PoDCall: Positive Droplet Caller for DNA Methylation ddPCR
Introduction
PoDCall (Positive Droplet Caller) is a package that aims to provide a robust calling of positive droplets in DNA methylation droplet digital PCR (ddPCR) experiments performed on the Bio-Rad platform. PoDCall provides functions that reads files exported from QuantaSoft or QX Manager containing amplitudes from a run of ddPCR (one 96 well plate), sets thresholds for both channels of each individual well and calculates concentrations and normalized concentration for each well.The resulting threshold table can optionally be written to file automatically by the main workflow function. PoDCall also offers functionality for plotting, both individual wells and multiple well plots. Plots for individual wells can be made and saved as .pdf-files as part of the main workflow function, or by calling the various plotting functions individually.
Gaussian Mixture Models
DdPCR experiments generate a mixture of droplets, positive droplets which contain the target that will be amplified, and negative droplets that does not contain the target and show no amplification. PoDCall relies on fitting Gaussian mixture models to set thresholds for each individual well that will be used to classify the droplets as either positive or negative. For more details on the concepts of PoDCall, see the application note.
Input Data
The input data is .csv-files exported from ‘QuantaSoft’ or ‘QX Manager, and each file contains the amplitude values of droplets from one well of a 96 well plate. The first two columns of the files should have headers ’Ch1 Amplitude’ and ‘Ch2 Amplitude’. To read in data, use the function importAmplitudeData, which will read all amplitude files in the directory given as argument. Each file will be stored as a named data frame in a list, where the name will be the well ID. For this reason, all raw data files in the directory given as argument should be from the same 96 well plate to avoid well coordinate duplicates.
Installation
PoDCall requires some packages to be installed, and if any required packages are not yet installed, the installation of PoDCall should take care of it (you will be prompted to install the packages that are missing).
The released version of PoDCall be installed from BIOCONDUCTOR, from GitHub or from a source file using:
## Install from Bioconductor
if (!requireNamespace("BiocManager", quietly = TRUE))
install.packages("BiocManager")
BiocManager::install("PoDCall")
## Install PoDCall from GitHub
install.packages("devtools")
devtools::install_github("HansPetterBrodal/PoDCall")
## Install PoDCall from source file
install.packages("remotes")
remotes::install_local("path/to/PoDCall_x.y.z.tar.gz")After installing PoDCall and the required packages, PoDCall can be loaded with:
Example / Usage
One step of setting thresholds includes a random sampling of droplets
to greatly reduce running time. To ensure reproducible results it is
recommended to set a seed using set.seed(). To run the full
PoDCall workflow, call the function podcallDdpcr():
Where “path/to/data/” is the path of the directory that contains amplitude files from a well plate, in which the files have names that end with “_wellID_amplitude.csv”. “software” is the software that was used to export the data (amplitude) files and the sample sheet. Must be either “QuantaSoft” or “QX Manager”. Since the different software versions format amplitude files and sample sheet differently, it is important to set the correct value as argument to ensure that data and sample sheet is read correctly.
Optional arguments
The following arguments have default values, but can be given other values if desired. For example to write results to file, which is disabled by default.
sampleSheetFile
Path to sample sheet file. Must be a .csv file and must include the
following columns: Well, Sample, TargetType and Target. The entries in
the column TargetType must be either ‘Ch1Unknown’ or ‘Ch2Unknown’, and
is used to extract rows with information from either channel 1 or
channel 2. An example file has been included in the package, which can
be found using
system.file("extdata", "Sample_names.csv", package="PoDCall")
B
The number of permutations used by the likelihood ratio test (LRT)
which decides the number of components in the model fitted from the
data. Default value B=200.
Q
A parameter used for calling outliers. Q is multiplied with the
interquartile range of the distribution of amplitude values to determine
if droplets of extreme amplitude values are to be considered outliers.
The default value is Q=9, which has been determined through
cell line experiments and testing. A higher Q will generally result in a
higher or more strict threshold. Q provides an option to adjust how
thresholds are set in a systematic and reproducible way. It is
recommended to try a few different values and visually inspect the
results.
refwell
The well used as reference when calculating the shift in baseline
between wells. By default refwell=1, but can be changed in
cases where the first well is not suited to be used.
nrChannels
If there is no control channel used, set nrChannels=1 to indicate only target channel used. Default value is 2.
targetChannel
When a multiplexed set up is used (more than a single target channel), what channel to be analysed is the target channel. Default targetChannel=1.
controlChannel
The channel used for control assay. Default controlChannel=2.
software
The Bio-Rad software the data was exported from. Must be either “QuantaSoft” or “QX Manager”, the latter being default. This is due to unequal number of lines that needs to be skipped in the raw amplitude data files.
resultsToFile
The user can choose to let PoDCall save the results table as a
.csv-file by setting resultsToFile=TRUE (default:
resultsToFile = FALSE). When resultsToFile is set to TRUE,
a results directory will be created where the result file will be saved.
