DAMEfinder Workflow
In DAMEfinder: Finds DAMEs - Differential Allelicly MEthylated regions

knitr::opts_chunk$set(cache = 0, fig.width = 6, 
                      fig.height = 7)

BiocStyle::markdown()

Introduction

What is allele-specific methylation?

The phenomenon occurs when there is an asymmetry in methylation between one specific allele and the alternative allele [@hu2013]. The best studied example of allele-specific methylation (ASM) is genomic imprinting. When a gene is imprinted, one of the parental alleles is hyper-methylated compared to the other allele, which leads to parent-allele-specific expression. This asymmetry is conferred in the gametes or very early in embryogenesis, and will remain for the lifetime of the individual [@kelsey2013]. ASM not related to imprinting, exhibits parental-specific methylation, but is not inherited from the germline [@hanna2017]. Another example of ASM is X chromosome inactivation in females. DAMEfinder detects ASM for several bisulfite-sequenced (BS-seq) samples in a cohort, and performs differential detection for regions that exhibit loss or gain of ASM.

Overview

We focus on any case of ASM in which there is an imbalance in the methylation level between two alleles, regardless of the allele of origin.

DAMEfinder runs in two modes: SNP-based (exhaustive-mode) and tuple-based (fast-mode), which converge when differential ASM is detected.

Why SNP-based?

This is the exhaustive mode because it extracts an ASM score for every CpG site in the reads containing the SNPs in a VCF file. Based on this score, DAMEs are detected. From a biological point of view, you might want to run this mode if you are interested in loss or gain of allele-specificity linked to somatic or germline heterozygous SNPs (sequence-dependent ASM). More specifically, you could detect genes that exhibit loss of imprinting (e.g. as in colorectal cancer [@cui2002]).

Why tuple-based?

To run the tuple-based mode you have to run methtuple[@hickey2015] first. The methtuple output is the only thing needed for this mode. I call this the fast-mode because you don't need SNP information. The assumption is that intermediate levels of methylation represent ASM along the genome. For example, we have shown (paper in prep) that the ASM score can distinguish females from males in the X chromosome. Using SNP information this wouldn't be possible.

Installation

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

BiocManager::install("DAMEfinder")

Get bam files

In order to run any of the two modes, you must obtain aligned bam files using bismark. Here we demonstrate how to generate these starting from paired-end fastq files of bisulfite-treated reads:

```{bash eval=FALSE}

Check quality of reads

fastqc -t 2 sample1_R1.fastq.gz sample1_R2.fastq.gz

Trim reads to remove bad quality regions and adapter sequence

trim_galore --paired sample1_R1.fastq.gz sample2_R2.fastq.gz

To trim the reads we use [`Trim
Galore`](https://github.com/FelixKrueger/TrimGalore) and specify the use of
paired reads. By default it will remove any adapter sequence it recognizes.
Please refer to the user guide for further specifications.

```{bash eval=FALSE}
#Build bisulfite reference 
bismark_genome_preparation <path_to_genome_folder>

#run Bismark
bismark -B sample1 --genome <path_to_genome_folder> 
    -1 sample1_R1_val_1.fq.gz 
    -2 sample1_R2_val_2.fq.gz

#deduplicate (optional)
deduplicate_bismark -p --bam sample1_pe.bam

#sort and index files
samtools sort -m 20G -O bam -T _tmp 
    -o sample1_pe.dedupl_s.bam sample1_pe.deduplicated.bam
samtools index file1_pe.dedupl_s.bam

Before the alignment, you must download a reference fasta file from Ensembl or Gencode, and generate a bisulfite converted reference. For this we use bismark_genome_preparation from the bismark suite, and specify the folder that contains the fasta file with its index file. Depending on the library type and kit used to obtain the reads, you may want to deduplicate your bam files (e.g. TruSeq). Please refer to the user guide for further explanation and specifications.

SNP-based (aka slow-mode)

To run the SNP-based mode, you need to additionally have a VCF file including the heterozygous SNPs per sample. If you do not have this, we recommend using the tuple-based mode, or running Bis-SNP to obtain variant calls from bisulfite-converted reads.

Example Workflow

In this example we use samples from two patients with colorectal cancer from a published dataset [@parker2018]. For each patient two samples were taken: NORM# corresponds to normal mucosa tissue and CRC# corresponds to the paired adenoma lesion. Each of these samples was sequenced using targeted BS-seq followed by variant calling using Bis-SNP.

