The Bioconductor Project

RAdelaide 2024

Dr Stevie Pederson

Black Ochre Data Labs
Telethon Kids Institute

July 11, 2024

The Bioconductor Project

Today’s Outline

• Not just to show how to perform an RNA-Seq analysis
• Understanding common file types
• Understanding Bioconductor objects, classes & methods
  • Has relevance beyond Bioconductor (e.g. map visualisation)
• Key concepts & resources

• Please ask questions!!! It’s the advantage of being here…

CRAN

• A Package is a collection of functions
• Associated with a given task/analysis/data-type
• The main repository is “The Comprehensive R Archive Network” aka CRAN

• We install packages using Tools > Install Packages...
  • Or by typing install.packages("pkg_name")
• Will only install packages from CRAN

CRAN

• Packages on CRAN cover everything & anything
  • GIS-based spatial data, stock trading, data visualisation etc
• Some packages are biological in focus, e.g. Seurat for scRNA

• Submission involves passing technical checks
  • Tests across OSX, Windows & Linux
  • Correct directory/file structures

Packages

• Many packages are written by the authors for the authors
• Decide to make them public in case others find them helpful

• Both Chris & I use ngsReports nearly every day (still…)

Bioconductor

• Bioconductor hosts packages focussed on biological research
  • www.bioconductor.org
  • Currently > 2,400 packages
• Created by Robert Gentleman (one of the “R”s in R) in 2001

• Packages checked for programming consistency \(\implies\) not methodology
  • Expected to integrate with other Bioconductor packages
• All packages require a vignette explaining how to use the package
  • Checked manually for clarity/helpfulness during package submission
• Packages tested nightly on OSX, Windows, Debian Linux + Arch Linux

Bioconductor

• Essentially represents a community of developers & users
• Strongly encourages & supports diversity
  • No longer on Twitter \(\implies\) not a safe space
• Has a Code of Conduct
• Consciously supporting those from emerging countries
  • Actively trying to translate resources to other languages
• Also tries to find ways to engage with & support both developers & users
  • support.bioconductor.org & slack.bioconductor.org
  • Members of R Core regularly post on the Bioconductor slack

Bioconductor Structure

• Core Team
  • All employed by Bioconductor
  • Primarily grant funded (NIH, NSF, CZI)
• Scientific Advisory Board (SAB)
  • Meet Annually.
  • External and Internal leaders who act as project advisors.
• Technical Advisory Board (TAB)
  • Meet monthly
  • Consider technical aspects of core infastructure and scientific direction of the project.
  • 15 members, 3 year term. Annual, open elections to rotate members.

Bioconductor Structure

• Community Advisory Board (CAB)
  • Meet monthly
  • Consider community outreach, events, education and training.
  • 15 members, 3 year term. Annual, open elections to rotate members.
• Multiple Working Groups
  • Code of Conduct committee
  • Conference Committees, Training & Education etc

Bioconductor Packages

• R generally has bi-annual releases (R 4.4.0 April 24^th, 2024)
  • Patch-fixes as needed \(\implies\) release 4.4.1 (14^th Jun, 2024)
• CRAN packages continually update

Bioconductor Packages

• Bioconductor has two releases per year
• Tested to a set R version (or later)
• Bug-fixes also released as needed
  • Generally new features added to packages at Bioc releases
• Latest Bioconductor release is 3.19 (May 1^st 2024)
  • Was tested on R 4.4.0

Bioconductor Package Installation

• BiocManager() is a CRAN package
• Enables installation from CRAN and Bioconductor
  • Also handles package installation from github

BiocManager::install(c("pkg1", "user/pkg2"))

Bioconductor Packages

• Many packages for specific analyses
  • DESeq2 & edgeR for bulk RNA-Seq Analysis
  • DiffBind & extraChIPs for ChIP-Seq Analysis
  • fgsea for GSEA within R

• Also multiple packages define object classes & general methods
  • e.g. GenomicRanges for working with GRanges objects
  • Is a foundational class many other packages build on
• New packages are expected to use existing classes where possible

Bioconductor Packages

Taken from https://carpentries-incubator.github.io/bioc-project/02-introduction-to-bioconductor.html

Bioconductor Packages

• The output from body(function_name) always has comments removed
• All Bioconductor packages have the source code available
  • https://code.bioconductor.org
• Code is always written as a set of R Scripts
• Inside the R directory
  • Will retain comments & formatting from the authors
  • Sometimes more helpful…

