Author: Felix G.M. Ernst

# RNAmodR: creating classes for additional modification detection from high throughput sequencing.

# Introduction

For users interested in the general aspect of any `RNAmodR` based package please have a look at the main vignette of the package.

This vignette is aimed at developers and researchers, who want to use the functionality of the `RNAmodR` package to develop a new modification strategy based on high throughput sequencing data.

    library(RNAmodR)

Two classes have to be considered to establish a new analysis pipeline using `RNAmodR`. These are the `SequenceData` and the `Modifier` class.

# A new `SequenceData` class

First, the `SequenceData` class has to be considered. Several classes are already implemented, which are:

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

If these cannot be reused, a new class can be implemented quite easily. First the DataFrame class, the Data class and a constructor has to defined. The only value, which has to be provided, is a default `minQuality` integer value and some basic information.

    setClass(Class = "ExampleSequenceDataFrame",
             contains = "SequenceDFrame")
    ExampleSequenceDataFrame <- function(df, ranges, sequence, replicate,
                                          condition, bamfiles, seqinfo){
      RNAmodR:::.SequenceDataFrame("Example",df, ranges, sequence, replicate,
                                   condition, bamfiles, seqinfo)
    }
    setClass(Class = "ExampleSequenceData",
             contains = "SequenceData",
             slots = c(unlistData = "ExampleSequenceDataFrame"),
             prototype = list(unlistData = ExampleSequenceDataFrame(),
                              unlistType = "ExampleSequenceDataFrame",
                              minQuality = 5L,
                              dataDescription = "Example data"))
    ExampleSequenceData <- function(bamfiles, annotation, sequences, seqinfo, ...){
      RNAmodR:::SequenceData("Example", bamfiles = bamfiles,
                             annotation = annotation, sequences = sequences,
                             seqinfo = seqinfo, ...)
    }

Second, the `getData` function has to be implemented. This is used to load the data from a bam file and must return a named list `IntegerList`, `NumericList` or `CompressedSplitDataFrameList` per file.

    setMethod("getData",
              signature = c(x = "ExampleSequenceData",
                            bamfiles = "BamFileList",
                            grl = "GRangesList",
                            sequences = "XStringSet",
                            param = "ScanBamParam"),
              definition = function(x, bamfiles, grl, sequences, param, args){
                ###
              }
    )

Third, the `aggregate` function has to be implemented. This function is used to aggregate data over replicates for all or one of the conditions. The resulting data is passed on to the `Modifier` class.

    setMethod("aggregateData",
              signature = c(x = "ExampleSequenceData"),
              function(x, condition = c("Both","Treated","Control")){
                ###
              }
    )

# A new `Modifier` class

A new `Modifier` class is probably the main class, which needs to be implemented. Three variable have to be set. `mod` must be a single element from the `Modstrings::shortName(Modstrings::ModRNAString())`. `score` is the default score, which is used for several function. A column with this name should be returned from the `aggregate` function. `dataType` defines the `SequenceData` class to be used. `dataType` can contain multiple names of a `SequenceData` class, which are then combined to form a `SequenceDataSet`.

    setClass("ModExample",
             contains = c("RNAModifier"),
             prototype = list(mod = "X",
                              score = "score",
                              dataType = "ExampleSequenceData"))
    ModExample <- function(x, annotation, sequences, seqinfo, ...){
      RNAmodR:::Modifier("ModExample", x = x, annotation = annotation,
                         sequences = sequences, seqinfo = seqinfo, ...)
    }

`dataType` can also be a `list` of `character` vectors, which leads then to the creation of `SequenceDataList`. However, for now this is a hypothetical case and should only be used, if the detection of a modification requires bam files from two or more different methods to be used to detect one modification.

The `settings<-` function can be amended to save specifc settings ( `.norm_example_args` must be defined seperatly to normalize input arguments in any way one sees fit).

    setReplaceMethod(f = "settings",
                     signature = signature(x = "ModExample"),
                     definition = function(x, value){
                       x <- callNextMethod()
                       # validate special setting here
                       x@settings[names(value)] <- unname(.norm_example_args(value))
                       x
                     })

The `aggregateData` function is used to take the aggregated data from the `SequenceData` object and to calculate the specific scores, which are then stored in the `aggregate` slot.

    setMethod(f = "aggregateData",
              signature = signature(x = "ModExample"),
              definition =
                function(x, force = FALSE){
                  # Some data with element per transcript
                }
    )

