# RNAmodR: analyzing high throughput sequencing data for post-transcriptional RNA modification footprints

Felix G.M. Ernst and Denis L.J. Lafontaine

#### 2024-10-30

#### Package

RNAmodR 1.20.0

# 1 Introduction

Post-transcriptional modifications can be found abundantly in rRNA and tRNA and can be detected classically via several strategies. However, difficulties arise if the identity and the position of the modified nucleotides is to be determined at the same time. Classically, a primer extension, a form of reverse transcription (RT), would allow certain modifications to be accessed by blocks during the RT or changes in the cDNA sequences. Other modification would need to be selectively treated by chemical reactions to influence the outcome of the reverse transcription.

With the increased availability of high throughput sequencing, these classical methods were adapted to high throughput methods allowing more RNA molecules to be accessed at the same time. With these advances post-transcriptional modifications were also detected on mRNA. Among these high throughput techniques are for example Pseudo-Seq (Carlile et al. 2014), RiboMethSeq (Birkedal et al. 2015) and AlkAnilineSeq (Marchand et al. 2018) each able to detect a specific type of modification from footprints in RNA-Seq data prepared with the selected methods.

Since similar pattern can be observed from some of these techniques, overlaps of the bioinformatical pipeline already are and will become more frequent with new emerging sequencing techniques.

`RNAmodR` implements classes and a workflow to detect post-transcriptional RNA modifications in high throughput sequencing data. It is easily adaptable to new methods and can help during the phase of initial method development as well as more complex screenings.

Briefly, from the `SequenceData`, specific subclasses are derived for accessing specific aspects of aligned reads, e.g. 5’-end positions or pileup data. With this a `Modifier` class can be used to detect specific patterns for individual types of modifications. The `SequenceData` classes can be shared by different `Modifier` classes allowing easy adaptation to new methods.

    ## Warning: replacing previous import 'utils::findMatches' by
    ## 'S4Vectors::findMatches' when loading 'ExperimentHubData'

    library(rtracklayer)
    library(Rsamtools)
    library(GenomicFeatures)
    library(txdbmaker)
    library(RNAmodR.Data)
    library(RNAmodR)

## 1.1 SequenceData

Each `SequenceData` object is created with a named character vector, which can be coerced to a `BamFileList`, or named `BamFileList`. The names must be either “treated” or “control” describing the condition the data file belongs to. Multiple files can be given per condition and are used as replicates.

    annotation <- GFF3File(RNAmodR.Data.example.gff3())
    sequences <- RNAmodR.Data.example.fasta()
    files <- c(Treated = RNAmodR.Data.example.bam.1(),
               Treated = RNAmodR.Data.example.bam.2(),
               Treated = RNAmodR.Data.example.bam.3())

For `annotation` and `sequences` several input are accepted. `annotation` can be a `GRangesList`, a `GFF3File` or a `TxDb` object. Internally, a `GFF3File` is converted to a `TxDb` object and a `GRangesList` is retrieved using the `exonsBy` function.

    seqdata <- End5SequenceData(files, annotation = annotation,
                                sequences = sequences)

    ## Import genomic features from the file as a GRanges object ... OK
    ## Prepare the 'metadata' data frame ... OK
    ## Make the TxDb object ... OK
    ## Loading 5'-end position data from BAM files ... OK

    seqdata

    ## End5SequenceData with 60 elements containing 3 data columns and 3 metadata columns
    ## - Data columns:
    ##  end5.treated.1 end5.treated.2 end5.treated.3
    ##       <integer>      <integer>      <integer>
    ## -  Seqinfo object with 84 sequences from an unspecified genome; no seqlengths:

`SequenceData` extends from a `CompressedSplitDataFrameList` and contains the data per transcript alongside the annotation information and the sequence. The additional data stored within the `SequenceData` can be accessed by several functions.

    names(seqdata) # matches the transcript names as returned by a TxDb object
    colnames(seqdata) # returns a CharacterList of all column names
    bamfiles(seqdata)
    ranges(seqdata) # generate from a TxDb object
    sequences(seqdata)
    seqinfo(seqdata)

Currently the following `SequenceData` classes are implemented:

- `End5SequenceData`
- `End3SequenceData`
- `EndSequenceData`
- `ProtectedEndSequenceData`
- `CoverageSequenceData`
- `PileupSequenceData`
- `NormEnd5SequenceData`
- `NormEnd3SequenceData`

The data types and names of the columns are different for most of the `SequenceData` classes. As a naming convenction a descriptor is combined with the condition as defined in the files input and the replicate number. For more details please have a look at the man pages, e.g. `?End5SequenceData`.