Obtain allele-based methylation calls

Similar to the bismark_methylation_extractor, we obtain methylation calls. However since we are interested in allele-specific methylation, we only extract methylation for CpG sites that fall within reads including a SNP. For every SNP in the VCF file an independent methylation call is performed by using extract_bams, which "extracts" reads from the bam file according to the alleles, and generates a list of GRangesLists:

suppressPackageStartupMessages({
  library(DAMEfinder)
  library(SummarizedExperiment)
  library(GenomicRanges)
  library(BSgenome.Hsapiens.UCSC.hg19)
  })

bam_files <- c(system.file("extdata", "NORM1_chr19_trim.bam", 
                           package = "DAMEfinder"),
               system.file("extdata", "CRC1_chr19_trim.bam", 
                           package = "DAMEfinder"))

vcf_files <- c(system.file("extdata", "NORM1.chr19.trim.vcf", 
                           package = "DAMEfinder"),
               system.file("extdata", "CRC1.chr19.trim.vcf", 
                           package = "DAMEfinder"))

sample_names <- c("NORM1", "CRC1")

#Use another reference file for demonstration, and fix the seqnames
genome <- BSgenome.Hsapiens.UCSC.hg19
seqnames(genome) <- gsub("chr","",seqnames(genome))
reference_file <- DNAStringSet(genome[[19]], use.names = TRUE)
names(reference_file) <- 19

#Extract reads and extract methylation according to allele
snp.list <- extract_bams(bam_files, vcf_files, sample_names, reference_file,
                       coverage = 2)

#CpG sites for first SNP in VCF file from sample NORM1
snp.list$NORM1[[1]]

#CpG sites for first SNP in VCF file from sample CRC1
snp.list$CRC1[[1]]

For demonstration, we include bam files from chromosome 19, and shortened VCF files. Typically we would run the function on an entire bam and VCF file, which would generate a large output.

The function also takes as input the reference file used to generate the alignments. For demonstration we use chromosome 19 of the GRCh37.91 reference fasta file.

Summarize methylation calls across samples

We use calc_derivedasm() to generate a RangedSummarizedExperiment from the large list we generated above:

derASM <- calc_derivedasm(snp.list)

derASM
assays(derASM)

Every row in the object is a single CpG site, and each column a sample. It contains 6 matrices in assays:

der.ASM: A derived SNP-based ASM defined as $abs(\frac{X^{r}_M}{X^{r}} - \frac{X^{a}_M}{X^{a}})$, where $X$ is the coverage in the reference $r$ or alternative allele $a$, and $X_M$ the number of methylated reads in $r$ or $a$. Basically, CpG sites with values of 1 or close to 1 have more allele-specificity. ASM of 1 represents the perfect scenario in which none of the reads belonging to one allele are methylated, and the reads of the other allele are completely methylated.
snp.table: Location of the SNP associated to the CpG site.
ref.cov: Coverage of the "reference" allele.
alt.cov: Covearage of the "alternative" allele.
ref.meth: Methylated reads from the "reference" allele.
alt.meth: Methylated reads from the "alternative" allele.

You can access these assays as:

x <- assay(derASM, "der.ASM")
head(x)

Find DAMEs

Now we detect regions that show differential ASM. The function find_dames() performs several steps:

Obtains a moderated t-statistic per CpG site using lmFit() and eBayes() from the limma package. The statistic reflects a measure of difference between the conditions being compared, in this case normal Vs cancer. The t-statistic is optionally smoothed (smooth parameter).

After this, two methods can be chosen (pvalAssign parameter):

Simes method:
1. (Default) Clusters of CpG sites are determined by closeness (maxGap), and a p-value for each cluster is calculated using the simes method, similar to the package csaw from @lun2014. With this approach, the p-value represents evidence against the null hypothesis that no sites are differential in the cluster.
Bumphunting method:
1. CpG sites with a t-statistic above and below a certain cutoff (set with Q), are grouped into segments (after being clustered). This is done with the regionFinder() function from bumphunter [@jaffe2012].
2. For each of these segments, a p-value is calculated empirically by permuting the groups (covariate) of interest. Depending on the number of samples, this can take longer than the Simes method. However the number of permutations can be controlled with maxPerms.

