Default plate type: ddpcr_plate

The most basic plate type is ddpcr_plate, and every plate type inherits from it, either directly or by inheriting from other plate types that are descendants of ddpcr_plate. This is handy because it means that all functionality that is common to all ddPCR types is implemented once for ddpcr_plate and can be used by all plate types, unless a plate type specifically removes or modifies some behaviour. Every plate type by default inherits all methods that ddpcr_plate has, which can be found by running methods(class = "ddpcr_plate"). If you create a new plate type and no parent is specified, or if you initialize a ddPCR plate without specifying a plate type, then ddpcr_plate is assumed.

Calling the analyze() function on any plate will result in running the ddPCR data through a series of steps that are defined for the given plate type, and the droplets in the data will be assigned to one of several clusters associated with the given plate type.

Plates of type ddpcr_plate have 4 possible clusters: UNDEFINED for any droplet that has not been assigned a cluster yet, FAILED for droplets in failed wells, OUTLIER for outlier droplets, and EMPTY for droplets without any template in them. This information can also be seen with the clusters() function.

Plates of type ddpcr_plate have 4 analysis steps: INITIALIZE, REMOVE_FAILURES, REMOVE_OUTLIERS, and REMOVE_EMPTY. This means that any plate that is created will perform these basic steps of removing failed wells, outlier droplets, and empty droplets. The exact functions used for each step can be seen with the steps() function. For example, new_plate(sample_data_dir()) %>% steps will reveal that the REMOVE_FAILURES steps calls the function remove_failures(), so you can view the algorithm by running ddpcr:::remove_failures.ddpcr_plate.

Plates of type ddpcr_plate also have a set of parameters that can be viewed with the params() function. Running new_plate(sample_data_dir()) %>% params %>% str will show all the default parameters that are used. Any of these parameters can be overridden in new plate types, and new parameters can be added.

Built-in plate types

The default plate type is the common base for all built-in types, but because it is not specific to any particular ddPCR data, it does not do any droplet gating, as that is highly dependent on the type of data. The default type is useful for pre-processing, exploring, and visualizing any ddPCR data. There are other built-in plate types available, and you can see a list through the plate_types variable. You can learn more about the built-in plate types and what kind of ddPCR data is suitable for each one by viewing its documentation with ?plate_types. If none of the built-in plate types are useful for your data, you can create a new plate type with custom analysis steps or parameters.

Functions to implement when adding a new plate type

If you want to add a new plate type, there are a few functions you need to implement. All these functions take one argument: a ddpcr_plate object. Note that all these functions are S3 generics, so you need to follow the S3 method naming scheme.

Example

As a simple exercise to show how easy it is to create a new plate type, this section will walk through the steps required to create a new plate type. The name of the new plate type is fam_border. In experiments of this type, we are interested in running the typical pre-processing (identify failed wells, outlier droplets, and empty droplets), followed by simply classifying the remaining droplets as either FAM-positive or FAM-negative, with the border between positive and negative being a predefined value. This plate type is clearly not very useful but it is good for demonstration purposes.

The first thing to do is to define the parent plate type, which in this case will be ddpcr_plate.

parent_plate_type.fam_border <- function(plate) {
  "ddpcr_plate"
}

The next step is to define the parameters of this plate type. ddPCR plates of this type are expected to have at least 14,000 droplets per well as a QA metric. If any well has fewer droplets, it is considered a failure. There is already a parameter for this, so we just need to modify it. We also add a new parameter for the border to use for gating the droplets, and give it a default value.

define_params.fam_border <- function(plate) {
  params <- NextMethod("define_params")
  
  new_params <- list(
    'REMOVE_FAILURES' = list(
      'TOTAL_DROPS_T' = 14000  # overwriting an existing parameter
    ),
    'GATE' = list(
      'FAM_BORDER' = 5000  # defining a new parameter
    )
  )
  params <- modifyList(params, new_params)
  
  params
}

Next we define the potential droplet clusters. We use the same clusters as the default plate type and add two more, to denote droplets in the FAM-positive and FAM-negative sections.

define_clusters.fam_border <- function(plate) {
  clusters <- NextMethod("define_clusters")
  
  c(clusters,
    'FAM_POSITIVE',
    'FAM_NEGATIVE'
  )
}

Next we define the analysis steps. We use all the default analysis steps (pre-processing steps), and add a gating step.