BiocViews

• BiocViews provide an overview of all Bioconductor packages
• Can be very helpful when looking for a resource
• Most packages are software (for analysis)
• Also annotation packages:
  • Genome sequences, gene to GO mappings etc
• Experimental data for demonstrating workflows
• Workflow packages are slowly growing

browseVignettes()

Object Classes

Object Classes

R has two common types of objects

• Built on top of (and including) vectors, lists etc.
• S3 are very common & old (1970s)
  • Usually list-type objects e.g. results from lm() or t.test()
• S4 introduced in ’90s
  • Focus on Object-Oriented Programming (OOP)
    
• Biconductor packages rely heavily on S4 objects
  • Also common in spatial packages on CRAN (i.e. making maps etc)

Objects and Methods

• Functions can be written to handle different types of input data
• Figuring out which version of the function to use
  \(\implies\)method dispatch

• A good example is the function summary()
  • Will return different results for a vector or data.frame

summary(letters)
summary(cars)

How does summary() know what to do for different data structures?

Objects and Methods

• If we try to look at the code used in summary() it’s a bit odd

body(summary)

UseMethod("summary")

• summary() uses different methods depending on the object class
• Sometimes they’re hidden (I don’t know why…)

Objects and Methods

methods(summary)

 [1] summary.aov                         summary.aovlist*                   
 [3] summary.aspell*                     summary.check_packages_in_dir*     
 [5] summary.connection                  summary.data.frame                 
 [7] summary.Date                        summary.default                    
 [9] summary.ecdf*                       summary.factor                     
[11] summary.glm                         summary.infl*                      
[13] summary.lm                          summary.loess*                     
[15] summary.manova                      summary.matrix                     
[17] summary.mlm*                        summary.nls*                       
[19] summary.packageStatus*              summary.POSIXct                    
[21] summary.POSIXlt                     summary.ppr*                       
[23] summary.prcomp*                     summary.princomp*                  
[25] summary.proc_time                   summary.rlang_error*               
[27] summary.rlang_message*              summary.rlang_trace*               
[29] summary.rlang_warning*              summary.rlang:::list_of_conditions*
[31] summary.srcfile                     summary.srcref                     
[33] summary.stepfun                     summary.stl*                       
[35] summary.table                       summary.tukeysmooth*               
[37] summary.warnings                   
see '?methods' for accessing help and source code

The class is given after the dot Those marked with an asterisk are hidden

Objects and Methods

• summary.data.frame()
  \(\implies\) used when summary() is called on a data.frame
• summary.lm()
  \(\implies\) for an object of class lm (produced by lm())
• summary.prcomp()
  \(\implies\) for an object of class prcomp (produced by prcomp())

• If no method is written for a class \(\implies\) summary.default()
• Look inside this using body(summary.default)
  • The last couple of lines were the output from summary(letters)

Objects and Methods

• Can also see what methods exist for a given class
• Before loading any packages ~56 methods exist for a data.frame

methods(class = "data.frame")

 [1] [             [[            [[<-          [<-           $<-           aggregate    
 [7] anyDuplicated anyNA         as.data.frame as.list       as.matrix     as.vector    
[13] by            cbind         coerce        dim           dimnames      dimnames<-   
[19] droplevels    duplicated    edit          format        formula       head         
[25] initialize    is.na         Math          merge         na.exclude    na.omit      
[31] Ops           plot          print         prompt        rbind         row.names    
[37] row.names<-   rowsum        show          slotsFromS3   sort_by       split        
[43] split<-       stack         str           subset        summary       Summary      
[49] t             tail          transform     type.convert  unique        unstack      
[55] within        xtfrm        
see '?methods' for accessing help and source code

Objects and Methods

• Loading a new package will often introduce new methods

library(tidyverse)
methods(class = "data.frame")

• Now we have ~170 methods for a data.frame

Objects and Methods

• Most classes have a print() method
• Determines what to print to the screen when calling an object
• Most common use case for me is print(my_tbl, n = 20)
  • Can use to override the default number of rows printed
  • Calls print.tbl (which is hidden)

S3 Objects

• Everything we’ve just seen applies to S3 objects
• Very common class type (data.frame, list, htest, lm etc)

• Sometimes classes have an explicit hierarchy
  • Best shown using is() instead of class()

is(band_members)

[1] "tbl_df"     "tbl"        "data.frame" "list"       "oldClass"   "vector"    

