Constructing the NanoMethResult object

To generate a methylation plot we need 3 components:

• methylation data in tabix format
• annotation of exons
• annotation of samples

The methylation information has been modified from the output of nanopolish/f5c. It has then been compressed and indexed using bgzip() and indexTabix() from the Rsamtools package.

# methylation data stored in tabix file
methy <- system.file(package = "NanoMethViz", "methy_subset.tsv.bgz")

# tabix is just a special gzipped tab-separated-values file
read.table(gzfile(methy), col.names = methy_col_names(), nrows = 6)

##               sample   chr       pos strand statistic
## 1  B6Cast_Prom_1_bl6 chr11 101463573      *     -0.33
## 2  B6Cast_Prom_1_bl6 chr11 101463573      *     -1.87
## 3  B6Cast_Prom_1_bl6 chr11 101463573      *     -4.19
## 4  B6Cast_Prom_1_bl6 chr11 101463573      *      0.10
## 5 B6Cast_Prom_1_cast chr11 101463573      *     -0.38
## 6 B6Cast_Prom_1_cast chr11 101463573      *     -0.84
##                              read_name
## 1 6cc38b35-6570-4b44-a1e3-2605fcf2ffe8
## 2 787f5f43-d144-4e15-ab7d-6b1474083389
## 3 c7ee7fb4-a915-4da7-9f36-da6ed5e68af2
## 4 bff8b135-0296-4495-9354-098242ea8cc4
## 5 11fe130b-8d48-4399-a9fa-2ca2860fa355
## 6 502fef95-c2f2-46ad-9bc5-fb3fc80b4245

The exon annotation was obtained from the Mus.musculus package, and joined into a single table. It is important that the chromosomes share the same convention as that found in the methylation data.

# helper function extracts exons from TxDb package
exon_tibble <- get_exons_mm10()

head(exon_tibble)

## # A tibble: 6 × 7
##   gene_id   chr   strand    start      end transcript_id symbol
##   <chr>     <chr> <chr>     <int>    <int>         <int> <chr> 
## 1 100009600 chr9  -      21062393 21062717         74536 Zglp1 
## 2 100009600 chr9  -      21062894 21062987         74536 Zglp1 
## 3 100009600 chr9  -      21063314 21063396         74536 Zglp1 
## 4 100009600 chr9  -      21066024 21066377         74536 Zglp1 
## 5 100009600 chr9  -      21066940 21067093         74536 Zglp1 
## 6 100009600 chr9  -      21062400 21062717         74538 Zglp1

We will defined the sample annotation ourselves. It is important that the sample names match those found in the methylation data.

sample <- c(
  "B6Cast_Prom_1_bl6", "B6Cast_Prom_1_cast",
  "B6Cast_Prom_2_bl6", "B6Cast_Prom_2_cast",
  "B6Cast_Prom_3_bl6", "B6Cast_Prom_3_cast"
)

group <- c("bl6", "cast", "bl6", "cast", "bl6", "cast")

sample_anno <- data.frame(sample, group, stringsAsFactors = FALSE)

sample_anno

##               sample group
## 1  B6Cast_Prom_1_bl6   bl6
## 2 B6Cast_Prom_1_cast  cast
## 3  B6Cast_Prom_2_bl6   bl6
## 4 B6Cast_Prom_2_cast  cast
## 5  B6Cast_Prom_3_bl6   bl6
## 6 B6Cast_Prom_3_cast  cast

For convenience we assemble these three pieces of data into a single object.

nmr <- NanoMethResult(methy, sample_anno, exon_tibble)

The genes we have available are

• Peg3
• Meg3
• Impact
• Xist
• Brca1
• Brca2

Basic Plotting

For demonstrative purposes we will plot Peg3.

plot_gene(nmr, "Peg3")

We can also load in some DMR results to highlight DMR regions.

# loading saved results from previous bsseq analysis
bsseq_dmr <- read.table(
    system.file(package = "NanoMethViz", "dmr_subset.tsv.gz"),
    sep = "\t",
    header = TRUE,
    stringsAsFactors = FALSE
)

plot_gene(nmr, "Peg3", anno_regions = bsseq_dmr)

Individual long reads can be visualised using the spaghetti argument.