The `findMod` function takes the aggregate data and searches for modifications, which are then returned as a GRanges object and stored in the `modifications` slot.

    setMethod("findMod",
              signature = c(x = "ModExample"),
              function(x){
                # an element per modification found.
              }
    )

## A new `ModifierSet` class

The `ModifierSet` class is implemented very easily by defining the class and the constructor. The functionality is defined by the `Modifier` class.

    setClass("ModSetExample",
             contains = "ModifierSet",
             prototype = list(elementType = "ModExample"))
    ModSetExample <- function(x, annotation, sequences, seqinfo, ...){
      RNAmodR:::ModifierSet("ModExample", x = x, annotation = annotation,
                            sequences = sequences, seqinfo = seqinfo, ...)
    }

# Visualization functions

Additional functions, which need to be implemented, are `getDataTrack` for the new `SequenceData` and new `Modifier` classes and `plotData`/`plotDataByCoord` for the new `Modifier` and `ModifierSet` classes. `name` defines a transcript name found in `names(ranges(x))` and `type` is the data type typically found as a column in the `aggregate` slot.

    setMethod(
      f = "getDataTrack",
      signature = signature(x = "ExampleSequenceData"),
      definition = function(x, name, ...) {
        ###
      }
    )
    setMethod(
      f = "getDataTrack",
      signature = signature(x = "ModExample"),
      definition = function(x, name, type, ...) {
      }
    )
    setMethod(
      f = "plotDataByCoord",
      signature = signature(x = "ModExample", coord = "GRanges"),
      definition = function(x, coord, type = "score", window.size = 15L, ...) {
      }
    )
    setMethod(
      f = "plotData",
      signature = signature(x = "ModExample"),
      definition = function(x, name, from, to, type = "score", ...) {
      }
    )
    setMethod(
      f = "plotDataByCoord",
      signature = signature(x = "ModSetExample", coord = "GRanges"),
      definition = function(x, coord, type = "score", window.size = 15L, ...) {
      }
    )
    setMethod(
      f = "plotData",
      signature = signature(x = "ModSetExample"),
      definition = function(x, name, from, to, type = "score", ...) {
      }
    )

If unsure, how to modify these functions, have a look a the code in the `Modifier-Inosine-viz.R` file of this package.

# Summary

As suggested directly above, for a more detailed example have a look at the `ModInosine` class source code found in the `Modifier-Inosine-class.R` and `Modifier-Inosine-viz.R` files of this package.

# Sessioninfo

    sessionInfo()