`SequenceData` objects can be subset like a `CompressedSplitDataFrameList`. Elements are returned as a `SequenceDataFrame` dependent of the type of `SequenceData` used. For each `SequenceData` class a matching `SequenceDataFrame` is implemented.

    seqdata[1]

    ## End5SequenceData with 1 elements containing 3 data columns and 3 metadata columns
    ## - Data columns:
    ##  end5.treated.1 end5.treated.2 end5.treated.3
    ##       <integer>      <integer>      <integer>
    ## -  Seqinfo object with 84 sequences from an unspecified genome; no seqlengths:

    sdf <- seqdata[[1]]
    sdf

    ## End5SequenceDataFrame with 1649 rows and 3 columns
    ##      end5.treated.1 end5.treated.2 end5.treated.3
    ##           <integer>      <integer>      <integer>
    ## 1                 1              4              0
    ## 2                 0              2              0
    ## 3                 0              0              0
    ## 4                 0              0              0
    ## 5                 0              0              0
    ## ...             ...            ...            ...
    ## 1645              0              0              0
    ## 1646              0              0              0
    ## 1647              0              0              0
    ## 1648              0              0              0
    ## 1649              0              0              0
    ##
    ## containing a GRanges object with 1 range and 3 metadata columns:
    ##             seqnames    ranges strand |   exon_id   exon_name exon_rank
    ##                <Rle> <IRanges>  <Rle> | <integer> <character> <integer>
    ##   [1] Q0020_15S_RRNA    1-1649      + |         1       Q0020         1
    ##   -------
    ##   seqinfo: 60 sequences from an unspecified genome; no seqlengths
    ##
    ## and a 1649-letter RNAString object
    ## seq: GUAAAAAAUUUAUAAGAAUAUGAUGUUGGUUCAGAU...UGCGGUGGGCUUAUAAAUAUCUUAAAUAUUCUUACA

The `SequenceDataFrame` objects retains some accessor functions from the `SequenceData` class.

    names(sdf) # this returns the columns names of the data
    ranges(sdf)
    sequences(sdf)

Subsetting of a `SequenceDataFrame` returns a `SequenceDataFrame` or `DataFrame`, depending on whether it is subset by a column or row, respectively. The `drop` argument is ignored for column subsetting.

    sdf[,1:2]

    ## End5SequenceDataFrame with 1649 rows and 2 columns
    ##      end5.treated.1 end5.treated.2
    ##           <integer>      <integer>
    ## 1                 1              4
    ## 2                 0              2
    ## 3                 0              0
    ## 4                 0              0
    ## 5                 0              0
    ## ...             ...            ...
    ## 1645              0              0
    ## 1646              0              0
    ## 1647              0              0
    ## 1648              0              0
    ## 1649              0              0
    ##
    ## containing a GRanges object with 1 range and 3 metadata columns:
    ##             seqnames    ranges strand |   exon_id   exon_name exon_rank
    ##                <Rle> <IRanges>  <Rle> | <integer> <character> <integer>
    ##   [1] Q0020_15S_RRNA    1-1649      + |         1       Q0020         1
    ##   -------
    ##   seqinfo: 60 sequences from an unspecified genome; no seqlengths
    ##
    ## and a 1649-letter RNAString object
    ## seq: GUAAAAAAUUUAUAAGAAUAUGAUGUUGGUUCAGAU...UGCGGUGGGCUUAUAAAUAUCUUAAAUAUUCUUACA

    sdf[1:3,]

