Methrix tutorial
Introduction
Methrix provides set of function which allows easy
importing of various flavors of bedgraphs generated by methylation
callers, and many downstream analysis to be performed on large
matrices.
This vignette describes basic usage of the package intended to process several large bedgraph files in R. In addition, a detailed exemplary complete data analysis with steps from reading in to annotation and differential methylation calling can be found in our WGBS best practices workflow
Overview and usage functions of the package
Installation
if (!requireNamespace("BiocManager", quietly = TRUE))
install.packages("BiocManager")
#Installing stable version from BioConductor
BiocManager::install("methrix")
#Installing developmental version from GitHub
BiocManager::install("CompEpigen/methrix")NOTE
Installation from BioConductor requires the BioC and R versions to be the newest. This arises from the restrictions imposed by BioConductor community which might cause package incompatibilities with the earlier versions of R (for e.g; R < 4.0). In that case installing from GitHub might be easier since it is much more merciful with regards to versions.
Reading bedgraph files
read_bedgraphs function is a versatile bedgraph reader
intended to import bedgraph files generated virtually by any sort of
methylation calling program. It requires user to provide indices for
chromosome names, start position and other required fields. There are
also presets available to import bedgraphs from most common
programs such as Bismark, MethylDackel, and
MethylcTools.
#Genome of your preference to work with
if (!requireNamespace("BiocManager", quietly = TRUE))
install.packages("BiocManager")
library(BiocManager)
if(!requireNamespace("BSgenome.Hsapiens.UCSC.hg19")) {
BiocManager::install("BSgenome.Hsapiens.UCSC.hg19")
}
library(BSgenome.Hsapiens.UCSC.hg19) #Example bedgraph files
bdg_files <- list.files(
path = system.file('extdata', package = 'methrix'),
pattern = "*bedGraph\\.gz$",
full.names = TRUE
)
print(basename(bdg_files))
#> [1] "C1.bedGraph.gz" "C2.bedGraph.gz" "N1.bedGraph.gz" "N2.bedGraph.gz"
#Generate some sample annotation table
sample_anno <- data.frame(
row.names = gsub(
pattern = "\\.bedGraph\\.gz$",
replacement = "",
x = basename(bdg_files)
),
Condition = c("cancer", 'cancer', "normal", "normal"),
Pair = c("pair1", "pair2", "pair1", "pair2"),
stringsAsFactors = FALSE
)
print(sample_anno)
#> Condition Pair
#> C1 cancer pair1
#> C2 cancer pair2
#> N1 normal pair1
#> N2 normal pair2We can import bedgraph files with the function
read_bedgraphs which reads in the bedgraphs, adds CpGs
missing from the reference set, and creates a methylation/coverage
matrices. Once the process is complete - it returns an object of class
methrix which in turn inherits SummarizedExperiment
class. methrix object contains ‘methylation’ and ‘coverage’
matrices (either in-memory or as on-disk HDF5 arrays) along with
pheno-data and other basic info. This object can be passed to all
downstream functions for various analysis.
#First extract genome wide CpGs from the desired reference genome
hg19_cpgs <- suppressWarnings(methrix::extract_CPGs(ref_genome = "BSgenome.Hsapiens.UCSC.hg19"))
#>
#> Attaching package: 'Biostrings'
#> The following object is masked from 'package:base':
#>
#> strsplit
#>
#> Attaching package: 'rtracklayer'
#> The following object is masked from 'package:BiocIO':
#>
#> FileForFormat
#> -Extracting CpGs
#> -Done. Extracted 28,217,448 CpGs from 25 contigs.#Read the files
meth <- methrix::read_bedgraphs(
files = bdg_files,
ref_cpgs = hg19_cpgs,
chr_idx = 1,
start_idx = 2,
M_idx = 3,
U_idx = 4,
stranded = FALSE,
zero_based = FALSE,
collapse_strands = FALSE,
coldata = sample_anno
)
#> ----------------------------
#> -Preset: Custom
#> --Missing beta and coverage info. Estimating them from M and U values
#> -CpGs raw: 28,217,448 (total reference CpGs)
#> -CpGs retained: 28,217,448(reference CpGs from contigs of interest)
#> ----------------------------
#> -Processing: C1.bedGraph.gz
#> --CpGs missing: 28,216,771 (from known reference CpGs)
#> -Processing: C2.bedGraph.gz
#> --CpGs missing: 28,216,759 (from known reference CpGs)
#> -Processing: N1.bedGraph.gz
#> --CpGs missing: 28,216,746 (from known reference CpGs)
#> -Processing: N2.bedGraph.gz
#> --CpGs missing: 28,216,747 (from known reference CpGs)
#> -Finished in: 41.8s elapsed (33.6s cpu)Note: Use the argument pipeline if your bedgraphs are
generated with “Bismark”, “MethylDeckal”, or “MethylcTools”. This will
automatically figure out the file formats for you, and you dont have to
use the arguments chr_idx start_idx and
so..