The results directory will have the same name as the data directory with
“_results” added: “path/to/data_results/
plots
The user can choose to make plots that are written to file by setting
plots=TRUE (default: plots=FALSE). Plots will
be saved to the results directory created when
resultsToFile=TRUE. The results directory will also be
created if only plots=TRUE.
resPath
Optional argument to specify a directory for writing results file(s)
to other than the results directory created by default. Requires
resultsToFile=TRUE.
Threshold table columns
The table that is returned when running podcall_ddpcr()
contains columns with more or less self-explanatory column names, and
well ID (well coordinates) as rownames:
sample_id
If a sample sheet file is provided, this will have the sample ID from the sample sheet. Otherwise empty
target_ch
The channel used for target assay
thr_target
the threshold set for selected target channel
thr_ctrl
The threshold set for control channel
pos_dr_target
The number of positive droplets in selected target channel
pos_dr_ctrl
The number of positive droplets in control channel
tot_droplets
Number of droplets.
c_target
Concentration of target, calculated by the formula $-\log\dfrac{\dfrac{\text{neg_drop_tar}}{\text{tot_droplets}}}{0.000851}$ where neg_drop_tar is number of negative droplets in channel 1 (target).
c_ctrl
Concentration of control, calculated by the formula $-\log\dfrac{\dfrac{\text{neg_drop_ctrl}}{\text{tot_droplets}}}{0.000851}$ where neg_drop_ctrl is number of negative droplets in channel 2 (control).
c_norm_4Plex
Normalized concentration with 4Plex as control, calculated by the formula $\dfrac{\text{c_target}}{\text{c_ctrl}}\cdot400$
c_norm_sg
Normalized concentration with single gene as control, calculated by the formula $\dfrac{\text{c_target}}{\text{c_ctrl}}\cdot100$
q
The value used for Q
target_assay
The assay used for target channel, provided via sample sheet.
ctrl_assay
The assay used for control channel, provided via sample sheet.
ref_well
The well used as reference well for setting threshold.
PoDCall functions
podcallDdpcr() is the main wrapper function that returns
a table with the results of PoDCall to the user. This function uses a
set of functions that read the amplitude data from file, set thresholds
and make plots. All functions involved can be used individually should
the user only want to use some of the functionality of PoDCall. Also see
help files for more details about the functions and their arguments.
importAmplitudeData()
Reads .csv-files with amplitude data outputted from QuantaSoft or QX Manager and store the data in a list, one data frame per well. Each element in the list will be named using it’s well ID (coordinate) of the 96 well plate that the sample belong to.
## Path to example data files included in PoDCall
path <- system.file("extdata", "Amplitudes/", package="PoDCall")
## Read in data files
dataList <- importAmplitudeData(dataDirectory=path, skipLines=0)
str(dataList)
#> List of 5
#> $ A04:'data.frame': 18739 obs. of 2 variables:
#> ..$ Ch1: num [1:18739] 940 971 971 976 985 ...
#> ..$ Ch2: num [1:18739] 11795 7868 8377 10007 9523 ...
#> $ B04:'data.frame': 16933 obs. of 2 variables:
#> ..$ Ch1: num [1:16933] 980 995 1002 1007 1014 ...
#> ..$ Ch2: num [1:16933] 9524 7999 7686 7799 9510 ...
#> $ D04:'data.frame': 11713 obs. of 2 variables:
#> ..$ Ch1: num [1:11713] 1042 1070 1094 1112 1112 ...
#> ..$ Ch2: num [1:11713] 7826 9934 7698 7605 7743 ...
#> $ D05:'data.frame': 12642 obs. of 2 variables:
#> ..$ Ch1: num [1:12642] 1045 1057 1063 1068 1079 ...
#> ..$ Ch2: num [1:12642] 9722 7752 9103 7716 7738 ...
#> $ H05:'data.frame': 19638 obs. of 2 variables:
#> ..$ Ch1: num [1:19638] 1043 1094 1098 1104 1119 ...
#> ..$ Ch2: num [1:19638] 7231 7063 7161 6863 7416 ...importSampleSheet()
Reads a .csv-file outputted from QuantaSoft or QX Manager to get information about the samples: Sample name/id, Assay for target and control.
