Introducing the raer package
2024-10-29
Package
raer 1.4.0
Contents
1 Introduction
The raer (RNA Adenosine editing in R) package provides tools to characterize A-to-I editing in single cell and bulk RNA-sequencing datasets. Both novel and known editing sites can be detected and quantified beginning with BAM alignment files. At it’s core the raer package uses the pileup routines from the HTSlib C library (Bonfield et al. (2021)) to identify candidate RNA editing sites, and leverages the annotation resources in the Bioconductor ecosystem to further characterize and identify high-confidence RNA editing sites.
Here we demonstrate how to use the raer package to a) quantify RNA editing sites in droplet scRNA-seq dataset, b) identify editing sites with condition specific editing in bulk RNA-seq data, and c) predict novel editing sites from bulk RNA-seq.
2 Installation
The raer package can be installed from Bioconductor using BiocManager.
if (!require("BiocManager", quietly = TRUE)) {
install.packages("BiocManager")
}
BiocManager::install("raer")Alternatively raer can be installed from github using
BiocManager::install("rnabioco/raer").
3 Characterizing RNA editing sites in scRNA-seq data
Here we will use the raer package to examine RNA editing in droplet-based
single cell RNA-seq data. pileup_cells() enables quantification of edited and
non-edited bases at specified sites from scRNA-seq data.
For this example we will examine a scRNA-seq dataset from human PBMC cells provided by 10x Genomics. The single cell data was aligned and processed using the 10x Genomics cellranger pipeline.
The PBMC scRNA-seq dataset from 10x Genomics, along with other
needed files will downloaded and cached using pbmc_10x()from the raerdata
ExperimentHub package. For this vignette, the BAM file was subset to retain
2 million alignments that overlap human RNA editing sites on chromosome 16.
pbmc_10x() returns a list containing a BamFile object, a GRanges object
with known RNA editing sites from the REDIportal database Mansi et al. (2021), and
a SingleCellExperiment populated with the gene expression data and cell type
annotations.
library(raer)
library(raerdata)
pbmc <- pbmc_10x()
pbmc_bam <- pbmc$bam
editing_sites <- pbmc$sites
sce <- pbmc$sceThis dataset contains T-cell, B-cells, and monocyte cell populations.
library(scater)
library(SingleCellExperiment)
plotUMAP(sce, colour_by = "celltype")
3.1 Specifying sites to quantify
Next we’ll select editing sites to quantify. For this analysis we will use RNA editing sites cataloged in the REDIportal database Mansi et al. (2021).
editing_sites## GRanges object with 15638648 ranges and 0 metadata columns:
## seqnames ranges strand
## <Rle> <IRanges> <Rle>
## [1] chr1 87158 -
## [2] chr1 87168 -
## [3] chr1 87171 -
## [4] chr1 87189 -
## [5] chr1 87218 -
## ... ... ... ...
## [15638644] chrY 56885715 +
## [15638645] chrY 56885716 +
## [15638646] chrY 56885728 +
## [15638647] chrY 56885841 +
## [15638648] chrY 56885850 +
## -------
## seqinfo: 44 sequences from hg38 genome; no seqlengthsThe sites to quantify are specified using a custom formatted GRanges object
with 1 base intervals, a strand (+ or -), and supplemented with metadata
columns named REF and ALT containing the reference and alternate base to
query. In this case we are only interested in A->I editing, so we set the ref
and alt to A and G. Note that the REF and ALT bases are in reference to
strand. For a - strand interval the bases should be the complement of the +
strand bases. Also note that these bases can be stored as traditional character
vectors or as Rle() objects to save memory.
editing_sites$REF <- Rle("A")
editing_sites$ALT <- Rle("G")
editing_sites## GRanges object with 15638648 ranges and 2 metadata columns:
## seqnames ranges strand | REF ALT
## <Rle> <IRanges> <Rle> | <Rle> <Rle>
## [1] chr1 87158 - | A G
## [2] chr1 87168 - | A G
## [3] chr1 87171 - | A G
## [4] chr1 87189 - | A G
## [5] chr1 87218 - | A G
## ... ... ... ... . ... ...
## [15638644] chrY 56885715 + | A G
## [15638645] chrY 56885716 + | A G
## [15638646] chrY 56885728 + | A G
## [15638647] chrY 56885841 + | A G
## [15638648] chrY 56885850 + | A G
## -------
## seqinfo: 44 sequences from hg38 genome; no seqlengths3.2 Quantifying sites in single cells using pileup_cells
pileup_cells() quantifies edited and non-edited UMI counts per cell barcode,
then organizes the site counts into a SingleCellExperiment object.
pileup_cells() accepts a FilterParam() object that specifies parameters for
multiple read-level and site-level filtering and processing options. Note that
pileup_cells() is strand sensitive by default, so it is important to ensure
that the strand of the input sites is correctly annotated, and that the
library-type is set correctly for the strandedness of the sequencing library.
For 10x Genomics data, the library type is set to fr-second-strand,
indicating that the strand of the BAM alignments is the same strand as the RNA.