    ## DataFrame with 3 rows and 3 columns
    ##   end5.treated.1 end5.treated.2 end5.treated.3
    ##        <integer>      <integer>      <integer>
    ## 1              1              4              0
    ## 2              0              2              0
    ## 3              0              0              0

## 1.2 Modifier

Whereas, the `SequenceData` classes are used to hold the data, `Modifier` classes are used to detect certain features within high throughput sequencing data to assign the presence of specific modifications for an established pattern. The `Modifier` class (and its nucleotide specific subclasses `RNAModifier` and `DNAModifier`) is virtual and can be addapted to individual methods. For example mapped reads can be analyzed using the `ModInosine` class to reveal the presence of I by detecting a A to G conversion in normal RNA-Seq data. Therefore, `ModInosine` inherits from `RNAModifier`.

To fix the data processing and detection strategy, for each type of sequencing method a `Modifier` class can be developed alongside to detect modifications. For more information on how to develop such a class and potentially a new corresponding `SequenceData` class, please have a look at the vignette for creating a new analysis.

For now three `Modifier` classes are available:

- `ModInosine`
- `ModRiboMethSeq` from the `RNAmodR.RiboMethSeq` package
- `ModAlkAnilineSeq` from the `RNAmodR.AlkAnilineSeq` package

`Modifier` objects can use and wrap multiple `SequenceData` objects as elements of a `SequenceDataSet` class. The elements of this class are different types of `SequenceData`, which are required by the specific `Modifier` class. However, they are required to contain data for the same annotation and sequence data.

`Modifier` objects are created with the same arguments as `SequenceData` objects and will start loading the necessary `SequenceData` objects from these. In addition they will automatically start to calculate any additional scores (aggregation) and then start to search for modifications, if the optional argument `find.mod` is not set to `FALSE`.

    mi <- ModInosine(files, annotation = annotation, sequences = sequences)

    ## Import genomic features from the file as a GRanges object ... OK
    ## Prepare the 'metadata' data frame ... OK
    ## Make the TxDb object ... OK
    ## Loading Pileup data from BAM files ... OK
    ## Aggregating data and calculating scores ... Starting to search for 'Inosine' ... done.

(Hint: If you use an artificial genome, name the chromosomes chr1-chrN. It will make some things easier for subsequent visualization, which relies on the `Gviz` package)

Since the `Modifier` class wraps a `SequenceData` object the accessors to data contained within work similarly to the `SequenceData` accessors described above. What type of conditions the `Modifier` class expects/supports is usually described in the man pages of the Modifier class.

    names(mi) # matches the transcript names as returned by a TxDb object
    bamfiles(mi)
    ranges(mi) # generated from a TxDb object
    sequences(mi)
    seqinfo(mi)
    sequenceData(mi) # returns the SequenceData

### 1.2.1 Settings

The behavior of a `Modifier` class can be fine tuned using settings. The `settings()` function is a getter/setter for arguments used in the analysis and my differ between different `Modifier` classes depending on the particular strategy and whether they are implemented as flexible settings.

    settings(mi)

    ## $minCoverage
    ## [1] 10
    ##
    ## $minReplicate
    ## [1] 1
    ##
    ## $find.mod
    ## [1] TRUE
    ##
    ## $minScore
    ## [1] 0.4

    settings(mi,"minScore")

    ## [1] 0.4

    settings(mi) <- list(minScore = 0.5)
    settings(mi,"minScore")

    ## [1] 0.5

## 1.3 ModifierSet

Each `Modifier` object is able to represent one sample set with multiple replicates of data. To easily compare multiple sample sets the `ModifierSet` class is implemented.