Here we show an example with a pre-processed set of samples: 4 colorectal cancer samples, and their paired normal mucosa:

data(extractbams_output)

#The data loaded is an output from `split_bams()`, therefore we run 
#`calc_derivedasm` to get the SummarizedExperiment
derASM <- calc_derivedasm(extractbams_output, cores = 1, verbose = FALSE)

#We remove all CpG sites with any NA values, but not 0s
filt <- rowSums(!is.na(assay(derASM, "der.ASM"))) == 8 
derASM <- derASM[filt,]

#set the design matrix
grp <- factor(c(rep("CRC",4),rep("NORM",4)), levels = c("NORM", "CRC"))
mod <- model.matrix(~grp)
mod

#Run default
dames <- find_dames(derASM, mod)

head(dames)

#Run empirical method
dames <- find_dames(derASM, mod, pvalAssign = "empirical")

head(dames)

A significant p-value represent regions where samples belonging to one group (in this case the cancer samples), gain or lose allele-specificity compared to the other group (here the normal group).

tuple-based (aka fast-mode)

Before running the tuple-based mode, you must obtain files from the methtuple tool to input them in the read_tuples function.

Run Methtuple on bam files

Methtuple requires the input BAM files of paired-end reads to be sorted by query name. For more information on the options in methtuple, refer to the user guide. For example the --sc option combines strand information.

```{bash, eval=FALSE}

Sort bam file by query name

samtools sort -n -@ 10 -m 20G -O bam -T _tmp -o sample1_pe_sorted.bam sample1_pe.deduplicated.bam

Run methtuple

methtuple --sc --gzip -m 2 sample1_pe_sorted.bam

## Example Workflow 

### Read methtuple files 

We use the same samples as above to run `methtuple` and obtain `.tsv.gz` files.
We read in these files using `read_tuples` and obtain a list of `tibble`s, each
one for every sample:

```r
tuple_files <- c(system.file("extdata", "NORM1_chr19.qs.CG.2.tsv.gz", 
                             package = "DAMEfinder"),
                 system.file("extdata", "CRC1_chr19.qs.CG.2.tsv.gz", 
                             package = "DAMEfinder"))

sample_names <- c("NORM1", "CRC1")

tuple_list <- read_tuples(tuple_files, sample_names)

head(tuple_list$NORM1)

Each row in the tibble displays a tuple. The chromosome name and strand are shown followed by pos1 and pos2, which refer to the genomic positions of the first and second CpG in the tuple. The MM, MU, UM, and UU counts of the tuple are displayed where M stands for methylated and U for unmethylated. For example, UM shows the read counts for the instances where pos1 is unmethylated and pos2 is methylated. The coverage and distance between the two genomic positions in the tuple are shown under cov and inter_dist respectively.

Calculate ASM Score

The calc_asm function takes the output from read_tuples(), and as in the SNP-based mode, generates a RangedSummarizedExperiment where each row is a tuple and each column is a sample. The object contains 6 assays including the MM, MU, UM, and UU counts, as well as the total coverage and the tuple-based ASM score. This score is a measure of ASM calculated directly from the reads without the need of SNP information. Because of this, it is a lot quicker than the SNP-based ASM, and is useful for more explorative purposes.

Equations \@ref(eq:asmGeneral), \@ref(eq:asmWeight) and \@ref(eq:asmTheta) show how the score is calculated. The log odds ratio in equation \@ref(eq:asmGeneral) provides a higher score the more MM and UU counts the tuple has, whereas a higher UM and MU would indicate "random" methylation. The weight further adds allele-specificity where a rather balanced MM:UU increases the score.