See quantifying Smart-seq2 scRNA-seq libraries for an example of using
pileup_cells() to handle unstranded data and data from libraries that produce
1 BAM file for each cell.
To exclude duplicate reads derived from PCR, pileup_cells() can use a UMI
sequence, supplied via the umi_tag argument, to only count 1 read for each
CB-UMI pair at each editing site position. Note however that by default the
bam_flags argument for the FilterParam class is set to include duplicate
reads when using pileup_cells(). Droplet single cell libraries produce
multiple cDNA fragments from a single reverse transcription event. The cDNA
fragments have different alignment positions due to fragmentation despite being
derived from a single RNA molecule. scRNA-seq data processed by cellranger from
10x Genomics will set the “Not primary alignment” BAM flag for every read except
one read for each UMI. If duplicates are removed based on this BAM flag, then
only 1 representative fragment for a single UMI will be examined, which will
exclude many valid regions.
To reduce processing time many functions in the raer package operate in
parallel across multiple chromosomes. To enable parallel processing, a
BiocParallel backend can be supplied via the BPPARAM argument (e.g.
MultiCoreParam()).
outdir <- file.path(tempdir(), "sc_edits")
cbs <- colnames(sce)
params <- FilterParam(
min_mapq = 255, # required alignment MAPQ score
library_type = "fr-second-strand", # library type
min_variant_reads = 1
)
e_sce <- pileup_cells(
bamfile = pbmc_bam,
sites = editing_sites,
cell_barcodes = cbs,
output_directory = outdir,
cb_tag = "CB",
umi_tag = "UB",
param = params
)
e_sce## class: SingleCellExperiment
## dim: 3849 500
## metadata(0):
## assays(2): nRef nAlt
## rownames(3849): site_chr16_83540_1_AG site_chr16_83621_1_AG ...
## site_chr16_31453268_2_AG site_chr16_31454303_2_AG
## rowData names(2): REF ALT
## colnames(500): TGTTTGTCAGTTAGGG-1 ATCTCTACAAGCTACT-1 ...
## GGGCGTTTCAGGACGA-1 CTATAGGAGATTGTGA-1
## colData names(0):
## reducedDimNames(0):
## mainExpName: NULL
## altExpNames(0):The outputs from pileup_cells() are a SingleCellExperiment object populated
with nRef and nAlt assays containing the base counts for the reference
(unedited) and alternate (edited) alleles at each position.
The sparseMatrices are also written to files, at a directory specified by
output_directory, which can be loaded into R using the read_sparray()
function.
dir(outdir)## [1] "barcodes.txt.gz" "counts.mtx.gz" "sites.txt.gz"read_sparray(
file.path(outdir, "counts.mtx.gz"),
file.path(outdir, "sites.txt.gz"),
file.path(outdir, "barcodes.txt.gz")
)## class: SingleCellExperiment
## dim: 3849 500
## metadata(0):
## assays(2): nRef nAlt
## rownames(3849): site_chr16_83540_1_AG site_chr16_83621_1_AG ...
## site_chr16_31453268_2_AG site_chr16_31454303_2_AG
## rowData names(2): REF ALT
## colnames(500): TGTTTGTCAGTTAGGG-1 ATCTCTACAAGCTACT-1 ...
## GGGCGTTTCAGGACGA-1 CTATAGGAGATTGTGA-1
## colData names(0):
## reducedDimNames(0):
## mainExpName: NULL
## altExpNames(0):Next we’ll filter the single cell editing dataset to find sites with an editing
event in at least 5 cells and add the editing counts to the gene expression
SingleCellExperiment as an altExp().
e_sce <- e_sce[rowSums(assays(e_sce)$nAlt > 0) >= 5, ]
e_sce <- calc_edit_frequency(e_sce,
edit_from = "Ref",
edit_to = "Alt",
replace_na = FALSE
)
altExp(sce) <- e_sce[, colnames(sce)]With the editing sites added to the gene expression SingleCellExperiment we can use plotting and other methods previously developed for single cell analysis. Here we’ll visualize editing sites with the highest edited read counts.
to_plot <- rownames(altExp(sce))[order(rowSums(assay(altExp(sce), "nAlt")),
decreasing = TRUE
)]
lapply(to_plot[1:5], function(x) {
plotUMAP(sce, colour_by = x, by_exprs_values = "nAlt")
})## [[1]]
##
## [[2]]
##
## [[3]]
##
## [[4]]
##
## [[5]]
Alternatively we can view these top edited sites as a Heatmap, showing the average number of edited reads per site in each cell type.
altExp(sce)$celltype <- sce$celltype
plotGroupedHeatmap(altExp(sce),
features = to_plot[1:25],
group = "celltype",
exprs_values = "nAlt"
)
raer provides additional tools to examine cell type specific editing.
find_scde_sites()will perform statistical testing to identify sites with different editing frequencies between clusters/cell types.calc_scAEI()will calculate the Alu Editing Index (AEI) metric in single cells.
If the editing sites of interest are not known, we recommend the following
approach. First, treat the single cell data as a bulk RNA-seq experiment, and
follow approaches described in the Novel editing site detection to
identify putative editing sites. Then query these sites in single cell mode
using pileup_cells()
3.3 Quantifying sites in Smart-seq2 libaries
pileup_cells() can also process Smart-seq2 single cell libraries. These
datasets typically store data from each cell in separate BAM files and the
library type for these alignments are generally unstranded. To process these
datasets the library-type should be set to unstranded, and the reference
editing sites need to be reported all on the + strand.
For example, the editing sites on the minus strand will need to be complemented
(set as T -> C rather than A -> G). Additionally the umi_tag and cb_tag
arguments should be set as follows to disable UMI and cell barcode detection.