The `ModifierSet` object is created from a named list of named character vectors or `BamFileList` objects. Each element in the list is a sample type with a corresponding name. Each entry in the character vector/`BamFileList` is a replicate (Alternatively a `ModifierSet` can also be created from a `list` of `Modifier` objects, if they are of the same type).

    sequences <- RNAmodR.Data.example.AAS.fasta()
    annotation <- GFF3File(RNAmodR.Data.example.AAS.gff3())
    files <- list("SampleSet1" = c(treated = RNAmodR.Data.example.wt.1(),
                                   treated = RNAmodR.Data.example.wt.2(),
                                   treated = RNAmodR.Data.example.wt.3()),
                  "SampleSet2" = c(treated = RNAmodR.Data.example.bud23.1(),
                                   treated = RNAmodR.Data.example.bud23.2()),
                  "SampleSet3" = c(treated = RNAmodR.Data.example.trm8.1(),
                                   treated = RNAmodR.Data.example.trm8.2()))

    msi <- ModSetInosine(files, annotation = annotation, sequences = sequences)

    ## Import genomic features from the file as a GRanges object ... OK
    ## Prepare the 'metadata' data frame ... OK
    ## Make the TxDb object ... OK
    ## 2. ModInosine analysis 'SampleSet2':
    ## Loading Pileup data from BAM files ... OK
    ## Aggregating data and calculating scores ... Starting to search for 'Inosine' ... done.
    ##
    ## 1. ModInosine analysis 'SampleSet1':
    ## Loading Pileup data from BAM files ... OK
    ## Aggregating data and calculating scores ... Starting to search for 'Inosine' ... done.
    ##
    ## 3. ModInosine analysis 'SampleSet3':
    ## Loading Pileup data from BAM files ... OK
    ## Aggregating data and calculating scores ... Starting to search for 'Inosine' ... done.

The creation of the `ModifierSet` will itself trigger the creation of a `Modifier` object each containing data from one sample set. This step is parallelized using the `BiocParallel` package. If a `Modifier` class itself uses parallel computing for its analysis, it is switched off unless `internalBP = TRUE` is set. In this case each `Modifier` object is created in sequence, allowing parallel computing during the creation of each object.

    names(msi)

    ## [1] "SampleSet1" "SampleSet2" "SampleSet3"

    msi[[1]]

    ## A ModInosine object containing PileupSequenceData with 11 elements.
    ## | Input files:
    ##    - treated: F:\biocbuild\ExperimentHub_cache/176818f6b50_2544
    ##    - treated: F:\biocbuild\ExperimentHub_cache/1768645e46e6_2546
    ##    - treated: F:\biocbuild\ExperimentHub_cache/176818bc4113_2548
    ## | Nucleotide - Modification type(s):  RNA  -  I
    ## | Modifications found: yes (6)
    ## | Settings:
    ##   minCoverage minReplicate  find.mod  minScore
    ##     <integer>    <integer> <logical> <numeric>
    ##            10            1      TRUE       0.4

Again accessors remain mostly the same as described above for the `Modifier` class returning a list of results, one element for each `Modifier` object.

    bamfiles(msi)
    ranges(msi) # generate from a TxDb object
    sequences(msi)
    seqinfo(msi)

# 2 Analysis of detected modifications

Found modifications can be retrieved from a `Modifier` or `ModifierSet` object via the `modifications()` function. The function returns a `GRanges` or `GRangesList` object, respectively, which contains the coordinates of the modifications with respect to the genome used. For example if a transcript starts at position 100 and contains a modified nucleotide at position 50 of the transcript, the returned coordinate will 150.

    mod <- modifications(msi)
    mod[[1]]

    ## GRanges object with 6 ranges and 5 metadata columns:
    ##       seqnames    ranges strand |         mod      source        type     score
    ##          <Rle> <IRanges>  <Rle> | <character> <character> <character> <numeric>
    ##   [1]     chr2        34      + |           I     RNAmodR      RNAMOD  0.900932
    ##   [2]     chr4        35      + |           I     RNAmodR      RNAMOD  0.899622
    ##   [3]     chr6        34      + |           I     RNAmodR      RNAMOD  0.984035
    ##   [4]     chr7        67      + |           I     RNAmodR      RNAMOD  0.934553
    ##   [5]     chr9         7      + |           I     RNAmodR      RNAMOD  0.709758
    ##   [6]    chr11        35      + |           I     RNAmodR      RNAMOD  0.874027
    ##            Parent
    ##       <character>
    ##   [1]           2
    ##   [2]           4
    ##   [3]           6
    ##   [4]           7
    ##   [5]           9
    ##   [6]          11
    ##   -------
    ##   seqinfo: 11 sequences from an unspecified genome; no seqlengths