\begin{equation} ASM^{(i)} = log{ \Big{ \frac{X_{MM}^{(i)} \cdot X_{UU}^{(i)}}{X_{MU}^{(i)} \cdot X_{UM}^{(i)}} \Big} \cdot w_i } (#eq:asmGeneral) \end{equation}

\begin{equation} w_i = P(0.5-\epsilon < \theta < 0.5+\epsilon~|~ X_{MM}^{(i)}, X_{UU}^{(i)}, \beta_1, \beta_2) (#eq:asmWeight) \end{equation}

\begin{equation} \theta^{(i)} | X_{MM}^{(i)}, X_{UU}^{(i)},\beta_1, \beta_2 \sim Beta(\beta_1+X_{MM}^{(i)}, \beta_2+X_{UU}^{(i)}) (#eq:asmTheta) \end{equation}

where $\theta^{(i)}$ represents the moderated proportion of MM to MM+UU alleles. The weight, $w_i$ is set such that the observed split between MM and UU alleles can depart somewhat from 50/50, while fully methylated or unmethylated tuples, which represents evidence for absence of allele-specificity, are attenuated to 0. The degree of allowed departure can be set according to $\epsilon$, the deviation from 50/50 allowed and the level of moderation, $\beta_1$ and $\beta_2$.

ASM_mat <- calc_asm(tuple_list)
ASM_mat

Find DAMEs

As above, the RangedSummarizedExperiment is used to detect differential ASM. Here we show an example with a pre-processed set of samples: 3 colorectal cancer samples, an 2 normal mucosa samples

#load package data
data(readtuples_output)

#run calc_asm and filter object
ASMscore <- calc_asm(readtuples_output)
filt <- rowSums(!is.na(assay(ASMscore, "asm"))) == 5 #filt to avoid warnings
ASMscore <- ASMscore[filt,]

#make design matrix (or specify a contrast)
grp <- factor(c(rep("CRC",3),rep("NORM",2)), levels = c("NORM", "CRC"))
mod <- model.matrix(~grp)

#run default and increase maxGap to get longer, more sparse regions
dames <- find_dames(ASMscore, mod, maxGap = 300)

head(dames)

#run alternative mode
dames <- find_dames(ASMscore, mod,  maxGap = 300, pvalAssign = "empirical")

head(dames)

Visualization

DAME tracks

After detecting a set of DAMEs you want to look at them individually. We do this with the function dame_track.

Depending on which object I used to obtain my DAMEs (tuple or SNP mode), I choose which SummarizedExperiment to input in the field ASM (for tuple), or derASM (for SNP). Either way, the SummarizedExperiment must have the columns group and samples in the colData field:

#Here I will use the tuple-ASM SummExp
colData(ASMscore)$group <- grp
colData(ASMscore)$samples <- colnames(ASMscore)

#Set a DAME as a GRanges. I choose a one from the tables we obtained above
dame <- GRanges(19,IRanges(323736,324622))

dame_track(dame = dame,
           ASM = ASMscore)

Because we used the tuple-ASM object, we get by default two tracks: the ASM score, and the marginal methylation (aka beta-value).

The shaded square delimits the DAME we defined to plot. We can look at the flanking regions of the DAME by changing window or positions. With window we specify the number of CpG positions we want to add to the plot up and down-stream. With positions we specify the number of base pairs.

dame_track(dame = dame,
           ASM = ASMscore,
           window = 2)

If we use the SNP-ASM as input we get different tracks:

dame <- GRanges(19,IRanges(387966,387983))

grp <- factor(c(rep("CRC",4),rep("NORM",4)), levels = c("NORM", "CRC"))
colData(derASM)$group <- grp

dame_track(dame = dame,
           derASM = derASM)

Here we get three tracks: the SNP-ASM score, and the methylation levels for each allele. Since the ASM score here depends on SNPs, we can see what SNPs are involved in the ASM calculation at each CpG position:

dame_track(dame = dame,
           derASM = derASM,
           plotSNP = TRUE)

We see that the SNP located at chr19:388,065 was the one used to split the allele methylation.

If you put both SummarizedExperiments with a single DAME, you would get all the tracks:

dame_track(dame = dame,
           derASM = derASM,
           ASM = ASMscore)

Notice that the first two tracks depend on the tuple-ASM, hence each point represents the midpoint between a pair of CpG sites.

If you think plotting all the samples separately is difficult to see, you can use the function dame_track_mean to summarize:

dame_track_mean(dame = dame,
           derASM = derASM,
           ASM = ASMscore)

As you can see, this region is not a very good DAME.

Methyl-circle plot

A typical way of visualizing ASM is to look at the reads overlapping a particular SNP, and the methylation state of the CpG sites in those reads (black circles for methylated and white for unmethylated, see @shoemaker2010 for examples). Here we offer this option with the function methyl_circle_plot(). As input it takes a GRanges with the SNP of interest, and the bam, VCF and reference files as in the extract_bams() function.