To illustrate this functionality, we will reprocess the 10x Genomics pbmc dataset, treating the data as mock Smart-seq2 data from 3 cells.
is_minus <- strand(editing_sites) == "-"
editing_sites[is_minus]$REF <- "T"
editing_sites[is_minus]$ALT <- "C"
strand(editing_sites[is_minus]) <- "+"
fp <- FilterParam(
library_type = "unstranded",
min_mapq = 255,
min_variant_reads = 1
)
ss2_bams <- c(pbmc_bam, pbmc_bam, pbmc_bam)
cell_ids <- c("cell1", "cell2", "cell3")
pileup_cells(
bamfiles = ss2_bams,
cell_barcodes = cell_ids,
sites = editing_sites,
umi_tag = NULL, # no UMI tag in most Smart-seq2 libraries
cb_tag = NULL, # no cell barcode tag
param = fp,
output_directory = outdir
)## class: SingleCellExperiment
## dim: 53971 3
## metadata(0):
## assays(2): nRef nAlt
## rownames(53971): site_chr16_22107_1_TC site_chr16_22140_1_TC ...
## site_chr16_31454310_1_TC site_chr16_31454314_1_TC
## rowData names(2): REF ALT
## colnames(3): cell1 cell2 cell3
## colData names(0):
## reducedDimNames(0):
## mainExpName: NULL
## altExpNames(0):4 Quantifying RNA editing sites in bulk RNA-Seq
Next we will perform a reanalysis of a published bulk RNA-seq dataset using the
raer package. The pileup_sites() function enable quantification of base
counts from bulk RNA-seq data and can be used to identify novel sites
(see Novel editing site detection).
For this reanalysis, we will examine a bulk RNA-seq dataset from accession GSE99249, which consists of RNA-seq data from ADAR1 mutants and control human cell lines, conditionally treated with Interferon-Beta. We will examine data from two genotypes, ADAR1 WT and KO, both treated with Interferon-B, with triplicate samples.
Aligned BAM files and other necessary files have been preprocessed for this
vignette and are available using GSE99249() from the raerdata package.
Calling GSE99249() will downloaded and cache the necessary files and return
a list containing the data.
ifnb <- GSE99249()
names(ifnb)## [1] "bams" "fasta" "sites"bams contains a vector of BamFile objects with the paths to each BAM file.
These BAM files are a subset of the full BAM files, containing alignments from
chromosome 18.
bam_files <- ifnb$bams
names(bam_files)## [1] "SRR5564260" "SRR5564261" "SRR5564269" "SRR5564270" "SRR5564271"
## [6] "SRR5564277"To quantify editing sites we will need a FASTA file to compare read alignments to the reference sequence. For space reasons we’ll use a FASTA file containing only chromosome 18 for this demo.
fafn <- ifnb$fastaWe will again use the database of known human editing sites from REDIPortal,
only processing those from chr18.
editing_sites <- ifnb$sites
chr_18_editing_sites <- keepSeqlevels(editing_sites, "chr18",
pruning.mode = "coarse"
)4.1 Generate editing site read counts using pileup_sites
The pileup_sites() function will process BAM files and calculate base counts
at each supplied position. The FilterParam() will again be used to specify
parameters to exclude reads and bases based on commonly used filters for
detecting RNA-editing events. Specific regions can also be queried using the
region argument which accepts a samtools style region specification string
(e.g. chr or chr:start-end).
fp <- FilterParam(
only_keep_variants = TRUE, # only report sites with variants
trim_5p = 5, # bases to remove from 5' or 3' end
trim_3p = 5,
min_base_quality = 30, # minimum base quality score
min_mapq = 255, # minimum MAPQ read score
library_type = "fr-first-strand", # library type
min_splice_overhang = 10 # minimum required splice site overhang
)
rse <- pileup_sites(bam_files,
fasta = fafn,
sites = chr_18_editing_sites,
chroms = "chr18",
param = fp
)
rse## class: RangedSummarizedExperiment
## dim: 6192 6
## metadata(0):
## assays(7): ALT nRef ... nC nG
## rownames(6192): site_chr18_178100_1 site_chr18_184553_1 ...
## site_chr18_80172518_2 site_chr18_80174441_2
## rowData names(4): REF rpbz vdb sor
## colnames(6): SRR5564260 SRR5564261 ... SRR5564271 SRR5564277
## colData names(1): samplePileup data is stored in a RangedSummarizedExperiment object which facilitates
comparisons across samples and conveniently stores genomic coordinate
information. The rowData() and rowRanges() slots are populated with the
reference base (REF) and information related to each editing site, and
similarly the colData() slot can be used to store sample metadata.
The base counts and other information are stored in different assays within the
object. REF and ALT bases and base count data are all provided in a stand
specific fashion depending on the supplied library-type parameter. The REF
and ALT bases are in reference to the strand.
assays(rse)## List of length 7
## names(7): ALT nRef nAlt nA nT nC nGassay(rse, "nA")[1:5, ]## SRR5564260 SRR5564261 SRR5564269 SRR5564270 SRR5564271
## site_chr18_178100_1 2 0 0 1 1
## site_chr18_184553_1 0 1 1 2 3
## site_chr18_184659_1 2 0 1 1 2
## site_chr18_184747_1 1 4 2 4 3
## site_chr18_185203_1 1 0 1 1 1
## SRR5564277
## site_chr18_178100_1 2
## site_chr18_184553_1 3
## site_chr18_184659_1 1
## site_chr18_184747_1 2
## site_chr18_185203_1 0assay(rse, "nG")[1:5, ]## SRR5564260 SRR5564261 SRR5564269 SRR5564270 SRR5564271
## site_chr18_178100_1 0 0 0 0 1
## site_chr18_184553_1 0 0 0 0 1
## site_chr18_184659_1 0 0 0 0 1
## site_chr18_184747_1 0 0 0 0 0
## site_chr18_185203_1 0 0 0 0 0
## SRR5564277
## site_chr18_178100_1 0
## site_chr18_184553_1 0
## site_chr18_184659_1 0
## site_chr18_184747_1 1
## site_chr18_185203_1 0Next we’ll add sample information which will be needed for identify sites with differential editing frequencies across genotypes.