# warnings have been turned off in this vignette, but this will generally
# generate many warnings as the smoothing for many reads will fail
plot_gene(nmr, "Peg3", anno_regions = bsseq_dmr, spaghetti = TRUE)

Alternatively the individual read information can be represented by a heatmap.

plot_gene(nmr, "Peg3", anno_regions = bsseq_dmr, heatmap = TRUE)

Data example

The conversion can be done using the create_tabix_file() function. We provide example data of nanopolish output within the package, we can look inside to see how the data looks coming out of nanopolish

methy_calls <- system.file(package = "NanoMethViz",
    c("sample1_nanopolish.tsv.gz", "sample2_nanopolish.tsv.gz"))

# have a look at the first 10 rows of methy_data
methy_calls_example <- read.table(
    methy_calls[1], sep = "\t", header = TRUE, nrows = 6)

methy_calls_example

##   chromosome strand     start       end                            read_name
## 1       chr1      - 127732476 127732476 e648c4e3-ca6a-4671-af17-86dab4c819eb
## 2      chr11      - 115423144 115423144 726dd8b5-1531-4279-9cf0-a7e4d5ea0478
## 3      chr11      +  69150806  69150814 34f9ee3e-4b27-4d2d-a203-4067f0662044
## 4       chr1      + 170484965 170484965 d8309c06-375f-4dfe-b22e-0c47af888cd9
## 5       chrY      -   4082060   4082060 f68940f6-4236-4f0f-9af7-a81b5c2911b6
## 6       chr8      + 120733312 120733312 13ae181f-b88b-4d6c-a815-553ff2e25312
##   log_lik_ratio log_lik_methylated log_lik_unmethylated num_calling_strands
## 1         -5.91            -100.38               -94.47                   1
## 2         -8.07            -115.21              -107.13                   1
## 3         -1.65            -183.12              -181.47                   1
## 4          2.74            -112.14              -114.88                   1
## 5         -1.78            -135.09              -133.32                   1
## 6          5.02            -129.31              -134.33                   1
##   num_motifs            sequence
## 1          1         CATTACGTTTC
## 2          1         AACTTCGTTGA
## 3          2 GGTCACGGGAATCCGGTTC
## 4          1         AGAAGCGCTAA
## 5          1         CTCACCGTATA
## 6          1         TCTGACGTTGA

We then create a temporary path to store a converted file, this will be deleted once you exit your R session. Once create_tabix_file() is run, it will create a .bgz file along with its tabix index. Because we have a small amount of data, we can read in a small portion of it for inspection, do not do this with large datasets as it decompresses all the data and will take very long to run.

Importing data

# create a temporary file to store the converted data
methy_tabix <- file.path(tempdir(), "methy_data.bgz")
samples <- c("sample1", "sample2")

# you should see messages when running this yourself
create_tabix_file(methy_calls, methy_tabix, samples)

# you don't need to do this with real data
# we have to use gzfile to tell R that we have a gzip compressed file
methy_data <- read.table(
    gzfile(methy_tabix), col.names = methy_col_names(), nrows = 6)

methy_data

##    sample  chr      pos strand statistic                            read_name
## 1 sample2 chr1  5141051      -      6.93 3818f2e2-d520-4305-bbab-efad891f67f2
## 2 sample1 chr1  6283068      -      1.05 36e3c55f-c41f-4bd6-b371-54368d013008
## 3 sample1 chr1  7975279      -      1.39 6f6cbc59-af4c-4dfa-8e48-ef4ac4eeb13b
## 4 sample1 chr1 10230293      -      2.19 fbe53b38-e264-4c7a-824e-2651c22f8ea6
## 5 sample1 chr1 13127128      -      2.51 7660ba1f-9b44-4783-b901-ed79b2f0481b
## 6 sample1 chr1 13127135      -      2.51 7660ba1f-9b44-4783-b901-ed79b2f0481b

Now methy_tabix will be the path to a tabix object that is ready for use with NanoMethViz.

Using BAM files with modification tags

In the latest versions of megalodon and other ONT software, the modifications are stored in the BAM file. This can be used directly with NanoMethViz using the ModBamResult class as a substitute for NanoMethResult.

This requires a ModBamFiles object, which is a list of paths to the BAM files and the sample names. The sample names must match the sample names in the sample_anno data frame.

Because the BAM files contain significantly more data than the tabix files, it may be useful to convert the BAM files to tabix files using the modbam_to_tabix() function for sharing the data.

In modbam files the modification calls are made along the read, allowing sequencing errors to produce modifications calls where there is none in the genome. This may lead to noise impacting the averaging results. The NanoMethViz.site_filter option can be used to remove sites with low coverage, by default it is set to 3 to remove any sites with coverage less than 3. See the “Site filtering” section for more information.

# construct with a ModBamFiles object as the methylation data
mbr <- ModBamResult(
    methy = ModBamFiles(
        samples = "sample1",
        system.file(package = "NanoMethViz", "peg3.bam")
    ),
    samples = data.frame(
        sample = "sample1",
        group = 1
    ),
    exons = exon_tibble
)

# use in the same way as you would a NanoMethResult object
plot_gene(mbr, "Peg3", heatmap = TRUE)

Exporting data

The methylation data can be exported into formats appropriate for bsseq, DSS, or edgeR.