HTML QC report
Get basic summary statistics of the methrix object with
methrix_report function which produces an interactive html
report
Click here for an example report.
Filtering
Remove uncovered loci
Usual task in analysis involves removing uncovered CpGs. i.e, those
loci which are not covered across all sample (in other words covered
only in subset of samples resulting NA for rest of the
samples ).
Remove SNPs
One can also remove CpG sites overlaping with common SNPs based on minor allele frequencies.
if(!require(MafDb.1Kgenomes.phase3.hs37d5)) {
BiocManager::install("MafDb.1Kgenomes.phase3.hs37d5")}
if(!require(GenomicScores)) {
BiocManager::install("GenomicScores")} library(MafDb.1Kgenomes.phase3.hs37d5)
#> Loading required package: GenomicScores
#>
#> Attaching package: 'GenomicScores'
#> The following object is masked from 'package:utils':
#>
#> citation
#> Warning: replacing previous import 'utils::findMatches' by
#> 'S4Vectors::findMatches' when loading 'MafDb.1Kgenomes.phase3.hs37d5'
library(GenomicScores)
meth_snps_filtered <- methrix::remove_snps(m = meth)
#> Used SNP database: MafDb.1Kgenomes.phase3.hs37d5.
#> Number of SNPs removed:
#> chr N
#> <char> <int>
#> 1: chr21 42
#> 2: chr22 39
#> Sum:
#> [1] 81
#> -Finished in: 3.622s elapsed (2.114s cpu)Basic operations
Extract methylation/coverage matrices
#Example data bundled, same as the previously generated meth
data("methrix_data")
#Coverage matrix
coverage_mat <- methrix::get_matrix(m = methrix_data, type = "C")
head(coverage_mat)
#> C1 C2 N1 N2
#> [1,] 13 7 9 10
#> [2,] NA 2 3 NA
#> [3,] 9 10 3 5
#> [4,] 11 8 12 8
#> [5,] 6 7 17 8
#> [6,] 13 6 6 14#Methylation matrix
meth_mat <- methrix::get_matrix(m = methrix_data, type = "M")
head(meth_mat)
#> C1 C2 N1 N2
#> [1,] 0.1538462 0.2857143 0.5555556 0.3000000
#> [2,] NA 0.5000000 0.0000000 NA
#> [3,] 0.5555556 0.7000000 0.3333333 0.8000000
#> [4,] 0.1818182 0.2500000 0.5833333 0.2500000
#> [5,] 0.6666667 1.0000000 0.8823529 0.8750000
#> [6,] 0.8461538 1.0000000 0.8333333 0.9285714#If you prefer you can attach loci info to the matrix and output in GRanges format
meth_mat_with_loci <- methrix::get_matrix(m = methrix_data, type = "M", add_loci = TRUE, in_granges = TRUE)
meth_mat_with_loci
#> GRanges object with 743 ranges and 4 metadata columns:
#> seqnames ranges strand | C1 C2 N1
#> <Rle> <IRanges> <Rle> | <numeric> <numeric> <numeric>
#> [1] chr21 27866423-27866424 * | 0.153846 0.285714 0.555556
#> [2] chr21 27866575-27866576 * | NA 0.500000 0.000000
#> [3] chr21 27866921-27866922 * | 0.555556 0.700000 0.333333
#> [4] chr21 27867197-27867198 * | 0.181818 0.250000 0.583333
#> [5] chr21 27867248-27867249 * | 0.666667 1.000000 0.882353
#> ... ... ... ... . ... ... ...
#> [739] chr22 49007313-49007314 * | 1.000000 0.714286 0.857143
#> [740] chr22 49007329-49007330 * | 1.000000 0.428571 1.000000
#> [741] chr22 49007347-49007348 * | 0.666667 0.166667 0.875000
#> [742] chr22 49007375-49007376 * | 0.333333 0.125000 1.000000
#> [743] chr22 49007398-49007399 * | 1.000000 0.600000 1.000000
#> N2
#> <numeric>
#> [1] 0.300
#> [2] NA
#> [3] 0.800
#> [4] 0.250
#> [5] 0.875
#> ... ...
