Fast, accurate, epiallele-aware
methylation caller and reporter

Introduction

epialleleR is an R package for calling and reporting cytosine DNA methylation. Developed to help identify and quantify epimutations (aberrant DNA methylation events), it has now acquired multiple additional functions to dissect DNA methylation in many useful ways. But the main feature of the package is to report frequencies of epimutations (variant epiallele frequencies, VEF) at the level of genomic regions or individual cytosines. All you need in order to use it, is a binary alignment map (BAM) file from basically any next-generation (methylation or native) sequencing experiment.

Features

very fast!
reference-free
designed with epimutation studies in mind
probably, the most accurate tool for conventional cytosine methylation reporting

For details, see the related publication and vignette.

Input Data

short-read and long-read (native)
paired-end and single-end
whole-genome, genome-wide (e.g., hybridization capture or adaptive sampling), and narrowly targeted (e.g., amplicon panels)

Capabilities

call cytosine methylation and save calls in a new BAM file (callMethylation)
create sample BAM files from scratch given mandatory and optional BAM fields (simulateBam)
create conventional reports of cytosine methylation (generateCytosineReport)
evaluate epimutation frequencies both at the level of genomic regions (generate[Bed|Amplicon|Capture]Report) and individual cytosines (generateCytosineReport)
evaluate linearised Methylated Haplotype Load (lMHL,
generateMhlReport*)
extract methylation patterns for genomic region of interest (extractPatterns)
visualise methylation patterns (plotPatterns)
test for the association between epiallele methylation status and sequence variations (generateVcfReport)
assess the distribution of per-read beta values for genomic regions of interest (generateBedEcdf)

Recent improvements

v1.20 [BioC 3.23]

using genomic coordinates of targets, only a subset of BAM reads or only fragments of BAM reads that are overlapping the targets can now be loaded
disrupting changes in all generate*Report functions (from version 1.19.1 onwards):
- cytosine.context parameter instead of threshold.context/haplotype.context
- new parameter filter.reads regulates filtering of reads with too few cytosines or presumable incomplete conversion of cytosines
- reads with too few within-the-context cytosines (less than min.context.sites) or out-of-context cytosine methylation higher than max.outofcontext.beta are filtered out (discarded) instead of being counted as hypomethylated reads (as was in v1.19.0 and earlier)
- new default value of 0 for min.context.sites

v1.14 [BioC 3.20]

creates pretty plots of methylation patterns

v1.12 [BioC 3.19]

inputs long-read sequencing alignments
full support for short-read sequencing alignments by Illumina DRAGEN, Bismark, bwa-meth, BSMAP
RRBS-specific options
lower memory usage

v1.10 [BioC 3.18]

inputs both single-end and paired-end sequencing alignments
makes and stores methylation calls
creates sample BAM files
reports linearised MHL

v1.4 [BioC 3.15]

significant speed-up
method to extract and visualize methylation patterns

v1.2 [BioC 3.14]

even faster and more memory-efficient BAM loading (by means of HTSlib)
min.baseq parameter to reduce the effect of low quality bases on methylation or SNV calling (in v1.0 the output of generateVcfReport was equivalent to the one of samtools mpileup -Q 0 ...)

check out NEWS for more!

Installation

install via Bioconductor

if (!requireNamespace("BiocManager", quietly = TRUE))
    install.packages("BiocManager")

BiocManager::install("epialleleR")

Install the latest version via install_github

library(devtools)
install_github("BBCG/epialleleR", build_vignettes=FALSE,
  repos=BiocManager::repositories(),
  dependencies=TRUE, type="source")

Using the package

Please read epialleleR vignette at GitHub pages or within the R environment: vignette("epialleleR", package="epialleleR"), or consult the function’s help pages for the extensive information on usage, parameters and output values.

