library(ELMER.data) library(ELMER) library(DT) library(dplyr) library(BiocStyle)
This step is to identify enriched motif in a set of probes which is carried out by
function get.enriched.motif
.
In order to identify enriched motifs and potential upstream regulatory TFs, all probes with occurring in significant probe-gene pairs are combined for motif enrichment analysis. HOMER (Hypergeometric Optimization of Motif EnRichment) [@heinz2010simple] is used to find motif occurrences in a $\pm 250bp$ region around each probe, using HOCOMOCO (HOmo sapiens COmprehensive MOdel COllection) v11 [@kulakovskiy2016hocomoco]. Transcription factor (TF) binding models are available at http://hocomoco.autosome.ru/downloads. HOCOMOCO is the most comprehensive TFBS database and is consistently updated, marking an improvement over ELMER version 1.
For each probe set tested (i.e. the set of all probes occurring in significant probe-gene pairs), we quantify enrichments using Fisher's exact test (where $a$ is the number of probes within the selected probe set that contains one or more motif occurrences; $b$ is the number of probes within the selected probe set that do not contain a motif occurrence; $c$ and $d$ are the same counts within the entire array probe set drawn from the same set of distal-only probes using the same definition as the primary analysis) and multiple testing correction with the Benjamini-Hochberg procedure [@fisher].
A probe set was considered significantly enriched
for a particular motif if the 95\% confidence interval of the Odds Ratio was greater than $1.1$ (specified by option lower.OR
, $1.1$ is default), the motif
occurred at least 10 times (specified by option min.incidence
, $10$ is default) in
the probe set and $FDR < 0.05$.
# Load results from previous sections mae <- get(load("mae.rda")) sig.diff <- read.csv("result/getMethdiff.hypo.probes.significant.csv") pair <- read.csv("result/getPair.hypo.pairs.significant.csv") head(pair) # significantly hypomethylated probes with putative target genes # Identify enriched motif for significantly hypomethylated probes which # have putative target genes. enriched.motif <- get.enriched.motif(data = mae, probes = pair$Probe, dir.out = "result", label = "hypo", min.incidence = 10, lower.OR = 1.1)
names(enriched.motif) # enriched motifs head(enriched.motif[names(enriched.motif)[1]]) ## probes in the given set that have the first motif. # get.enriched.motif automatically save output files. # getMotif.hypo.enriched.motifs.rda contains enriched motifs and the probes with the motif. # getMotif.hypo.motif.enrichment.csv contains summary of enriched motifs. dir(path = "result", pattern = "getMotif") # motif enrichment figure will be automatically generated. dir(path = "result", pattern = "motif.enrichment.pdf")
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