• R looks for print.tbl_df() \(\rightarrow\) print.tbl() \(\rightarrow\) print.data.frame() etc
• Will use the first one found
• If none found \(\implies\) print.default()

S4 Objects

Many Bioconductor Packages define S4 objects

• Very strict controls on data structure
• Can be frustrating at first
• Use the @ symbol for “slots” as well as $ for list elements
  • Slots are strictly defined components
• Methods are also strictly defined

S4 Objects

• Can be a little more challenging to interact with the tidyverse
• Bioconductor pre-dates the tidyverse by > 10 years
• tidyomics is an active area of Bioconductor development
  • Led by Stefano Mangiola from SAiGENCI

S4 Objects

• Some packages use S4 implementations of S3 objects
  • data.frame (S3) Vs DataFrame (S4)
  • list (S3) Vs List (S4)
  • vector (S3) Vs Vector (S4)
  • rle (S3) Vs Rle (S4)

• Many are written for memory efficiency
• Look and behave similarly, but can ocassionally trip you over
  • Object may require a DataFrame and you have a data.frame
  • Coercion is usually relatively simple between base-level classes

Many S4 objects & methods were developed in the days when compute resources were limited

Rle Vectors

• These are Run-Length Encoded vectors

library(S4Vectors)
test <- c(rep("X", 10), rep("Y", 5))
test

 [1] "X" "X" "X" "X" "X" "X" "X" "X" "X" "X" "Y" "Y" "Y" "Y" "Y"

Rle(test)

character-Rle of length 15 with 2 runs
  Lengths:  10   5
  Values : "X" "Y"

• Can encode millions of chromosomes with minimal memory
  • Sorting can help keep memory useage down

Variations on data.frame Objects

data.frame Objects

• data.frame
  1. Can set rownames
  2. Dumps all data to your screen
  3. Cannot have column names with spaces

• tibble aka tbl_df
  1. rownames are always 1:nrow(df)
  2. Prints a summary with rownames hidden
  3. Column names with spaces permitted
  4. My preferred data.frame type

DataFrame objects

?DataFrame

• An S4 version
  • Doesn’t work with the tidyverse (dplyr, ggplot2 etc)
  • Still missing from tidyomics

• Until 2021 couldn’t coerce to a tibble directly
• I hated that so wrote as_tibble() for DataFrame objects
  • In the package extraChIPs
  • Enables passing S4 objects to ggplot()
  • Please test & find any bugs I haven’t found yet

DataFrame objects

• S3 Methods from dplyr will not work on DataFrame objects
• Some equivalents exist (most pre-date the tidyverse)
  • subset() pre-dates dplyr::filter()
  • rbind() and combineRows() \(\implies\) bind_rows()
  • cbind(), combineCols() and merge() \(\implies\) joins
  • sort() \(\implies\) arrange()
  • unique() \(\implies\) distinct()
• No simple equivalent for mutate(), summarise(), across(), pivot_*()

DataFrame objects

• Can have columns of lists (so can tbl_df objects)
  • e.g. a CharacterList() from IRanges
  • S4 lists can be typed \(\implies\) memory efficiency
  • List objects can exist in a compressed form \(\implies\) memory efficiency
• DataFrame objects can have S4 objects as columns
  • S3 data frames (including tibbles) cannot

By typing a list we only need to record the type once, instead of once for each element. Can make a big difference with large objects

DataFrame objects

library(IRanges)
genes <- c("A", "B")
transcripts <- CharacterList(
  c("A1", "A2", "A3"), c("B1", "B2")
)
transcripts

CharacterList of length 2
[[1]] A1 A2 A3
[[2]] B1 B2

DF <- DataFrame(Gene = genes, Transcripts = transcripts)
DF

DataFrame with 2 rows and 2 columns
         Gene     Transcripts
  <character> <CharacterList>
1           A        A1,A2,A3
2           B           B1,B2

library(extraChIPs)
as_tibble(DF)

# A tibble: 2 × 2
  Gene  Transcripts
  <chr> <list>     
1 A     <chr [3]>  
2 B     <chr [2]>  

DataFrame objects

• Object-level metadata can be added to DataFrame objects
• Must be a list

metadata(DF) <- list(details = "Created for RAdelaide 2024")
glimpse(DF)