colData(rse)$treatment <- "Interferon beta"
colData(rse)$genotype <- factor(rep(c("ADAR1KO", "Wildtype"), each = 3))
colData(rse)## DataFrame with 6 rows and 3 columns
## sample treatment genotype
## <character> <character> <factor>
## SRR5564260 SRR5564260 Interferon beta ADAR1KO
## SRR5564261 SRR5564261 Interferon beta ADAR1KO
## SRR5564269 SRR5564269 Interferon beta ADAR1KO
## SRR5564270 SRR5564270 Interferon beta Wildtype
## SRR5564271 SRR5564271 Interferon beta Wildtype
## SRR5564277 SRR5564277 Interferon beta Wildtype4.2 Prepare for differential editing
raer provides the calc_edit_frequency function to calculate the editing
percentage and read depth at each position. With the drop = TRUE argument we
will also exclude non-adenosine sites. The editing frequencies will not be used
for differential editing analysis, which will be conducted using the raw counts,
however these are useful for filtering and visualization. calc_edit_frequency
will add two additional assays to the object, the editing frequency
(edit_freq) and read depth, both computed based on the edit_to and
edit_from counts.
rse <- calc_edit_frequency(rse,
edit_from = "A",
edit_to = "G",
drop = TRUE
)We’ll next filter to exclude low frequency editing events. For this analysis we require that an editing site shows editing in at least 1 sample and has at least 5 counts in each sample.
has_editing <- rowSums(assay(rse, "edit_freq") > 0) >= 1
has_depth <- rowSums(assay(rse, "depth") >= 5) == ncol(rse)
rse <- rse[has_editing & has_depth, ]
rse## class: RangedSummarizedExperiment
## dim: 612 6
## metadata(0):
## assays(9): ALT nRef ... depth edit_freq
## rownames(612): site_chr18_204626_1 site_chr18_212426_1 ...
## site_chr18_79984359_2 site_chr18_79984760_2
## rowData names(4): REF rpbz vdb sor
## colnames(6): SRR5564260 SRR5564261 ... SRR5564271 SRR5564277
## colData names(5): sample treatment genotype n_sites edit_idxOnce the object has been filtered, we will transform it into an alternative data
structure for differential editing analysis that contains an assay with read
counts of both the ALT and REF alleles in a single matrix.
deobj <- make_de_object(rse, min_prop = 0.05, min_samples = 3)
assay(deobj, "counts")[1:3, c(1, 7, 2, 8)]## SRR5564260_ref SRR5564260_alt SRR5564261_ref SRR5564261_alt
## site_chr18_691546_2 8 0 6 0
## site_chr18_691578_2 8 0 7 0
## site_chr18_692372_2 6 0 9 04.3 Run differential editing
At this stage, you can use the object to perform differential yourself or use
find_de_sites() to use edgeR or DESeq2 to identify condition specific
editing events. For differential editing, we use the design
design <- ~0 + condition:sample + condition:count and perform testing to
compare the edited read counts against unedited read counts.
deobj$sample <- factor(deobj$sample)
de_results <- find_de_sites(deobj,
test = "DESeq2",
sample_col = "sample",
condition_col = "genotype",
condition_control = "Wildtype",
condition_treatment = "ADAR1KO"
)This returns a list containing the dds object, the full results, the significant results, and the model matrix.
de_results$sig_results[1:5, ]## baseMean log2FoldChange lfcSE stat pvalue
## site_chr18_23296417_2 15.500000 -2.450652 0.8459822 -2.896813 0.003769742
## site_chr18_32070144_1 6.666667 -2.631946 1.0760283 -2.445983 0.014445798
## site_chr18_21632030_1 10.000000 -3.377617 1.4959882 -2.257783 0.023959185
## site_chr18_21631237_1 6.666667 -3.381984 1.5541279 -2.176130 0.029545546
## site_chr18_35263230_2 9.333333 -3.403508 1.4894626 -2.285057 0.022309459
## padj
## site_chr18_23296417_2 0.008216103
## site_chr18_32070144_1 0.024557856
## site_chr18_21632030_1 0.036474262
## site_chr18_21631237_1 0.044059148
## site_chr18_35263230_2 0.035633191library(ComplexHeatmap)
top_sites <- rownames(de_results$sig_results)[1:20]
Heatmap(assay(rse, "edit_freq")[top_sites, ],
name = "editing frequency",
column_labels = paste0(rse$genotype, "-", rse$treatment)
)
As anticipated the top identified sites are those with greatly reduced editing in the ADAR1KO samples.
4.4 Examine overall editing activites using the Alu Editing Index
For some studies it is informative to assess the overall ADAR editing activity in addition to examining individual editing sites. The Alu Editing Index (AEI), developed by Roth, Levanon, and Eisenberg (2019), is a metric that summarizes that amount of editing occurring at ALU elements which account for the vast majority of A-to-I editing (> 99%) in humans.
raer provides calc_AEI(), based on this approach, to calculate the AEI
metric. Many of the same parameters used for pileup_sites() are available in
calc_AEI().