## Path to example files included in PoDCall
path <- system.file("extdata", "Sample_names.csv", package="PoDCall")
## Select wells to get information for
well_id <- c("A04", "B04", "D04")
## Read in sample sheet information for selected wells
sampleSheet <- importSampleSheet(sampleSheet=path, well_id=well_id,
software="QuantaSoft")
print(sampleSheet)
#> well_id sample_id target_assay ctrl_assay
#> 1 A04 SW1463 VIM new4Plex
#> 2 B04 SW403 VIM new4Plex
#> 3 D04 SW480 VIM new4PlexpodcallThresholds()
Takes a list of data frames, one for each well, as argument and sets
individual thresholds for each channel of each well. It returns a table
with thresholds, number of positive droplets, concentrations etc. The
number of permutations for likelihood ratio test is by default set to
B=400 as a compromise between run time and stability of the
results. The parameter for calling outliers is by default set to
Q=9. Higher Q means more conservative (higher) thresholds,
lower Q will result in over all lower thresholds.
## Path to example data files included in PoDCall
path <- system.file("extdata", "Amplitudes/", package="PoDCall")
## Read in data files
ampData <- importAmplitudeData(dataDirectory=path, skipLines=0)
## Calculate thresholds, metrics, concentrations
thresholdTable <- podcallThresholds(plateData=ampData)
print(thresholdTable)podcallChannelPlot()
Takes the threshold and amplitude values corresponding to a channel of a well as arguments, calls functions that makes scatter plot and histogram and draws a plot with both.
## Read in data and threshold table
path <- system.file("extdata", "Amplitudes/", package="PoDCall")
ampData <- importAmplitudeData(dataDirectory=path, skipLines=0)
data("thrTable")
thresholdTable <- thrTable
## Select channel and well to plot
ch <- 1 # target channel
well_id <- names(ampData)[1] # First well in list
## Plot title
plotTitle <- paste0(well_id, ", Ch", ch)
## Create plot
podcallChannelPlot(channelData=ampData[[well_id]][,ch],
thr=thresholdTable[well_id, "thr_target"],
channel=ch,
plotId=plotTitle)
podcallScatterplot()
Takes the threshold and amplitude values corresponding to a channel of a well as argument and returns a scatter plot.
## Read in data and threshold table
path <- system.file("extdata", "Amplitudes/", package="PoDCall")
ampData <- importAmplitudeData(dataDirectory=path, skipLines=0)
thresholdTable <- thrTable
## Select channel and well to plot
ch <- 1 # target channel
well_id <- names(ampData)[1] # First well in list
## Plot title
plotTitle <- paste0(well_id, ", Ch", ch)
## Create plot
podcallScatterplot(channelData=ampData[[well_id]][,ch],
thr=thresholdTable[well_id, "thr_target"],
channel=ch,
plotId=plotTitle)
podcallHistogram()
Takes the threshold and amplitude values corresponding to a channel of a well as argument, and returns a histogram.
## Read in data and threshold table
path <- system.file("extdata", "Amplitudes/", package="PoDCall")
ampData <- importAmplitudeData(dataDirectory=path, skipLines=0)
thresholdTable <- thrTable
## Select channel and well to plot
ch <- 1 # target channel
well_id <- names(ampData)[1] # First well in list
## Plot title
plotTitle <- paste0(well_id, ", Ch", ch)
## Create plot
podcallHistogram(channelData=ampData[[well_id]][,ch],
thr=thresholdTable[well_id, "thr_target"],
channel=ch,
plotId=plotTitle)
podcallMultiplot()
takes a list of data frames with amplitude data, one per well, and their respective thresholds as arguments and returns faceted scatter plots suitable for comparing wells.
## Read in data and threshold table
path <- system.file("extdata", "Amplitudes/", package="PoDCall")
ampData <- importAmplitudeData(dataDirectory=path, skipLines=0)
thresholdTable <- thrTable
## Channel to plot
ch <- "target"
## Create comparison plot
podcallMultiplot(plateData=ampData,
thresholds=thresholdTable[names(ampData),],
channel=ch, colCh=1)
PoDCall shiny application
PoDCall does also include an application powered by shiny that launches in a web browser. The application provides a user friendly and interactive interface to the functionality of PoDCall. To start the app:
PodCall example data
There are some amplitude files and a sample sheet included in the package that are intended to be used to run examples and to try out the functionality of PoDCall. The data files are from a real experiment performed with cell line samples. There is also a threshold table computed from the example data included. PoDCall takes a few minutes to run due to bootstrapping, and this table is used in examples for functions where threshold is an argument.
