' that map to the same CpG site. Since DMRcate requires a 1-to-1 relationship between rows of methylation measurements and CpG loci, lest the kernel become biased towards sites with multiple mappings, we need to attenuate our \textit{M}-value matrix to maintain this relationship.  \texttt{cpg.annotate()} now does this when \texttt{arraytype = "EPICv2"}, with user options specified by the argument \texttt{epicv2Filter}. The default is to take the \texttt{mean} of each replicate group (with row name represented by the first probe in each group that appears in the manifest). However, we can also select one probe from each replicate group based on its performance from cross-platform comparisons with EPICv1 and WGBS data (see Peters \textit{et al.} 2024)\cite{Peters2024}. Probe robustness is measured in terms of either: 

\begin{itemize}

\item its sensitivity to methylation change and measurement precision, relative to a platform consensus obtained from matched EPICv1 and whole genome bisulphite sequencing (WGBS) values\cite{Peters2019}.

\item minimum root mean squared error (RMSE) with WGBS (mainly for probes that don't appear on EPICv1)

\end{itemize}

In some cases, a replicated probe will prove superior in both sensitivity and precision, however in other cases superior sensitivity and precision is split between two probes in a replicate group. Alternatively, an individual probe may have superior sensitivity in a group, but superior precision is incurred by the group mean. Hence we give the user the option of choosing \texttt{sensitivity} or \texttt{precision}, based on their appetite for potential Type I error. In cases where there is insufficient evidence for superiority, a replicate probe is taken at random from the group. This strategy is generalised to all replicate groups for the \texttt{random} option. Figure 1 shows the differences in probe selection based on the \texttt{epicv2Filter} argument. In practice, the number of differentially methylated CpGs (and hence DMRs) is unlikely to vary greatly depending on\texttt{epicv2Filter}, but we provide this optionality to make use of all available probes on the array, and to potentially update the manifest with more rigorous probe benchmarking as usage proliferates.


\begin{figure}[htbp!]
  \caption{Variation in probe selection based on the \texttt{epicv2Filter} argument from \texttt{cpg.annotate()}. Values in the ``Random'' group may differ when reproduced.
}
  \centering
    \includegraphics[width=\textwidth]{ProbeVenn.pdf}
\end{figure}

The main event, however, is calling DMRs. Let's take our replicate-averaged \textit{M}-value matrix and specify our hypothesis to call DMRs between T cell and B cell ALL samples:

<<hypothesis>>=
type <- gsub("_.*", "", colnames(ALLMs.noSNPs))
type
design <- model.matrix(~type)
design
@


Now for \texttt{cpg.annotate()}. In addition to \texttt{epicv2Filter}, we have also added an extra logical argument \texttt{epicv2Remap} giving the user the option of reassigning probes to an off-target CpG site where there is evidence (based on both \textit{in silico} alignment and concordance with WGBS data) that the probe is preferentially hybridising to that site. As stated previously, this will only apply when you have specified \texttt{rmcrosshyb=FALSE} in \texttt{rmSNPandCH()}. 

<<cpg.annotate>>=
myannotation <- cpg.annotate("array", object=ALLMs.noSNPs, what = "M", 
                             arraytype = "EPICv2", epicv2Filter = "mean", 
                             epicv2Remap = TRUE,  analysis.type="differential", 
                             design=design, coef=2, fdr=0.001)
@
                             
And then, as usual, call DMRs and annotate them. Unlike previous arrays, the EPICv2 manifest is in hg38:

<<dmrcate>>=
dmrcoutput <- dmrcate(myannotation, lambda=1000, C=2)
results.ranges <- extractRanges(dmrcoutput, genome = "hg38")
results.ranges
@

And finally, plotting one of the top DMRs. 

<<plot>>=
groups <- c(BALL="forestgreen", TALL="orange")
cols <- groups[as.character(type)]
DMR.plot(ranges=results.ranges, dmr=3, CpGs=myannotation, 
         what = "Beta", arraytype = "EPICv2", phen.col=cols, genome = "hg38") 
@
 
<<sessionInfo>>=       
sessionInfo()
@


\begin{thebibliography}{99}

\bibitem{Peters2024}
  Peters, T.J., Meyer, B., Ryan, L., Achinger-Kawecka, J., Song, J., 
  Campbell, E.M., Qu, W., Nair, S., Loi-Luu, P., Stricker, P., Lim, E., 
  Stirzaker, C., Clark, S.J. and Pidsley, (2024). Characterisation and 
  reproducibility of the HumanMethylationEPIC v2.0 BeadChip for DNA methylation 
  profiling. \emph{BMC Genomics} 25, 251. 
  doi:10.1186/s12864-024-10027-5.


\bibitem{Noguera}
  Noguera-Castells A, Garcia-Prieto CA, Alvarez-Errico D, Esteller, M. 
  (2023). Validation of the new EPIC DNA methylation microarray (900K EPIC v2) 
  for high-throughput profiling of the human DNA methylome. \emph{Epigenetics}
  18(1), 2185742.

\bibitem{Peters2019}
  Peters TJ, French HJ, Bradford ST, Pidsley R, Stirzaker C, Varinli H, 
  Nair, S, Qu W, Song J, Giles KA, Statham AL, Speirs H, Speed TP, Clark, SJ. 
  (2019). Evaluation of cross-platform and interlaboratory concordance via 
  consensus modelling of genomic measurements. \emph{Bioinformatics}, 2019 
  35(4), 560-570.



\end{thebibliography}


\end{document}