#> [739] 1.0
#> [740] 1.0
#> [741] 1.0
#> [742] 0.6
#> [743] 1.0
#> -------
#> seqinfo: 2 sequences from an unspecified genome; no seqlengthsCoverage filter
Furthermore if you prefer you can filter sites based on coverage conditions.
#e.g; Retain all loci which are covered at-least in two sample by 3 or more reads
methrix::coverage_filter(m = methrix_data, cov_thr = 3, min_samples = 2)
#> -Retained 600 of 743 sites
#> -Finished in: 0.883s elapsed (0.883s cpu)
#> An object of class methrix
#> n_CpGs: 600
#> n_samples: 4
#> is_h5: FALSE
#> Reference: hg19Subset operations
Subset operations in methrix make use of
data.tables fast
binary search which is several orders faster than bsseq
or other similar packages.
Subset by chromosome
Subset by genomic regions
Regions can be data.table or GRanges format.
#e.g; Retain sites only in TP53 loci
target_loci <- GenomicRanges::GRanges("chr21:27867971-27868103")
print(target_loci)
#> GRanges object with 1 range and 0 metadata columns:
#> seqnames ranges strand
#> <Rle> <IRanges> <Rle>
#> [1] chr21 27867971-27868103 *
#> -------
#> seqinfo: 1 sequence from an unspecified genome; no seqlengths
methrix::subset_methrix(m = methrix_data, regions = target_loci)
#> -Subsetting by genomic regions
#> An object of class methrix
#> n_CpGs: 4
#> n_samples: 4
#> is_h5: FALSE
#> Reference: hg19Subset by samples
methrix::subset_methrix(m = methrix_data, samples = "C1")
#> Subsetting by samples
#> An object of class methrix
#> n_CpGs: 743
#> n_samples: 1
#> is_h5: FALSE
#> Reference: hg19
#Or you could use [] operator to subset by index
methrix_data[,1]
#> An object of class methrix
#> n_CpGs: 743
#> n_samples: 1
#> is_h5: FALSE
#> Reference: hg19Summary statsitcis
Basic summaries
meth_stats <- get_stats(m = methrix_data)
#> -Finished in: 0.963s elapsed (0.962s cpu)
print(meth_stats)
#> Chromosome Sample_Name mean_meth median_meth sd_meth mean_cov
#> <fctr> <char> <list> <list> <list> <list>
#> 1: chr21 C1 0.560004.... 0.651515.... 0.400401.... 4.745967....
#> 2: chr21 C2 0.493499.... 0.5 0.389620.... 5.047524....
#> 3: chr21 N1 0.524541.... 0.6125 0.420522.... 4.978682....
#> 4: chr21 N2 0.533344.... 0.666666.... 0.422757.... 5.055662....
#> 5: chr22 C1 0.739242.... 0.857142.... 0.311536.... 4.657458....
#> 6: chr22 C2 0.577809.... 0.651515.... 0.367876.... 5.5
#> 7: chr22 N1 0.844555.... 1 0.227311.... 5.505376....
#> 8: chr22 N2 0.852069.... 1 0.220972.... 5.866666....
#> median_cov sd_cov
#> <list> <list>
#> 1: 4 2.990217....
#> 2: 4 3.294072....
#> 3: 4 3.214882....
#> 4: 5 3.148071....
#> 5: 4 2.813422....
#> 6: 5 3.046093....
#> 7: 5 3.270254....
#> 8: 5 3.166514....
PCA
mpca <- methrix_pca(m = methrix_data, do_plot = FALSE)
#Plot PCA results
plot_pca(pca_res = mpca, show_labels = TRUE)
Plotting
Converting methrix to BSseq
If you prefer to work with bsseq
object, you can generate bsseq object from methrix with the
methrix2bsseq.