Cell Line Amplitude Data
The cell line amplitude data files can be found in the “extdata”
subdirectory of the package directory and can be found using
system.file():
## Path to files
path <- system.file("extdata", "Amplitudes/", package="PoDCall")
## List files
list.files(path)
#> [1] "VIM_4Plex_A04_Amplitude.csv" "VIM_4Plex_B04_Amplitude.csv"
#> [3] "VIM_4Plex_D04_Amplitude.csv" "VIM_4Plex_D05_Amplitude.csv"
#> [5] "VIM_4Plex_H05_Amplitude.csv"The control assay used for the samples in the example data files is an assay developed in-house called 4Plex H. Pharo et al.
Calculated Threshold Table
The already calculated threshold table is instantly available when
PoDCall is loaded, and is available as an object called
thrTable. See ?thrTable for help file with
documentation on the table.
## The threshold table
thrTable
#> sample_id target_ch thr_target thr_ctrl pos_dr_target pos_dr_ctrl
#> A04 SW1463 Ch1 2761 9128 2479 12905
#> B04 SW403 Ch1 2739 8632 660 7471
#> D04 SW480 Ch1 2863 8489 44 8348
#> D05 IVDZ_bisulf Ch1 2818 8473 1675 6584
#> H05 NTC Ch1 2823 7910 0 0
#> tot_droplets c_target c_ctrl c_norm_4Plex c_norm_sg q target_assay
#> A04 18739 166.700 1371.0 48.64 12.16 9 VIM
#> B04 16933 46.720 683.9 27.33 6.831 9 VIM
#> D04 11713 4.423 1466.0 1.207 0.3017 9 VIM
#> D05 12642 167.000 864.4 77.28 19.32 9 VIM
#> H05 19638 0.000 0.0 No DNA No DNA 9 VIM
#> ctrl_assay ref_well
#> A04 new4Plex A04
#> B04 new4Plex A04
#> D04 new4Plex A04
#> D05 new4Plex A04
#> H05 new4Plex A04Session info
Here is the output of sessionInfo() on the system on
which this document was compiled
sessionInfo()
#> R version 4.4.1 (2024-06-14)
#> Platform: x86_64-pc-linux-gnu
#> Running under: Ubuntu 24.04.1 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=C
#> [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] stats graphics grDevices utils datasets methods base
#>
#> other attached packages:
#> [1] PoDCall_1.15.0 BiocStyle_2.35.0
#>
#> loaded via a namespace (and not attached):
#> [1] sass_0.4.9 utf8_1.2.4 hms_1.1.3
#> [4] digest_0.6.37 magrittr_2.0.3 evaluate_1.0.1
#> [7] grid_4.4.1 fastmap_1.2.0 jsonlite_1.8.9
#> [10] mclust_6.1.1 promises_1.3.0 gridExtra_2.3
#> [13] BiocManager_1.30.25 purrr_1.0.2 fansi_1.0.6
#> [16] scales_1.3.0 jquerylib_0.1.4 rlist_0.4.6.2
#> [19] cli_3.6.3 shiny_1.9.1 crayon_1.5.3
#> [22] rlang_1.1.4 bit64_4.5.2 LaplacesDemon_16.1.6
#> [25] munsell_0.5.1 withr_3.0.2 cachem_1.1.0
#> [28] yaml_2.3.10 tools_4.4.1 parallel_4.4.1
#> [31] tzdb_0.4.0 colorspace_2.1-1 ggplot2_3.5.1
#> [34] httpuv_1.6.15 DT_0.33 mime_0.12
#> [37] buildtools_1.0.0 vctrs_0.6.5 R6_2.5.1
#> [40] lifecycle_1.0.4 bit_4.5.0 htmlwidgets_1.6.4
#> [43] vroom_1.6.5 shinyjs_2.1.0 pkgconfig_2.0.3
#> [46] later_1.3.2 pillar_1.9.0 bslib_0.8.0
#> [49] gtable_0.3.6 Rcpp_1.0.13 glue_1.8.0
#> [52] data.table_1.16.2 highr_0.11 tidyselect_1.2.1
#> [55] xfun_0.48 tibble_3.2.1 sys_3.4.3
#> [58] knitr_1.48 farver_2.1.2 xtable_1.8-4
#> [61] htmltools_0.5.8.1 labeling_0.4.3 rmarkdown_2.28
#> [64] maketools_1.3.1 readr_2.1.5 compiler_4.4.1
#> [67] diptest_0.77-1