First we will use the AnnotationHub package to obtain coordinates for ALU
elements in the human genome. For this example we will only examine a subset of
ALUs on chr18. We will also use a SNPlocs package, based on the dbSNP
database, to exclude any SNPs overlapping the ALU elements from the AEI
calculation. The SNP coordinates are NCBI based, whereas the ALU elements
are based on hg38, we will therefore convert between the two as needed to
obtain SNP and ALU element coordinates based on hg38.
library(AnnotationHub)
library(SNPlocs.Hsapiens.dbSNP144.GRCh38)
ah <- AnnotationHub()
rmsk_hg38 <- ah[["AH99003"]]
alus <- rmsk_hg38[rmsk_hg38$repFamily == "Alu", ]
alus <- alus[seqnames(alus) == "chr18", ]
alus <- keepStandardChromosomes(alus)
alus <- alus[1:1000, ]
seqlevelsStyle(alus) <- "NCBI"
genome(alus) <- "GRCh38.p2"
alu_snps <- get_overlapping_snps(alus, SNPlocs.Hsapiens.dbSNP144.GRCh38)
seqlevelsStyle(alu_snps) <- "UCSC"
alu_snps[1:3, ]## UnstitchedGPos object with 3 positions and 0 metadata columns:
## seqnames pos strand
## <Rle> <integer> <Rle>
## [1] chr18 21651 *
## [2] chr18 21654 *
## [3] chr18 21667 *
## -------
## seqinfo: 25 sequences (1 circular) from hg38 genomeseqlevelsStyle(alus) <- "UCSC"
alus[1:3, ]## GRanges object with 3 ranges and 11 metadata columns:
## seqnames ranges strand | swScore milliDiv milliDel milliIns
## <Rle> <IRanges> <Rle> | <integer> <numeric> <numeric> <numeric>
## [1] chr18 21645-21819 + | 1319 114 0 0
## [2] chr18 26052-26327 + | 1539 199 7 0
## [3] chr18 31708-32021 + | 2192 140 0 3
## genoLeft repName repClass repFamily repStart repEnd
## <integer> <character> <character> <character> <integer> <integer>
## [1] -80351466 AluSq2 SINE Alu 136 310
## [2] -80346958 AluJr SINE Alu 1 278
## [3] -80341264 AluSp SINE Alu 1 313
## repLeft
## <integer>
## [1] -3
## [2] -34
## [3] 0
## -------
## seqinfo: 25 sequences (1 circular) from hg38 genomecalc_AEI() returns a matrix containing the AEI calculated for all allelic
combinations and a more detailed table containing values for each chromosome.
alu_index <- calc_AEI(bam_files,
fasta = fafn,
snp_db = alu_snps,
alu_ranges = alus,
param = fp
)
names(alu_index)## [1] "AEI" "AEI_per_chrom"Heatmap(alu_index$AEI,
name = "AEI",
row_labels = rse$genotype[match(rownames(alu_index$AEI), rse$sample)]
)
The AEI in the Wildtype samples is highest for A-to-G, and sharply
reduced in the ADAR1KO samples as expected.
5 Novel RNA editing site detection
Next we will demonstrate how to identify novel RNA editing sites using the raer package. It is best practice to have a matched DNA sequencing dataset to exclude sample specific genetic variation and common alignment artifacts. However, high confidence editing sites can also be identified if the dataset contains many samples and there are high coverage SNP databases for the organism queried. Additionally high confidence editing sites can also be identified if a dataset contains a sample with reduced or absent ADAR activity. A false-positive rate estimate can be obtained by examining the proportion of A->I editing sites recovered, relative to other variants, (e.g. G->C, C->A).
In this analysis a published RNA-seq and whole genome sequencing dataset will be
analyzed. High coverage whole-genome sequencing was conducted ERR262997 along
with paired-end RNA-seq SRR1258218
in a human cell line (NA12878).
Aligned BAM files, a genome FASTA file, and a GRanges object containing SNPs
corresponding to the first 1Mb region of chr4 have been prepared for this
vignette and can be downloaded and cached using NA12878().
rna_wgs <- NA12878()
names(rna_wgs)## [1] "bams" "fasta" "snps"Additionally we will use the following additional annotation resources:
A database of known SNPs, for example from the
SNPlocs.Hsapiens.dbSNP144.GRCh38package. Due to space and memory constraints in this vignette we will only examine SNPs from the first 1Mb region of chr4.TxDb.Hsapiens.UCSC.hg38.knownGene, a database of transcript models. Alternatively these can be generated from a.gtffile usingmakeTxDbFromGRanges()from thetxdbmakerpackage.RepeatMasker annotations, which can be obtained from the
AnnotationHub()for hg38, as shown in the bulk RNA-seq tutorial.
library(TxDb.Hsapiens.UCSC.hg38.knownGene)
txdb <- TxDb.Hsapiens.UCSC.hg38.knownGene
chr4snps <- rna_wgs$snpsThe pileup_sites() function accept multiple BAM files, here we supply one
from RNA-seq, and one from whole genome sequencing. A subset of the filtering
parameters (FilterParam()) can accept multiple arguments matched to each of
the input BAM files. This allows us to have distinct settings for the WGS and
RNA-seq BAM files.