SessionInfo
sessionInfo()
#> R version 4.4.1 (2024-06-14)
#> Platform: x86_64-pc-linux-gnu
#> Running under: Ubuntu 24.04.1 LTS
#>
#> Matrix products: default
#> BLAS: /usr/lib/x86_64-linux-gnu/openblas-pthread/libblas.so.3
#> LAPACK: /usr/lib/x86_64-linux-gnu/openblas-pthread/libopenblasp-r0.3.26.so; LAPACK version 3.12.0
#>
#> locale:
#> [1] LC_CTYPE=en_US.UTF-8 LC_NUMERIC=C
#> [3] LC_TIME=en_US.UTF-8 LC_COLLATE=C
#> [5] LC_MONETARY=en_US.UTF-8 LC_MESSAGES=en_US.UTF-8
#> [7] LC_PAPER=en_US.UTF-8 LC_NAME=C
#> [9] LC_ADDRESS=C LC_TELEPHONE=C
#> [11] LC_MEASUREMENT=en_US.UTF-8 LC_IDENTIFICATION=C
#>
#> time zone: Etc/UTC
#> tzcode source: system (glibc)
#>
#> attached base packages:
#> [1] stats4 stats graphics grDevices utils datasets methods
#> [8] base
#>
#> other attached packages:
#> [1] bsseq_1.43.0 MafDb.1Kgenomes.phase3.hs37d5_3.10.0
#> [3] GenomicScores_2.17.0 BSgenome.Hsapiens.UCSC.hg19_1.4.3
#> [5] BSgenome_1.75.0 rtracklayer_1.65.0
#> [7] BiocIO_1.17.0 Biostrings_2.75.0
#> [9] XVector_0.45.0 methrix_1.21.0
#> [11] SummarizedExperiment_1.35.5 Biobase_2.67.0
#> [13] GenomicRanges_1.57.2 GenomeInfoDb_1.41.2
#> [15] IRanges_2.39.2 S4Vectors_0.43.2
#> [17] BiocGenerics_0.53.0 MatrixGenerics_1.17.1
#> [19] matrixStats_1.4.1 data.table_1.16.2
#>
#> loaded via a namespace (and not attached):
#> [1] DBI_1.2.3 bitops_1.0-9
#> [3] permute_0.9-7 rlang_1.1.4
#> [5] magrittr_2.0.3 compiler_4.4.1
#> [7] RSQLite_2.3.7 DelayedMatrixStats_1.29.0
#> [9] png_0.1-8 vctrs_0.6.5
#> [11] pkgconfig_2.0.3 crayon_1.5.3
#> [13] fastmap_1.2.0 dbplyr_2.5.0
#> [15] labeling_0.4.3 utf8_1.2.4
#> [17] Rsamtools_2.21.2 rmarkdown_2.28
#> [19] UCSC.utils_1.1.0 bit_4.5.0
#> [21] xfun_0.48 zlibbioc_1.51.2
#> [23] cachem_1.1.0 jsonlite_1.8.9
#> [25] blob_1.2.4 highr_0.11
#> [27] rhdf5filters_1.17.0 DelayedArray_0.33.1
#> [29] Rhdf5lib_1.27.0 BiocParallel_1.41.0
#> [31] parallel_4.4.1 R6_2.5.1
#> [33] RColorBrewer_1.1-3 bslib_0.8.0
#> [35] limma_3.61.12 jquerylib_0.1.4
#> [37] Rcpp_1.0.13 knitr_1.48
#> [39] R.utils_2.12.3 Matrix_1.7-1
#> [41] tidyselect_1.2.1 abind_1.4-8
#> [43] yaml_2.3.10 codetools_0.2-20
#> [45] curl_5.2.3 lattice_0.22-6
#> [47] tibble_3.2.1 withr_3.0.2
#> [49] KEGGREST_1.45.1 evaluate_1.0.1
#> [51] BiocFileCache_2.15.0 pillar_1.9.0
#> [53] BiocManager_1.30.25 filelock_1.0.3
#> [55] generics_0.1.3 RCurl_1.98-1.16
#> [57] BiocVersion_3.21.1 ggplot2_3.5.1
#> [59] sparseMatrixStats_1.17.2 munsell_0.5.1
#> [61] scales_1.3.0 gtools_3.9.5
#> [63] glue_1.8.0 maketools_1.3.1
#> [65] tools_4.4.1 AnnotationHub_3.15.0
#> [67] sys_3.4.3 locfit_1.5-9.10
#> [69] GenomicAlignments_1.41.0 buildtools_1.0.0
#> [71] XML_3.99-0.17 rhdf5_2.49.0
#> [73] grid_4.4.1 AnnotationDbi_1.69.0
#> [75] colorspace_2.1-1 GenomeInfoDbData_1.2.13
#> [77] HDF5Array_1.33.8 restfulr_0.0.15
#> [79] cli_3.6.3 rappdirs_0.3.3
#> [81] fansi_1.0.6 S4Arrays_1.5.11
#> [83] dplyr_1.1.4 gtable_0.3.6
#> [85] R.methodsS3_1.8.2 sass_0.4.9
#> [87] digest_0.6.37 SparseArray_1.5.45
#> [89] rjson_0.2.23 farver_2.1.2
#> [91] memoise_2.0.1 htmltools_0.5.8.1
#> [93] R.oo_1.26.0 lifecycle_1.0.4
#> [95] httr_1.4.7 statmod_1.5.0
#> [97] bit64_4.5.2