To retrieve the coordinates with respect to the transcript boundaries, use the optional argument `perTranscript = TRUE`. In the example provided here, this will yield the same coordinates, since a custom genome was used for mapping of the example, which does not contain transcripts on the negative strand and per transcript chromosomes.

    mod <- modifications(msi, perTranscript = TRUE)
    mod[[1]]

    ## GRanges object with 6 ranges and 5 metadata columns:
    ##       seqnames    ranges strand |         mod      source        type     score
    ##          <Rle> <IRanges>  <Rle> | <character> <character> <character> <numeric>
    ##   [1]     chr2        34      * |           I     RNAmodR      RNAMOD  0.900932
    ##   [2]     chr4        35      * |           I     RNAmodR      RNAMOD  0.899622
    ##   [3]     chr6        34      * |           I     RNAmodR      RNAMOD  0.984035
    ##   [4]     chr7        67      * |           I     RNAmodR      RNAMOD  0.934553
    ##   [5]     chr9         7      * |           I     RNAmodR      RNAMOD  0.709758
    ##   [6]    chr11        35      * |           I     RNAmodR      RNAMOD  0.874027
    ##            Parent
    ##       <character>
    ##   [1]           2
    ##   [2]           4
    ##   [3]           6
    ##   [4]           7
    ##   [5]           9
    ##   [6]          11
    ##   -------
    ##   seqinfo: 11 sequences from an unspecified genome; no seqlengths

## 2.1 Compairing results

To compare results between samples, a `ModifierSet` as well as a definition of positions to compare are required. To construct a set of positions, we will use the intersection of all modifications found as an example.

    mod <- modifications(msi)
    coord <- unique(unlist(mod))
    coord$score <- NULL
    coord$sd <- NULL
    compareByCoord(msi,coord)

    ## DataFrame with 6 rows and 6 columns
    ##   SampleSet1 SampleSet2 SampleSet3    names positions         mod
    ##    <numeric>  <numeric>  <numeric> <factor>  <factor> <character>
    ## 1   0.900932   0.998134   0.953651       2         34           I
    ## 2   0.899622   0.856241   0.976928       4         35           I
    ## 3   0.984035   0.992012   0.993128       6         34           I
    ## 4   0.934553   0.942905   0.943773       7         67           I
    ## 5   0.709758   0.766484   0.681451       9         7            I
    ## 6   0.874027   0.971474   0.954782       11        35           I

The result can also be plotted using `plotCompareByCoord`, which accepts an optional argument `alias` to allow transcript ids to be converted to other identifiers. For this step it is probably helpful to construct a `TxDb` object right at the beginning and use it for constructing the `Modifier`/`ModifierSet` object as the `annotation` argument.

    txdb <- makeTxDbFromGFF(annotation)

    ## Import genomic features from the file as a GRanges object ... OK
    ## Prepare the 'metadata' data frame ... OK
    ## Make the TxDb object ... OK

    alias <- data.frame(tx_id = names(id2name(txdb)),
                        name = id2name(txdb))

    plotCompareByCoord(msi, coord, alias = alias)

Figure 1: Heatmap for identified Inosine positions

Additionally, the order of sample sets can be adjusted, normalized to any of the sample sets and the numbering of positions shown per transcript.

    plotCompareByCoord(msi[c(3,1,2)], coord, alias = alias, normalize = "SampleSet3",
                       perTranscript = TRUE)

Figure 2: Heatmap for identified Inosine positions with normalized scores

The calculated scores and data can be visualized along the transcripts or chunks of the transcript. With the optional argument `showSequenceData` the plotting of the sequence data in addition to the score data can be triggered by setting it to `TRUE`.

    plotData(msi, "2", from = 10L, to = 45L, alias = alias) # showSequenceData = FALSE

Figure 3: Scores along a transcript containing a A to G conversion indicating the presence of Inosine

    plotData(msi[1:2], "2", from = 10L, to = 45L, showSequenceData = TRUE, alias = alias)

Figure 4: Scores along a transcript containing a A to G conversion indicating the presence of Inosine
This figure includes the detailed pileup sequence data.

