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

Felix G.M. Ernst

#### 2020-04-29

#### Package

RNAmodR 1.2.1

# 1 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.

# 2 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")){
                ###
              }
    )

# 3 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.
              }
    )

## 3.1 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, ...)
    }

# 4 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.

# 5 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.

# 6 Sessioninfo

    sessionInfo()

    ## R version 4.0.0 (2020-04-24)
    ## Platform: x86_64-apple-darwin17.0 (64-bit)
    ## Running under: macOS Mojave 10.14.6
    ##
    ## Matrix products: default
    ## BLAS:   /Library/Frameworks/R.framework/Versions/4.0/Resources/lib/libRblas.dylib
    ## LAPACK: /Library/Frameworks/R.framework/Versions/4.0/Resources/lib/libRlapack.dylib
    ##
    ## locale:
    ## [1] C/en_US.UTF-8/en_US.UTF-8/C/en_US.UTF-8/en_US.UTF-8
    ##
    ## attached base packages:
    ## [1] parallel  stats4    stats     graphics  grDevices utils     datasets
    ## [8] methods   base
    ##
    ## other attached packages:
    ##  [1] RNAmodR_1.2.1            GenomeInfoDbData_1.2.3   Modstrings_1.4.0
    ##  [4] RNAmodR.Data_1.1.2       ExperimentHubData_1.14.0 AnnotationHubData_1.18.0
    ##  [7] futile.logger_1.4.3      ExperimentHub_1.14.0     AnnotationHub_2.20.0
    ## [10] BiocFileCache_1.12.0     dbplyr_1.4.3             GenomicFeatures_1.40.0
    ## [13] AnnotationDbi_1.50.0     Biobase_2.48.0           Rsamtools_2.4.0
    ## [16] Biostrings_2.56.0        XVector_0.28.0           rtracklayer_1.47.0
    ## [19] GenomicRanges_1.40.0     GenomeInfoDb_1.24.0      IRanges_2.22.1
    ## [22] S4Vectors_0.26.0         BiocGenerics_0.34.0      BiocStyle_2.16.0
    ##
    ## loaded via a namespace (and not attached):
    ##   [1] RUnit_0.4.32                  tidyselect_1.0.0
    ##   [3] RSQLite_2.2.0                 htmlwidgets_1.5.1
    ##   [5] grid_4.0.0                    BiocParallel_1.22.0
    ##   [7] munsell_0.5.0                 codetools_0.2-16
    ##   [9] colorspace_1.4-1              OrganismDbi_1.30.0
    ##  [11] highr_0.8                     knitr_1.28
    ##  [13] rstudioapi_0.11               ROCR_1.0-7
    ##  [15] assertive.base_0.0-7          labeling_0.3
    ##  [17] optparse_1.6.6                bit64_0.9-7
    ##  [19] farver_2.0.3                  vctrs_0.2.4
    ##  [21] lambda.r_1.2.4                xfun_0.13
    ##  [23] biovizBase_1.36.0             R6_2.4.1
    ##  [25] assertive.sets_0.0-3          AnnotationFilter_1.12.0
    ##  [27] bitops_1.0-6                  DelayedArray_0.14.0
    ##  [29] assertthat_0.2.1              promises_1.1.0
    ##  [31] scales_1.1.0                  nnet_7.3-14
    ##  [33] gtable_0.3.0                  biocViews_1.56.0
    ##  [35] ensembldb_2.12.0              rlang_0.4.5
    ##  [37] splines_4.0.0                 lazyeval_0.2.2
    ##  [39] acepack_1.4.1                 dichromat_2.0-0
    ##  [41] checkmate_2.0.0               BiocManager_1.30.10
    ##  [43] yaml_2.2.1                    reshape2_1.4.4
    ##  [45] backports_1.1.6               httpuv_1.5.2
    ##  [47] Hmisc_4.4-0                   RBGL_1.64.0
    ##  [49] tools_4.0.0                   bookdown_0.18
    ##  [51] ggplot2_3.3.0                 ellipsis_0.3.0
    ##  [53] gplots_3.0.3                  assertive.strings_0.0-3
    ##  [55] RColorBrewer_1.1-2            assertive.reflection_0.0-4
    ##  [57] Rcpp_1.0.4.6                  plyr_1.8.6
    ##  [59] base64enc_0.1-3               progress_1.2.2
    ##  [61] zlibbioc_1.34.0               purrr_0.3.4
    ##  [63] RCurl_1.98-1.2                prettyunits_1.1.1
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    ##  [81] xtable_1.8-4                  XML_3.99-0.3
    ##  [83] jpeg_0.1-8.1                  gridExtra_2.3
    ##  [85] compiler_4.0.0                biomaRt_2.44.0
    ##  [87] tibble_3.0.1                  assertive.datetimes_0.0-2
    ##  [89] KernSmooth_2.23-17            crayon_1.3.4
    ##  [91] htmltools_0.4.0               later_1.0.0
    ##  [93] assertive_0.3-5               Formula_1.2-3
    ##  [95] DBI_1.1.0                     formatR_1.7
    ##  [97] assertive.files_0.0-2         rappdirs_0.3.1
    ##  [99] assertive.numbers_0.0-2       Matrix_1.2-18
    ## [101] getopt_1.20.3                 assertive.types_0.0-3
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    ## [105] gdata_2.18.0                  Gviz_1.32.0
    ## [107] pkgconfig_2.0.3               GenomicAlignments_1.24.0
    ## [109] foreign_0.8-79                assertive.data.us_0.0-2
    ## [111] stringdist_0.9.5.5            AnnotationForge_1.30.0
    ## [113] BiocCheck_1.24.0              stringr_1.4.0
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    ## [117] graph_1.66.0                  assertive.code_0.0-3
    ## [119] rmarkdown_2.1                 htmlTable_1.13.3
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