Formal class 'DFrame' [package "S4Vectors"] with 6 slots
  ..@ rownames       : NULL
  ..@ nrows          : int 2
  ..@ elementType    : chr "ANY"
  ..@ elementMetadata: NULL
  ..@ metadata       :List of 1
  .. ..$ details: chr "Created for RAdelaide 2024"
  ..@ listData       :List of 2
  .. ..$ Gene       : chr [1:2] "A" "B"
  .. ..$ Transcripts:Formal class 'CompressedCharacterList' [package "IRanges"] with 5 slots

• Notice where rownames are and how the “columns” are stored

Point out the @ structure

DataFrame objects

• Also enable the addition of column-specific metadata \(\implies\) mcols()
  • Used heavily in Bioconductor

mcols(DF) <- DataFrame(meta =  c("Made-up genes", "Made-up transcripts"))
mcols(DF)

DataFrame with 2 rows and 1 column
                           meta
                    <character>
Gene              Made-up genes
Transcripts Made-up transcripts

glimpse(DF) # This is in the @elementMetadata slot

Formal class 'DFrame' [package "S4Vectors"] with 6 slots
  ..@ rownames       : NULL
  ..@ nrows          : int 2
  ..@ elementType    : chr "ANY"
  ..@ elementMetadata:Formal class 'DFrame' [package "S4Vectors"] with 6 slots
  ..@ metadata       :List of 1
  .. ..$ details: chr "Created for RAdelaide 2024"
  ..@ listData       :List of 2
  .. ..$ Gene       : chr [1:2] "A" "B"
  .. ..$ Transcripts:Formal class 'CompressedCharacterList' [package "IRanges"] with 5 slots

S4 Object Structure

• S4 objects have slots denoted with @
  • Subtly different to list elements
• These are fixed and mandatory for every S4 class
  • Can still be empty (NULL) objects
  • Can be S3 or S4 objects
• Enforces a strict structure with checks as objects formed
  • Saves time performing checks within functions
  • Makes structure strict, rigid & hard to break

S3 objects are easy to break. Just change the class attribute…

S4 Object Structure

• We can’t lapply our way through these objects
• Can access each slot using shortcuts object@slotName
  • More formally using slot(object, "slotName")

DF@listData

$Gene
[1] "A" "B"

$Transcripts
CharacterList of length 2
[[1]] A1 A2 A3
[[2]] B1 B2

slot(DF, "listData")

$Gene
[1] "A" "B"

$Transcripts
CharacterList of length 2
[[1]] A1 A2 A3
[[2]] B1 B2

S4 Object Structure

• Slot names can be found using slotNames(object)

slotNames(DF)

[1] "rownames"        "nrows"           "elementType"     "elementMetadata" "metadata"       
[6] "listData"       

• The full type description can also be found

getSlots("DFrame")

           rownames               nrows         elementType     elementMetadata            metadata 
"character_OR_NULL"           "integer"         "character" "DataFrame_OR_NULL"              "list" 
           listData 
             "list" 

S4 Methods

• S3 method dispatch uses the method.class syntax
• S4 is very different but has some similarities

• S4 objects almost always have hierarchical classes
  • Increasingly common for S3 objects
  • Each level extends the lower level class

• Methods are strictly defined by package authors
  • A Generic function must be defined for each method/class
  • The hierarchy is traversed until a method is found

S4 Methods

• To check the class hierarchy of an object use is()

is(DF)

 [1] "DFrame"            "DataFrame"         "SimpleList"        "RectangularData"  
 [5] "List"              "DataFrame_OR_NULL" "Vector"            "list_OR_List"     
 [9] "Annotated"         "vector_OR_Vector" 

• Logical tests can be applied

is(DF, "DataFrame")

[1] TRUE

is(DF, "data.frame")

[1] FALSE

methods(class = "DataFrame")

S4 Methods

• The function body() will return standardGeneric()
  • Slightly different to UseMethod()

• To show code within a function

getMethod(f = "nrow", signature = "DataFrame")

Method Definition:

function (x) 
x@nrows
<bytecode: 0x55649b071c30>
<environment: namespace:S4Vectors>

Signatures:
        x          
target  "DataFrame"
defined "DataFrame"

Recap

• The Bioconductor Project extends back to the early days of R
  • Also the early days of bioinformatics
  • Has a genuine community aspect . . .
• S4 object classes are common
  • Foundational classes enabling package inter-operability
  • Much less common in CRAN packages (spatial/GIS)
• Method dispatch is handled differently

• Can play badly with the tidyverse
  • An area of active development