# 3 Performance measurements

Since the detection of modifications from high throughput sequencing data relies usually on thresholds for calling modifications, there is considerable interest in analyzing the performance of the method based on scores chosen and available samples. To analyse the performance, the function `plotROC()` is implemented, which is a wrapper around the functionality of the `ROCR` package (Sing et al. 2005)(#References).

For the example data used in this vignette, the information gained is rather limited and the following figure should be regarded just as a proof of concept. In addition, the use of found modifications sites as an input for `plotROC` is strongly discouraged, since defeats the purpose of the test. Therefore, please regard this aspect of the next chunk as proof of concept as well.

    plotROC(msi, coord)

Figure 5: TPR vs. FPR plot

Please have a look at `?plotROC` for additional details. Most of the functionality from the `ROCR` package is available via additional arguments, thus the output of `plotROC` can be heavily customized.

# 4 Additional informations

To have a look at metadata of reads for an analysis with `RNAmodR` the function `stats()` can be used. It can be used with a bunch of object types: `SequenceData`, `SequenceDataList`, `SequenceDataSet`, `Modifier` or `ModifierSet`. For `SequenceData*` objects, the `BamFile` to be analyzed must be provided as well, which automatically done for `Modifier*` objects. For more details have a look at `?stats`.

    stats <- stats(msi)
    stats

    ## List of length 3
    ## names(3): SampleSet1 SampleSet2 SampleSet3

    stats[["SampleSet1"]]

    ## DataFrameList of length 3
    ## names(3): treated treated treated

    stats[["SampleSet1"]][["treated"]]

    ## DataFrame with 12 rows and 6 columns
    ##     seqnames seqlength    mapped  unmapped          used     used_distro
    ##     <factor> <integer> <numeric> <numeric> <IntegerList>          <List>
    ## 1       chr1      1800    197050         0        159782 83,1252,860,...
    ## 2       chr2        85      5863         0          2459     2,16,16,...
    ## 3       chr3        76     76905         0         63497 35,478,4106,...
    ## 4       chr4        77      8299         0          6554     6,27,36,...
    ## 5       chr5        74     11758         0          8818  520,105,93,...
    ## ...      ...       ...       ...       ...           ...             ...
    ## 8      chr8         75    144293         0        143068    14,44,48,...
    ## 9      chr9         75     13790         0          9753     1,49,43,...
    ## 10     chr10        85     19861         0         17729    35,21,10,...
    ## 11     chr11        77     10532         0          9086   53,92,185,...
    ## 12     *             0         0    961095            NA              NA

The data returned by `stats()` is a `DataFrame`, which can be wrapped as a `DataFrameList` or a `SimpleList` depending on the input type. Analysis of the data must be manually done and can be used to produced output like the following plot for distribution of lengths for reads analyzed.

Figure 6: Distribution of lengths for reads used in the analysis

# 5 Further development

The development of `RNAmodR` will continue. General ascpects of the analysis workflow will be addressed in the `RNAmodR` package, whereas additional classes for new sequencing techniques targeted at detecting post-transcriptional will be wrapped in individual packages. This will allow general improvements to propagate upstream, but not hinder individual requirements of each detection strategy.

For an example have a look at the `RNAmodR.RiboMethSeq` and `RNAmodR.AlkAnilineSeq` packages.

Features, which might be added in the future:

- interaction with our packages for data aggregation (for example meta gene aggregation)
- interaction with our packages for downstream analysis for visualization

We welcome contributions of any sort.