bams <- rna_wgs$bams
names(bams) <- c("rna", "dna")
fp <- FilterParam(
min_depth = 1, # minimum read depth across all samples
min_base_quality = 30, # minimum base quality
min_mapq = c(255, 30), # minimum MAPQ for each BAM file
library_type = c("fr-first-strand", "unstranded"), # sample library-types
trim_5p = 5, # bases to trim from 5' end of alignment
trim_3p = 5, # bases to trim from 3' end of alignment
indel_dist = 4, # ignore read if contains an indel within distance from site
min_splice_overhang = 10, # required spliced alignment overhang
read_bqual = c(0.25, 20), # fraction of the read with base quality
only_keep_variants = c(TRUE, FALSE), # report site if rnaseq BAM has variant
report_multiallelic = FALSE, # exclude sites with multiple variant alleles
)
rse <- pileup_sites(bams,
fasta = rna_wgs$fasta,
chroms = "chr4",
param = fp
)
rse## class: RangedSummarizedExperiment
## dim: 1035 2
## metadata(0):
## assays(7): ALT nRef ... nC nG
## rownames(1035): site_chr4_40244_2 site_chr4_44338_2 ...
## site_chr4_995145_1 site_chr4_998975_1
## rowData names(4): REF rpbz vdb sor
## colnames(2): rna dna
## colData names(1): sampleNext we filter to keep those sites with a variant in the RNA-seq, but no variant
in the DNA-seq, and a minimum of 5 reads covering the site in the DNA-seq. The
DNA-seq data is unstranded, and therefore will be reported on the “+” strand
whereas the RNA-seq data will be reported on expressing RNA strand.
We therefore use subsetByOverlaps(..., ignore.strand = TRUE) to retain sites
passing these DNA-seq based filters independent of strand.
to_keep <- (assay(rse, "nRef")[, "dna"] >= 5 &
assay(rse, "ALT")[, "dna"] == "-")
rse <- subsetByOverlaps(rse, rse[to_keep, ], ignore.strand = TRUE)
nrow(rse)## [1] 339Next we filter to remove any multiallelic sites. These sites are stored as
comma-separated strings in the ALT assay (e.g. G,C). Non-variant sites are
stored as -. filter_multiallelic() will remove any sites that have multiple
variants in the samples present in the summarizedExperiment object. It will
add a new column to the rowData() to indicate the variant for each site, and
will calculate an edit_freq assay with variant allele frequencies for each
sample.
rse <- filter_multiallelic(rse)
rse <- calc_edit_frequency(rse)
rowData(rse)## DataFrame with 260 rows and 5 columns
## REF rpbz vdb sor ALT
## <character> <numeric> <numeric> <numeric> <character>
## site_chr4_124551_1 G -1.138153 Inf 1.42563 A
## site_chr4_124611_1 T -0.266758 Inf 1.60944 G
## site_chr4_124781_1 T -1.656293 Inf 1.45499 G
## site_chr4_124820_1 C -0.600205 Inf 1.51787 A
## site_chr4_124940_1 C -0.754898 Inf 1.24398 G
## ... ... ... ... ... ...
## site_chr4_992535_1 G -0.661008 Inf 1.499465 T
## site_chr4_993342_1 A 0.802887 Inf 0.260455 G
## site_chr4_994239_1 A 1.412126 Inf 1.425634 G
## site_chr4_995144_1 A 0.387298 Inf 1.491655 G
## site_chr4_995145_1 A 0.522233 Inf 1.464766 GNext we’ll remove sites in simple repeat regions. We will add repeat information
to the rowData() using the annot_from_gr() function.
# subset both to chromosome 4 to avoid warning about different seqlevels
seqlevels(rse, pruning.mode = "coarse") <- "chr4"
seqlevels(rmsk_hg38, pruning.mode = "coarse") <- "chr4"
rse <- annot_from_gr(rse,
rmsk_hg38,
cols_to_map = c("repName", "repClass", "repFamily")
)
rowData(rse)[c("repName", "repFamily")]## DataFrame with 260 rows and 2 columns
## repName repFamily
## <Rle> <Rle>
## site_chr4_124551_1 NA NA
## site_chr4_124611_1 NA NA
## site_chr4_124781_1 NA NA
## site_chr4_124820_1 NA NA
## site_chr4_124940_1 NA NA
## ... ... ...
## site_chr4_992535_1 NA NA
## site_chr4_993342_1 NA NA
## site_chr4_994239_1 AluJr Alu
## site_chr4_995144_1 NA NA
## site_chr4_995145_1 NA NArse <- rse[!rowData(rse)$repFamily %in% c("Simple_repeat", "Low_complexity")]Next we’ll remove sites adjacent to other sites with different variant types. For example if an A->G variant is located proximal to a C->T variant then the variants will be removed.
seqlevels(txdb, pruning.mode = "coarse") <- "chr4"
rse <- filter_clustered_variants(rse, txdb, variant_dist = 100)
rse## class: RangedSummarizedExperiment
## dim: 159 2
## metadata(0):
## assays(9): ALT nRef ... depth edit_freq
## rownames(159): site_chr4_124940_1 site_chr4_126885_1 ...
## site_chr4_995144_1 site_chr4_995145_1
## rowData names(8): REF rpbz ... repClass repFamily
## colnames(2): rna dna
## colData names(3): sample n_sites edit_idxNext, we’ll annotate if the site is a known SNP and remove any known SNPs. If
using a SNPlocs package you can use the annot_snps() function, which also
allows one to compare the variant base to the SNP variant base. Here we will
use the annot_from_gr() function to annotate using the chr4snps object
and coarsely remove any editing sites overlapping the same position as a SNP.