nmr <- load_example_nanomethresult()
bss <- methy_to_bsseq(nmr)

bss

## An object of type 'BSseq' with
##   4778 methylation loci
##   6 samples
## has not been smoothed
## All assays are in-memory

gene_regions <- exons_to_genes(NanoMethViz::exons(nmr))
edger_mat <- bsseq_to_edger(bss, gene_regions)

edger_mat

##        B6Cast_Prom_1_bl6_Me B6Cast_Prom_1_bl6_Un B6Cast_Prom_1_cast_Me
## 12189                  3259                 2033                  2628
## 12190                  2384                 1349                  1604
## 16210                  2696                 1173                  1564
## 17263                  1752                 1672                  2156
## 18616                  1812                  595                  1436
## 213742                 1264                  803                   848
##        B6Cast_Prom_1_cast_Un B6Cast_Prom_2_bl6_Me B6Cast_Prom_2_bl6_Un
## 12189                   1970                 3380                 1764
## 12190                   1627                 2564                 1853
## 16210                   1788                 2544                  895
## 17263                   1831                 2027                 1698
## 18616                   1184                 1690                  573
## 213742                  1465                 1012                  596
##        B6Cast_Prom_2_cast_Me B6Cast_Prom_2_cast_Un B6Cast_Prom_3_bl6_Me
## 12189                   2663                  2043                 3658
## 12190                   1860                  1573                 2110
## 16210                   1630                  1693                 1955
## 17263                   1349                  1153                 1787
## 18616                   1442                  1520                 3011
## 213742                  1072                  1040                 1432
##        B6Cast_Prom_3_bl6_Un B6Cast_Prom_3_cast_Me B6Cast_Prom_3_cast_Un
## 12189                  2341                  2565                  1968
## 12190                  1502                  1884                  1663
## 16210                   769                  1466                  1787
## 17263                  1880                  1883                  1694
## 18616                  1331                   948                   931
## 213742                  728                   653                  1189

Importing Annotations

Annotations can be simplified if full exon and isoform information is not required. For example, gene-body annotation can be represented as single exon genes. For example we can take the example dataset and transform the isoform annotations of Peg3 into a single gene-body block. The helper function exons_to_genes() can help with this common conversion.

nmr <- load_example_nanomethresult()

plot_gene(nmr, "Peg3")

new_exons <- NanoMethViz::exons(nmr) %>%
    exons_to_genes() %>%
    mutate(transcript_id = gene_id)

NanoMethViz::exons(nmr) <- new_exons

plot_gene(nmr, "Peg3")

Preparing data for dimensionality reduction

It is assumed that users of this package have imported the data into the gzipped tabix format. From there, further processing is required to create the log-methylation-ratio matrix used in dimensionality reduction. Namely we go through the BSseq format as it is easily coerced into the desired matrix and is itself useful for various other analyses.

# convert to bsseq
bss <- methy_to_bsseq(nmr)
bss

## An object of type 'BSseq' with
##   4778 methylation loci
##   6 samples
## has not been smoothed
## All assays are in-memory

We can generate the log-methylation-ratio based on individual methylation sites or computed over genes, or other feature types. Aggregating over features will generally provide more stable and robust results, here we will use genes.

# create gene annotation from exon annotation
gene_anno <- exons_to_genes(NanoMethViz::exons(nmr))

# create log-methylation-ratio matrix
lmr <- bsseq_to_log_methy_ratio(bss, regions = gene_anno)

NanoMethViz currently provides two options, a MDS plot based on the limma implementation of MDS, and a PCA plot using BiocSingular.

plot_mds(lmr) +
    ggtitle("MDS Plot")

plot_pca(lmr) +
    ggtitle("PCA Plot")

Additional coloring and labeling options can be provided via arguments to either function. Further customisations can be done using typical ggplot2 commands.

new_labels <- gsub("B6Cast_Prom_", "", colnames(lmr))
new_labels <- gsub("(\\d)_(.*)", "\\2 \\1", new_labels)
groups <- gsub(" \\d", "", new_labels)

plot_mds(lmr, labels = new_labels, groups = groups) +
    ggtitle("MDS Plot") +
    scale_colour_brewer(palette = "Set1")

Points can also be plotted without labels by setting labels = NULL.

plot_mds(lmr, labels = NULL, groups = groups) +
    ggtitle("MDS Plot") +
    scale_colour_brewer(palette = "Set1")

Site filtering

NanoMethViz offers a site filtering option to remove sites with low coverage. This is particularly useful for modbam files as the modifications calls are made along the reads, allowing methylation to be called at miscalled sites. The site filter is set to 3 by default, to filter any sites with coverage less than 3. This can be changed using the option NanoMethViz.site_filter. The following will remove any sites with coverage less than 5 from queries and plots.

options("NanoMethViz.site_filter" = 5)

Region annotation colours

By default the anno_regions argument in plotting functions will create bands coloured grey, specifically grey50. This can be changed globally across the package using the option NanoMethViz.highlight_col. For example the following will change the colour to red.

options("NanoMethViz.highlight_col" = "red")