    ## R version 4.6.0 (2026-04-24)
    ## Platform: x86_64-pc-linux-gnu
    ## Running under: Ubuntu 24.04.4 LTS
    ##
    ## Matrix products: default
    ## BLAS:   /usr/lib/x86_64-linux-gnu/openblas-pthread/libblas.so.3
    ## LAPACK: /usr/lib/x86_64-linux-gnu/openblas-pthread/libopenblasp-r0.3.26.so;  LAPACK version 3.12.0
    ##
    ## locale:
    ##  [1] LC_CTYPE=en_US.UTF-8       LC_NUMERIC=C
    ##  [3] LC_TIME=en_US.UTF-8        LC_COLLATE=en_US.UTF-8
    ##  [5] LC_MONETARY=en_US.UTF-8    LC_MESSAGES=en_US.UTF-8
    ##  [7] LC_PAPER=en_US.UTF-8       LC_NAME=C
    ##  [9] LC_ADDRESS=C               LC_TELEPHONE=C
    ## [11] LC_MEASUREMENT=en_US.UTF-8 LC_IDENTIFICATION=C
    ##
    ## time zone: Etc/UTC
    ## tzcode source: system (glibc)
    ##
    ## attached base packages:
    ## [1] stats4    stats     graphics  grDevices utils     datasets  methods
    ## [8] base
    ##
    ## other attached packages:
    ##  [1] RNAmodR_1.26.0       Modstrings_1.26.0    Biostrings_2.80.0
    ##  [4] XVector_0.52.0       GenomicRanges_1.64.0 Seqinfo_1.2.0
    ##  [7] IRanges_2.46.0       S4Vectors_0.50.0     BiocGenerics_0.58.0
    ## [10] generics_0.1.4       BiocStyle_2.40.0
    ##
    ## loaded via a namespace (and not attached):
    ##   [1] RColorBrewer_1.1-3          sys_3.4.3
    ##   [3] rstudioapi_0.18.0           jsonlite_2.0.0
    ##   [5] magrittr_2.0.5              GenomicFeatures_1.64.0
    ##   [7] farver_2.1.2                rmarkdown_2.31
    ##   [9] BiocIO_1.22.0               vctrs_0.7.3
    ##  [11] ROCR_1.0-12                 memoise_2.0.1
    ##  [13] Rsamtools_2.28.0            RCurl_1.98-1.18
    ##  [15] base64enc_0.1-6             htmltools_0.5.9
    ##  [17] S4Arrays_1.12.0             progress_1.2.3
    ##  [19] curl_7.1.0                  SparseArray_1.12.0
    ##  [21] Formula_1.2-5               sass_0.4.10
    ##  [23] bslib_0.10.0                htmlwidgets_1.6.4
    ##  [25] plyr_1.8.9                  Gviz_1.56.0
    ##  [27] httr2_1.2.2                 cachem_1.1.0
    ##  [29] buildtools_1.0.0            GenomicAlignments_1.48.0
    ##  [31] lifecycle_1.0.5             pkgconfig_2.0.3
    ##  [33] Matrix_1.7-5                R6_2.6.1
    ##  [35] fastmap_1.2.0               MatrixGenerics_1.24.0
    ##  [37] digest_0.6.39               colorspace_2.1-2
    ##  [39] AnnotationDbi_1.74.0        Hmisc_5.2-5
    ##  [41] RSQLite_2.4.6               filelock_1.0.3
    ##  [43] colorRamps_2.3.4            httr_1.4.8
    ##  [45] abind_1.4-8                 compiler_4.6.0
    ##  [47] bit64_4.8.0                 htmlTable_2.5.0
    ##  [49] S7_0.2.2                    backports_1.5.1
    ##  [51] BiocParallel_1.46.0         DBI_1.3.0
    ##  [53] biomaRt_2.68.0              rappdirs_0.3.4
    ##  [55] DelayedArray_0.38.0         rjson_0.2.23
    ##  [57] tools_4.6.0                 foreign_0.8-91
    ##  [59] nnet_7.3-20                 glue_1.8.1
    ##  [61] restfulr_0.0.16             grid_4.6.0
    ##  [63] checkmate_2.3.4             reshape2_1.4.5
    ##  [65] cluster_2.1.8.2             gtable_0.3.6
    ##  [67] BSgenome_1.80.0             ensembldb_2.36.0
    ##  [69] data.table_1.18.2.1         hms_1.1.4
    ##  [71] pillar_1.11.1               stringr_1.6.0
    ##  [73] dplyr_1.2.1                 BiocFileCache_3.2.0
    ##  [75] lattice_0.22-9              rtracklayer_1.72.0
    ##  [77] bit_4.6.0                   deldir_2.0-4
    ##  [79] biovizBase_1.60.0           tidyselect_1.2.1
    ##  [81] maketools_1.3.2             knitr_1.51
    ##  [83] gridExtra_2.3               ProtGenerics_1.44.0
    ##  [85] SummarizedExperiment_1.42.0 xfun_0.57
    ##  [87] Biobase_2.72.0              matrixStats_1.5.0
    ##  [89] stringi_1.8.7               UCSC.utils_1.8.0
    ##  [91] lazyeval_0.2.3              yaml_2.3.12
    ##  [93] evaluate_1.0.5              codetools_0.2-20
    ##  [95] cigarillo_1.2.0             interp_1.1-6
    ##  [97] tibble_3.3.1                BiocManager_1.30.27
    ##  [99] cli_3.6.6                   rpart_4.1.27
    ## [101] jquerylib_0.1.4             dichromat_2.0-0.1
    ## [103] Rcpp_1.1.1-1.1              GenomeInfoDb_1.48.0
    ## [105] dbplyr_2.5.2                png_0.1-9
    ## [107] XML_3.99-0.23               parallel_4.6.0
    ## [109] ggplot2_4.0.3               blob_1.3.0
    ## [111] prettyunits_1.2.0           latticeExtra_0.6-31
    ## [113] jpeg_0.1-11                 AnnotationFilter_1.36.0
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    ## [117] VariantAnnotation_1.58.0    scales_1.4.0
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    ## [121] KEGGREST_1.52.0