Comparison of beta, VEF and lMHL values for various use cases is given by the values vignette (vignette("values", package="epialleleR"))

Very brief synopsis:

library(epialleleR)

# make methylation calls if necessary
callMethylation(
  input.bam.file=system.file("extdata", "test", "dragen-se-unsort-xg.bam", package="epialleleR"),
  output.bam.file=tempfile(pattern="output-", fileext=".bam"),
  genome=system.file("extdata", "test", "reference.fasta.gz", package="epialleleR")
)

# make a sample BAM file from scratch
simulateBam(output.bam.file=tempfile(pattern="simulated-", fileext=".bam"),
            pos=c(1, 2), XM=c("ZZZzzZZZ", "ZZzzzzZZ"), XG=c("CT", "AG"))

# or use external files
amplicon.bam <- system.file("extdata", "amplicon010meth.bam",
                            package="epialleleR")
amplicon.bed <- system.file("extdata", "amplicon.bed", package="epialleleR")
amplicon.vcf <- system.file("extdata", "amplicon.vcf.gz", package="epialleleR")

# preload the data
bam.data <- preprocessBam(amplicon.bam)

# methylation patterns and their plot
patterns <- extractPatterns(bam=amplicon.bam, bed=amplicon.bed, bed.row=3)
plotPatterns(patterns)

# conventional cytosine report
cx.report <- generateCytosineReport(bam.data, filter.reads=FALSE,
                                    threshold.reads=FALSE, report.context="CX")

# CpG VEF report for individual bases
cg.vef.report <- generateCytosineReport(bam.data)

# BED-guided VEF report for genomic ranges
bed.report <- generateBedReport(bam=amplicon.bam, bed=amplicon.bed,
                                bed.type="capture")

# VCF report
vcf.report <- generateVcfReport(bam=amplicon.bam, bed=amplicon.bed,
                                vcf=amplicon.vcf, vcf.style="NCBI")

# lMHL report
mhl.report <- generateMhlReport(bam=amplicon.bam)

Citing the `epialleleR` package

Oleksii Nikolaienko, Per Eystein Lønning, Stian Knappskog, epialleleR: an R/Bioconductor package for sensitive allele-specific methylation analysis in NGS data. GigaScience, Volume 12, 2023, giad087, https://doi.org/10.1093/gigascience/giad087. Data: GSE201690

Our experimental studies that use the package

Per Eystein Lonning, Oleksii Nikolaienko, Kathy Pan, Allison W. Kurian, Hans Petter Petter Eikesdal, Mary Pettinger, Garnet L Anderson, Ross L Prentice, Rowan T. Chlebowski, and Stian Knappskog. Constitutional BRCA1 methylation and risk of incident triple-negative breast cancer and high-grade serous ovarian cancer. JAMA Oncology 2022. https://doi.org/10.1001/jamaoncol.2022.3846

Oleksii Nikolaienko, Hans P. Eikesdal, Elisabet Ognedal, Bjørnar Gilje, Steinar Lundgren, Egil S. Blix, Helge Espelid, Jürgen Geisler, Stephanie Geisler, Emiel A.M. Janssen, Synnøve Yndestad, Laura Minsaas, Beryl Leirvaag, Reidun Lillestøl, Stian Knappskog, Per E. Lønning. Prenatal BRCA1 epimutations contribute significantly to triple-negative breast cancer development. Genome Medicine 2023. https://doi.org/10.1186/s13073-023-01262-8. Data: GSE243966

Oleksii Nikolaienko, Garnet L Anderson, Rowan T Chlebowski, Su Yon Jung, Holly R Harris, Stian Knappskog, and Per E Lønning. MGMT epimutations and risk of incident cancer of the colon, glioblastoma multiforme, and diffuse large B-cell lymphomas. Clinical Epigenetics 2025. https://doi.org/10.1186/s13148-025-01835-x

`epialleleR` at Bioconductor

release, development version

License

Artistic License 2.0

Fast, accurate, epiallele-aware methylation caller and reporter