rse <- annot_from_gr(rse, chr4snps, "name")
rowData(rse)[c("name")]## DataFrame with 159 rows and 1 column
## name
## <Rle>
## site_chr4_124940_1 rs1361822847
## site_chr4_126885_1 NA
## site_chr4_126955_1 NA
## site_chr4_133310_1 NA
## site_chr4_133971_1 rs998760640
## ... ...
## site_chr4_992535_1 rs1714447589
## site_chr4_993342_1 NA
## site_chr4_994239_1 NA
## site_chr4_995144_1 rs1217215121
## site_chr4_995145_1 NArse <- rse[is.na(rowData(rse)$name), ]
rse## class: RangedSummarizedExperiment
## dim: 100 2
## metadata(0):
## assays(9): ALT nRef ... depth edit_freq
## rownames(100): site_chr4_126885_1 site_chr4_126955_1 ...
## site_chr4_994239_1 site_chr4_995145_1
## rowData names(9): REF rpbz ... repFamily name
## colnames(2): rna dna
## colData names(3): sample n_sites edit_idxLastly, we’ll further filter the edit sites to require that the editing frequency is > 0.05 and that at least 2 reads support the editing site.
to_keep <- assay(rse, "edit_freq")[, 1] > 0.05
rse <- rse[to_keep, ]
rse <- rse[assay(rse, "nAlt")[, 1] >= 2]With the above filtering approach we obtain a set of putative editing sites. The specificity of the filtering can be estimated by examining the number of A-to-G changes relative to other variants. A-to-I RNA editing is more common than other types of editing (e.g. C->U editing by APOBEC enzymes) in human datasets so the majority of the variants should by A-to-G. In this vignette data all of the identified sites are A-to-G.
rowRanges(rse)## GRanges object with 10 ranges and 9 metadata columns:
## seqnames ranges strand | REF rpbz
## <Rle> <IRanges> <Rle> | <character> <numeric>
## site_chr4_126885_1 chr4 126885 + | A 0.342818
## site_chr4_134353_1 chr4 134353 + | A -0.264728
## site_chr4_348458_1 chr4 348458 + | A -0.252166
## site_chr4_378548_1 chr4 378548 + | A 1.406115
## site_chr4_379540_1 chr4 379540 + | A -1.949107
## site_chr4_379558_1 chr4 379558 + | A -0.803727
## site_chr4_380392_1 chr4 380392 + | A -0.207575
## site_chr4_380573_1 chr4 380573 + | A -1.918805
## site_chr4_778739_2 chr4 778739 - | A 1.109806
## site_chr4_778780_2 chr4 778780 - | A -1.469323
## vdb sor ALT repName repClass repFamily
## <numeric> <numeric> <character> <Rle> <Rle> <Rle>
## site_chr4_126885_1 0.7000000 1.061473 G <NA> <NA> <NA>
## site_chr4_134353_1 0.4310148 0.252306 G <NA> <NA> <NA>
## site_chr4_348458_1 0.8000000 0.168623 G AluSx1 SINE Alu
## site_chr4_378548_1 0.2800000 2.833213 G <NA> <NA> <NA>
## site_chr4_379540_1 0.2200000 1.243978 G <NA> <NA> <NA>
## site_chr4_379558_1 0.0200000 0.446287 G <NA> <NA> <NA>
## site_chr4_380392_1 0.5689852 0.910414 G AluSq2 SINE Alu
## site_chr4_380573_1 0.1000000 2.285077 G AluSq2 SINE Alu
## site_chr4_778739_2 0.1918587 0.604657 G AluSg SINE Alu
## site_chr4_778780_2 0.0490877 2.821974 G AluSg SINE Alu
## name
## <Rle>
## site_chr4_126885_1 <NA>
## site_chr4_134353_1 <NA>
## site_chr4_348458_1 <NA>
## site_chr4_378548_1 <NA>
## site_chr4_379540_1 <NA>
## site_chr4_379558_1 <NA>
## site_chr4_380392_1 <NA>
## site_chr4_380573_1 <NA>
## site_chr4_778739_2 <NA>
## site_chr4_778780_2 <NA>
## -------
## seqinfo: 1 sequence from an unspecified genomeFinally once a set of sites has been identified, additional packages in the Bioconductor ecosystem, such as the VariantAnnotation package, can be used to determine the genomic context and potential molecular consequences of the editing event.