# 6 Sessioninfo

    sessionInfo()

    ## R version 4.4.1 (2024-06-14 ucrt)
    ## Platform: x86_64-w64-mingw32/x64
    ## Running under: Windows Server 2022 x64 (build 20348)
    ##
    ## Matrix products: default
    ##
    ##
    ## locale:
    ## [1] LC_COLLATE=C
    ## [2] LC_CTYPE=English_United States.utf8
    ## [3] LC_MONETARY=English_United States.utf8
    ## [4] LC_NUMERIC=C
    ## [5] LC_TIME=English_United States.utf8
    ##
    ## time zone: America/New_York
    ## tzcode source: internal
    ##
    ## attached base packages:
    ## [1] stats4    stats     graphics  grDevices utils     datasets  methods
    ## [8] base
    ##
    ## other attached packages:
    ##  [1] RNAmodR_1.20.0           Modstrings_1.22.0        RNAmodR.Data_1.19.0
    ##  [4] ExperimentHubData_1.32.0 AnnotationHubData_1.36.0 futile.logger_1.4.3
    ##  [7] ExperimentHub_2.14.0     AnnotationHub_3.14.0     BiocFileCache_2.14.0
    ## [10] dbplyr_2.5.0             txdbmaker_1.2.0          GenomicFeatures_1.58.0
    ## [13] AnnotationDbi_1.68.0     Biobase_2.66.0           Rsamtools_2.22.0
    ## [16] Biostrings_2.74.0        XVector_0.46.0           rtracklayer_1.66.0
    ## [19] GenomicRanges_1.58.0     GenomeInfoDb_1.42.0      IRanges_2.40.0
    ## [22] S4Vectors_0.44.0         BiocGenerics_0.52.0      BiocStyle_2.34.0
    ##
    ## loaded via a namespace (and not attached):
    ##   [1] BiocIO_1.16.0               bitops_1.0-9
    ##   [3] filelock_1.0.3              tibble_3.2.1
    ##   [5] graph_1.84.0                XML_3.99-0.17
    ##   [7] rpart_4.1.23                lifecycle_1.0.4
    ##   [9] httr2_1.0.5                 lattice_0.22-6
    ##  [11] ensembldb_2.30.0            OrganismDbi_1.48.0
    ##  [13] backports_1.5.0             magrittr_2.0.3
    ##  [15] Hmisc_5.2-0                 sass_0.4.9
    ##  [17] rmarkdown_2.28              jquerylib_0.1.4
    ##  [19] yaml_2.3.10                 RUnit_0.4.33
    ##  [21] Gviz_1.50.0                 DBI_1.2.3
    ##  [23] RColorBrewer_1.1-3          abind_1.4-8
    ##  [25] zlibbioc_1.52.0             purrr_1.0.2
    ##  [27] AnnotationFilter_1.30.0     biovizBase_1.54.0
    ##  [29] RCurl_1.98-1.16             nnet_7.3-19
    ##  [31] VariantAnnotation_1.52.0    rappdirs_0.3.3
    ##  [33] GenomeInfoDbData_1.2.13     AnnotationForge_1.48.0
    ##  [35] codetools_0.2-20            DelayedArray_0.32.0
    ##  [37] xml2_1.3.6                  tidyselect_1.2.1
    ##  [39] UCSC.utils_1.2.0            farver_2.1.2
    ##  [41] matrixStats_1.4.1           base64enc_0.1-3
    ##  [43] GenomicAlignments_1.42.0    jsonlite_1.8.9
    ##  [45] Formula_1.2-5               tools_4.4.1
    ##  [47] progress_1.2.3              snow_0.4-4
    ##  [49] stringdist_0.9.12           Rcpp_1.0.13
    ##  [51] glue_1.8.0                  gridExtra_2.3
    ##  [53] SparseArray_1.6.0           BiocBaseUtils_1.8.0
    ##  [55] xfun_0.48                   MatrixGenerics_1.18.0
    ##  [57] dplyr_1.1.4                 withr_3.0.2
    ##  [59] formatR_1.14                BiocManager_1.30.25
    ##  [61] fastmap_1.2.0               latticeExtra_0.6-30
    ##  [63] fansi_1.0.6                 digest_0.6.37
    ##  [65] R6_2.5.1                    mime_0.12
    ##  [67] colorspace_2.1-1            jpeg_0.1-10
    ##  [69] dichromat_2.0-0.1           biomaRt_2.62.0
    ##  [71] RSQLite_2.3.7               utf8_1.2.4
    ##  [73] generics_0.1.3              data.table_1.16.2
    ##  [75] prettyunits_1.2.0           httr_1.4.7
    ##  [77] htmlwidgets_1.6.4           S4Arrays_1.6.0
    ##  [79] pkgconfig_2.0.3             gtable_0.3.6
    ##  [81] blob_1.2.4                  htmltools_0.5.8.1
    ##  [83] bookdown_0.41               RBGL_1.82.0
    ##  [85] ProtGenerics_1.38.0         scales_1.3.0
    ##  [87] png_0.1-8                   colorRamps_2.3.4
    ##  [89] knitr_1.48                  lambda.r_1.2.4
    ##  [91] rstudioapi_0.17.1           reshape2_1.4.4
    ##  [93] rjson_0.2.23                checkmate_2.3.2
    ##  [95] curl_5.2.3                  biocViews_1.74.0
    ##  [97] cachem_1.1.0                stringr_1.5.1
    ##  [99] BiocVersion_3.20.0          parallel_4.4.1
    ## [101] foreign_0.8-87              restfulr_0.0.15
    ## [103] pillar_1.9.0                grid_4.4.1
    ## [105] vctrs_0.6.5                 cluster_2.1.6
    ## [107] htmlTable_2.4.3             evaluate_1.0.1
    ## [109] magick_2.8.5                tinytex_0.53
    ## [111] cli_3.6.3                   compiler_4.4.1
    ## [113] futile.options_1.0.1        rlang_1.1.4
    ## [115] crayon_1.5.3                labeling_0.4.3
    ## [117] interp_1.1-6                plyr_1.8.9
    ## [119] stringi_1.8.4               deldir_2.0-4
    ## [121] BiocParallel_1.40.0         BiocCheck_1.42.0
    ## [123] munsell_0.5.1               lazyeval_0.2.2
    ## [125] Matrix_1.7-1                BSgenome_1.74.0
    ## [127] hms_1.1.3                   bit64_4.5.2
    ## [129] ggplot2_3.5.1               KEGGREST_1.46.0
    ## [131] SummarizedExperiment_1.36.0 highr_0.11
    ## [133] ROCR_1.0-11                 memoise_2.0.1
    ## [135] bslib_0.8.0                 bit_4.5.0