#put SNP in GRanges (you can find the SNP with the dame_track function)
snp <- GRanges(19, IRanges(267039, width = 1)) #always set the width if your 
#GRanges has 1 site

snp

bam.file <- system.file("extdata", "CRC1_chr19_trim.bam", 
                        package = "DAMEfinder")

vcf.file <- system.file("extdata", "CRC1.chr19.trim.vcf", 
                        package = "DAMEfinder")

methyl_circle_plot(snp = snp, vcfFile = vcf.file, bamFile = bam.file, 
                   refFile = reference_file)

You can reduce the number of reads included with the option sampleReads, which performs a random sampling of the number of reads to be shows per allele. The number of reads can be specified with numReads.

If you are interested in a specific CpG site within this plot, you can include an extra GRanges with its location, and the triangle at the bottom will point to it:

cpgsite <- GRanges(19, IRanges(266998, width = 1))

methyl_circle_plot(snp = snp, vcfFile = vcf.file, bamFile = bam.file, 
                   refFile = reference_file, cpgsite = cpgsite)

If you are instead interested in reads overlapping a CpG site, you can use methyl_circle_plotCpG(), which is useful if you run the tuple-mode:

cpgsite <- GRanges(19, IRanges(266998, width = 1))

methyl_circle_plotCpG(cpgsite = cpgsite, bamFile = bam.file, 
                      refFile = reference_file)

You can also limit both the SNP plot and the CpG plot to a specific window of interest (to zoom in or out), or if you want to look at the specific DAME region:

#a random region
dame <- GRanges(19, IRanges(266998,267100))

methyl_circle_plot(snp = snp, vcfFile = vcf.file, bamFile = bam.file, 
                   refFile = reference_file, dame = dame)

MDS plot

To plot a multidimensional scaling plot (MDS), we provide a wrapper to plotMDS() from limma, which adjusts the ASM score to calculate the euclidean distances. The input is a SummarizedExperiment, and the vector of covariates to color the points by:

grp <- factor(c(rep("CRC",3),rep("NORM",2)), levels = c("NORM", "CRC"))
methyl_MDS_plot(ASMscore, group = grp)

Session Info

utils::sessionInfo()

References

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DAMEfinder documentation built on Nov. 8, 2020, 11:10 p.m.

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DAMEfinder
Finds DAMEs - Differential Allelicly MEthylated regions

DAMEfinder Workflow
In DAMEfinder: Finds DAMEs - Differential Allelicly MEthylated regions

Introduction

What is allele-specific methylation?

Overview

Why SNP-based?

Why tuple-based?

Installation

Get bam files

Check quality of reads

Trim reads to remove bad quality regions and adapter sequence

SNP-based (aka slow-mode)

Example Workflow

Obtain allele-based methylation calls

Summarize methylation calls across samples

Find DAMEs

tuple-based (aka fast-mode)

Run Methtuple on bam files

Sort bam file by query name

Run methtuple

Calculate ASM Score

Find DAMEs

Visualization

DAME tracks

Methyl-circle plot

MDS plot

Session Info

References

Try the DAMEfinder package in your browser

R Package Documentation

Browse R Packages

We want your feedback!

DAMEfinder Finds DAMEs - Differential Allelicly MEthylated regions

DAMEfinder Workflow In DAMEfinder: Finds DAMEs - Differential Allelicly MEthylated regions

Introduction

What is allele-specific methylation?

Overview

Why SNP-based?

Why tuple-based?

Installation

Get bam files

Check quality of reads

Trim reads to remove bad quality regions and adapter sequence

SNP-based (aka slow-mode)

Example Workflow

Obtain allele-based methylation calls

Summarize methylation calls across samples

Find DAMEs

tuple-based (aka fast-mode)

Run Methtuple on bam files

Sort bam file by query name

Run methtuple

Calculate ASM Score

Find DAMEs

Visualization

DAME tracks

Methyl-circle plot

MDS plot

Session Info

References

Try the DAMEfinder package in your browser

R Package Documentation

Browse R Packages

We want your feedback!

DAMEfinder
Finds DAMEs - Differential Allelicly MEthylated regions

DAMEfinder Workflow
In DAMEfinder: Finds DAMEs - Differential Allelicly MEthylated regions