define_steps.fam_border <- function(plate) {
  steps <- NextMethod("define_steps")
  
  c(steps,
    list(
      'GATE' = 'gate_droplets'
    ))
}

We need to create the gate_droplets function with the actual logic that will perform the step. Notice the use of the following helper functions that are especially useful when writing analysis step functions: step, check_step, step_begin, step_end, unanalyzed_clusters, and status. You can look up the documentation for each of these functions to learn more.

gate_droplets <- function(plate) {
  # make sure this step was not called prematurely
  current_step <- step(plate, 'GATE')
  check_step(plate, current_step)  
  
  # show an informative message to the user
  step_begin("Classifying droplets as FAM-positive or negative")
  
  data <- plate_data(plate)
  border <- params(plate, 'GATE', 'FAM_BORDER')
  
  # get a list of clusters that have not been considered yet in the analysis
  # this is useful so that we only look at droplets that have not yet been
  # assigned to a cluster
  unanalyzed_clusters <- unanalyzed_clusters(plate, 'FAM_POSITIVE')
  
  # get the indices of all droplets that are FAM-positive and negative
  unanalyzed_idx <- data$cluster %in% unanalyzed_clusters
  fam_pos <- unanalyzed_idx & data$FAM >= border
  fam_neg <- unanalyzed_idx & data$FAM < border
  
  # assign each droplet to its cluster
  data[fam_pos, 'cluster'] <- cluster(plate, 'FAM_POSITIVE')
  data[fam_neg, 'cluster'] <- cluster(plate, 'FAM_NEGATIVE')
  
  # update the data on the plate object
  plate_data(plate) <- data
  
  # record how many drops in each well are in each cluster
  # and add this info to the plate's metadata
  drops_per_cluster <- 
    plyr::ddply(data, ~ well, function(x) {
      data.frame(
        'drops_positive' = sum(x$cluster == cluster(plate, 'FAM_POSITIVE')),
        'drops_negative' = sum(x$cluster == cluster(plate, 'FAM_NEGATIVE'))
      )
    })
  plate_meta(plate) <-
    dplyr::left_join(
      plate_meta(plate),
      drops_per_cluster,
      by = "well"
    )
  
  # VERY IMPORTANT - do not forget to update the status of the plate
  status(plate) <- current_step
  step_end()
  
  plate
}

Now the new plate type is ready to be used. We can also add a plot function if there are any customizations to the plot that are specific to this plate type. The default plot function of ddPCR plates goes a long way, but one thing we can add is a line showing the division border.

plot.fam_border <- function(x, ..., show_border = FALSE) {
  # Plot a regular ddpcr plate
  p <- NextMethod("plot", x)
  
  # Show the custom thresholds
  if (show_border) {
    border <- params(x, 'GATE', 'FAM_BORDER')
    p <- p +
      ggplot2::geom_hline(yintercept = border)
  }
  
  p
}

The one last thing we can add is a convenient way for the user to set the border parameter.

`fam_border<-` <- function(plate, value) {
  params(plate, 'GATE', 'FAM_BORDER') <- value
  plate
}

Now we can create a new ddPCR plate with the new type and run a full analysis of it.

library(ddpcr)

## 
## Attaching package: 'ddpcr'

## The following object is masked from 'package:stats':
## 
##     step

plate <- new_plate(dir = sample_data_dir(), type = "fam_border")
fam_border(plate) <- 8000
plate <- analyze(plate)
plot(plate, show_drops_empty = TRUE)
plot(plate, col_drops_fam_negative = "red",
     col_drops_fam_positive = "blue", show_border = TRUE)
plate_meta(plate, only_used = TRUE)

##   well sample row col used    target_ch1     target_ch2 drops success
## 1  A01   Dean   A   1 TRUE Consensus_FAM WTspecific_HEX 15820    TRUE
## 2  C01   Mike   C   1 TRUE Consensus_FAM WTspecific_HEX 14256    TRUE
## 3  F05   Mary   F   5 TRUE Consensus_FAM WTspecific_HEX 15377    TRUE
## 4  A05   Dave   A   5 TRUE Consensus_FAM WTspecific_HEX 13165   FALSE
## 5  C05  Emily   C   5 TRUE Consensus_FAM WTspecific_HEX 14109   FALSE
##   drops_outlier drops_empty drops_non_empty drops_empty_fraction concentration
## 1             2       13690            2130                0.865           170
## 2             0       12879            1377                0.903           120
## 3             0       14126            1251                0.919            99
## 4             1          NA              NA                   NA            NA
## 5             0          NA              NA                   NA            NA
##   drops_positive drops_negative
## 1           1889            239
## 2           1281             96
## 3           1090            161
## 4              0              0
## 5              0              0