# References

Birkedal, Ulf, Mikkel Christensen-Dalsgaard, Nicolai Krogh, Radhakrishnan Sabarinathan, Jan Gorodkin, and Henrik Nielsen. 2015. “Profiling of Ribose Methylations in Rna by High-Throughput Sequencing.” Angewandte Chemie (International Ed. In English) 54 (2): 451–55. https://doi.org/10.1002/anie.201408362.

Carlile, Thomas M., Maria F. Rojas-Duran, Boris Zinshteyn, Hakyung Shin, Kristen M. Bartoli, and Wendy V. Gilbert. 2014. “Pseudouridine Profiling Reveals Regulated mRNA Pseudouridylation in Yeast and Human Cells.” Nature 515 (7525): 143–46.

Marchand, Virginie, Lilia Ayadi, Felix G. M. Ernst, Jasmin Hertler, Valérie Bourguignon-Igel, Adeline Galvanin, Annika Kotter, Mark Helm, Denis L. J. Lafontaine, and Yuri Motorin. 2018. “AlkAniline-Seq: Profiling of m7G and m3C Rna Modifications at Single Nucleotide Resolution.” Angewandte Chemie International Edition 57 (51): 16785–90. https://doi.org/10.1002/anie.201810946.

Sing, Tobias, Oliver Sander, Niko Beerenwinkel, and Thomas Lengauer. 2005. “ROCR: Visualizing Classifier Performance in R.” Bioinformatics (Oxford, England) 21 (20): 3940–1. https://doi.org/10.1093/bioinformatics/bti623.