6 R session information
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: /home/biocbuild/bbs-3.20-bioc/R/lib/libRblas.so
## LAPACK: /usr/lib/x86_64-linux-gnu/lapack/liblapack.so.3.12.0
##
## locale:
## [1] LC_CTYPE=en_US.UTF-8 LC_NUMERIC=C
## [3] LC_TIME=en_GB 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: America/New_York
## tzcode source: system (glibc)
##
## attached base packages:
## [1] grid stats4 stats graphics grDevices utils datasets
## [8] methods base
##
## other attached packages:
## [1] TxDb.Hsapiens.UCSC.hg38.knownGene_3.20.0
## [2] GenomicFeatures_1.58.0
## [3] AnnotationDbi_1.68.0
## [4] BSgenome.Hsapiens.UCSC.hg38_1.4.5
## [5] SNPlocs.Hsapiens.dbSNP144.GRCh38_0.99.20
## [6] BSgenome_1.74.0
## [7] rtracklayer_1.66.0
## [8] BiocIO_1.16.0
## [9] AnnotationHub_3.14.0
## [10] BiocFileCache_2.14.0
## [11] dbplyr_2.5.0
## [12] ComplexHeatmap_2.22.0
## [13] Rsamtools_2.22.0
## [14] Biostrings_2.74.0
## [15] XVector_0.46.0
## [16] scater_1.34.0
## [17] ggplot2_3.5.1
## [18] scuttle_1.16.0
## [19] SingleCellExperiment_1.28.0
## [20] SummarizedExperiment_1.36.0
## [21] Biobase_2.66.0
## [22] MatrixGenerics_1.18.0
## [23] matrixStats_1.4.1
## [24] GenomicRanges_1.58.0
## [25] GenomeInfoDb_1.42.0
## [26] IRanges_2.40.0
## [27] S4Vectors_0.44.0
## [28] BiocGenerics_0.52.0
## [29] raerdata_1.3.0
## [30] raer_1.4.0
## [31] BiocStyle_2.34.0
##
## loaded via a namespace (and not attached):
## [1] RColorBrewer_1.1-3 shape_1.4.6.1 jsonlite_1.8.9
## [4] magrittr_2.0.3 ggbeeswarm_0.7.2 magick_2.8.5
## [7] farver_2.1.2 rmarkdown_2.28 GlobalOptions_0.1.2
## [10] zlibbioc_1.52.0 vctrs_0.6.5 Cairo_1.6-2
## [13] memoise_2.0.1 RCurl_1.98-1.16 tinytex_0.53
## [16] htmltools_0.5.8.1 S4Arrays_1.6.0 curl_5.2.3
## [19] BiocNeighbors_2.0.0 SparseArray_1.6.0 sass_0.4.9
## [22] bslib_0.8.0 cachem_1.1.0 GenomicAlignments_1.42.0
## [25] iterators_1.0.14 mime_0.12 lifecycle_1.0.4
## [28] pkgconfig_2.0.3 rsvd_1.0.5 Matrix_1.7-1
## [31] R6_2.5.1 fastmap_1.2.0 clue_0.3-65
## [34] GenomeInfoDbData_1.2.13 digest_0.6.37 colorspace_2.1-1
## [37] DESeq2_1.46.0 irlba_2.3.5.1 ExperimentHub_2.14.0
## [40] RSQLite_2.3.7 beachmat_2.22.0 filelock_1.0.3
## [43] labeling_0.4.3 fansi_1.0.6 httr_1.4.7
## [46] abind_1.4-8 compiler_4.4.1 doParallel_1.0.17
## [49] bit64_4.5.2 withr_3.0.2 BiocParallel_1.40.0
## [52] viridis_0.6.5 DBI_1.2.3 highr_0.11
## [55] rappdirs_0.3.3 DelayedArray_0.32.0 rjson_0.2.23
## [58] tools_4.4.1 vipor_0.4.7 beeswarm_0.4.0
## [61] glue_1.8.0 restfulr_0.0.15 cluster_2.1.6
## [64] generics_0.1.3 gtable_0.3.6 BiocSingular_1.22.0
## [67] ScaledMatrix_1.14.0 utf8_1.2.4 foreach_1.5.2
## [70] ggrepel_0.9.6 BiocVersion_3.20.0 pillar_1.9.0
## [73] circlize_0.4.16 dplyr_1.1.4 lattice_0.22-6
## [76] bit_4.5.0 tidyselect_1.2.1 locfit_1.5-9.10
## [79] knitr_1.48 gridExtra_2.3 bookdown_0.41
## [82] xfun_0.48 pheatmap_1.0.12 UCSC.utils_1.2.0
## [85] yaml_2.3.10 evaluate_1.0.1 codetools_0.2-20
## [88] tibble_3.2.1 BiocManager_1.30.25 cli_3.6.3
## [91] munsell_0.5.1 jquerylib_0.1.4 Rcpp_1.0.13
## [94] png_0.1-8 XML_3.99-0.17 parallel_4.4.1
## [97] blob_1.2.4 bitops_1.0-9 viridisLite_0.4.2
## [100] scales_1.3.0 purrr_1.0.2 crayon_1.5.3
## [103] GetoptLong_1.0.5 rlang_1.1.4 cowplot_1.1.3
## [106] KEGGREST_1.46.0Bonfield, James K, John Marshall, Petr Danecek, Heng Li, Valeriu Ohan, Andrew Whitwham, Thomas Keane, and Robert M Davies. 2021. “HTSlib: C Library for Reading/Writing High-Throughput Sequencing Data.” Gigascience 10 (2). https://doi.org/10.1093/gigascience/giab007.
Mansi, Luigi, Marco Antonio Tangaro, Claudio Lo Giudice, Tiziano Flati, Eli Kopel, Amos Avraham Schaffer, Tiziana Castrignanò, Giovanni Chillemi, Graziano Pesole, and Ernesto Picardi. 2021. “REDIportal: Millions of Novel A-to-I RNA Editing Events from Thousands of RNAseq Experiments.” Nucleic Acids Res. 49 (D1): D1012–D1019. https://doi.org/10.1093/nar/gkaa916.
Roth, Shalom Hillel, Erez Y Levanon, and Eli Eisenberg. 2019. “Genome-Wide Quantification of ADAR Adenosine-to-Inosine RNA Editing Activity.” Nat. Methods 16 (11): 1131–8. https://doi.org/10.1038/s41592-019-0610-9.