Introduction to how to use Normalisation Package
Mark Cheng
09/11/2023
introduction.RmdSetting up
load the library/package from github.
if(!require("remotes"))install.packages("remotes",repos="http://cran.us.r-project.org")## Loading required package: remotes
remotes::install_github("TKMarkCheng/NormaliseForIC50",dependencies = TRUE, force = TRUE)## Using github PAT from envvar GITHUB_PAT. Use `gitcreds::gitcreds_set()` and unset GITHUB_PAT in .Renviron (or elsewhere) if you want to use the more secure git credential store instead.## Downloading GitHub repo TKMarkCheng/NormaliseForIC50@HEAD## ps (1.7.6 -> 1.7.7) [CRAN]## Installing 1 packages: ps## Installing package into '/home/runner/work/_temp/Library'
## (as 'lib' is unspecified)## ── R CMD build ─────────────────────────────────────────────────────────────────
## * checking for file ‘/tmp/Rtmpm907De/remotes181f60249af5/TKMarkCheng-NormaliseForIC50-f54d19a/DESCRIPTION’ ... OK
## * preparing ‘NormaliseForIC50’:
## * checking DESCRIPTION meta-information ... OK
## * checking for LF line-endings in source and make files and shell scripts
## * checking for empty or unneeded directories
## * building ‘NormaliseForIC50_2.2.0.tar.gz’## Installing package into '/home/runner/work/_temp/Library'
## (as 'lib' is unspecified)Loading sample, defining output file, and plate rotation (if any)
It is very important for you to change the following file paths and settings to fit your data. (Where are they stored? Where and what do you what the output excel file to be called? Are your plates rotated?)
input_file <- here::here("Validation/2022-09-04_reads/example1.xlsx") #CHANGE to example file in your directory for troubleshooting
input_directory = here::here("Validation/2022-09-04_reads/") #CHANGE this to directory of input .xlsx data files
output_file <- here::here("Validation/test.xlsx") # CHANGE directory and name for yourselfIt is also important that you communicate how your plates are setup, and they should be consistent for each folder that you use this script on. i.e. don’t have horizontal plates and vertical plates mixed in the same folder, and positive and negative controls should be in consistent columns in each folder this script is used on.
Getting the filenames from our directory
What are the names of all of our input files?
neut_raw_files <- Sys.glob(paste0(
paste0(input_directory,"/*.xlsx")) # find all .xlsx file in input directory
)
neut_raw_files## [1] "/home/runner/work/NormaliseForIC50/NormaliseForIC50/Validation/2022-09-04_reads//example1.xlsx"
## [2] "/home/runner/work/NormaliseForIC50/NormaliseForIC50/Validation/2022-09-04_reads//example10.xlsx"
## [3] "/home/runner/work/NormaliseForIC50/NormaliseForIC50/Validation/2022-09-04_reads//example2.xlsx"
## [4] "/home/runner/work/NormaliseForIC50/NormaliseForIC50/Validation/2022-09-04_reads//example3.xlsx"
## [5] "/home/runner/work/NormaliseForIC50/NormaliseForIC50/Validation/2022-09-04_reads//example4.xlsx"
## [6] "/home/runner/work/NormaliseForIC50/NormaliseForIC50/Validation/2022-09-04_reads//example5.xlsx"
## [7] "/home/runner/work/NormaliseForIC50/NormaliseForIC50/Validation/2022-09-04_reads//example6.xlsx"
## [8] "/home/runner/work/NormaliseForIC50/NormaliseForIC50/Validation/2022-09-04_reads//example7.xlsx"
## [9] "/home/runner/work/NormaliseForIC50/NormaliseForIC50/Validation/2022-09-04_reads//example8.xlsx"
## [10] "/home/runner/work/NormaliseForIC50/NormaliseForIC50/Validation/2022-09-04_reads//example9.xlsx"Example case: Plate 1
An inital view of plate 1
plate1 <- NormaliseForIC50::read_promega_plate_excel(input_promega_excel_file_path = input_file)
plate1## neg pos sample one sample one.1 sample two NA sample three
## A 90 376100 182200 229100 171100 186500 80
## B 80 277500 289200 292000 275900 245400 100
## C 100 287200 288400 292000 278800 277700 380
## D 110 301300 309500 301100 304800 313300 24090
## E 50 280600 293000 286400 309400 292500 99030
## F 90 272600 266000 289600 303000 309200 175700
## G 100 251200 295100 286500 311100 283200 244100
## H 50 348100 350900 345000 386400 364800 343600
## sample three.1 9 10 11 12
## A 90 133200 143600 55510 96660
## B 110 266000 269900 259500 298000
## C 830 291200 293900 275200 327900
## D 27390 314900 289400 314200 397400
## E 119800 300700 322300 293400 360300
## F 203000 302600 294500 293000 379000
## G 239600 287200 307500 295000 372400
## H 351700 360300 332200 350300 368300The Name assigned to each column can be any string of characters, as long as there’s no excessively weird symbols.
plate1_normalised <- NormaliseForIC50::final_func(neut_raw_files[1],control_neg_column = c(1),control_pos_column = c(2), rotation_deg_needed = rotate_by)## processing input file: /home/runner/work/NormaliseForIC50/NormaliseForIC50/Validation/2022-09-04_reads//example1.xlsx## negative control = 83.75## positive control = 299325## Finished processing: /home/runner/work/NormaliseForIC50/NormaliseForIC50/Validation/2022-09-04_reads//example1.xlsx
plate1_normalised## sample one sample one.1 sample two NA sample three sample three.1
## 1 39.141 23.468 42.850 37.704 100.001 99.998
## 2 3.384 2.448 7.828 18.021 99.995 99.991
## 3 3.651 2.448 6.859 7.227 99.901 99.751
## 4 -3.400 -0.593 -1.830 -4.670 91.978 90.875
## 5 2.114 4.319 -3.367 2.281 66.934 59.993
## 6 11.136 3.250 -1.228 -3.300 41.313 32.190
## 7 1.412 4.286 -3.935 5.389 18.455 19.959
## 8 -17.235 -15.264 -29.099 -21.880 -14.796 -17.503
## 9 10 11 12
## 1 55.515 52.040 81.478 67.726
## 2 11.136 9.833 13.309 0.443
## 3 2.715 1.813 8.062 -9.549
## 4 -5.205 3.317 -4.971 -32.775
## 5 -0.459 -7.678 1.980 -20.377
## 6 -1.094 1.612 2.114 -26.626
## 7 4.052 -2.732 1.445 -24.420
## 8 -20.377 -10.986 -17.035 -23.050
# openxlsx::write.xlsx(
# plate1_normalised,
# "../tests/testthat/test_data/example1_normalised_without_anomaly_detection.xlsx")Processing all the files in the directory
#create xlsx and populate df into separate sheets. CHANGE rotation_deg_needed if the plate was ROTATED when setting up.
library(openxlsx)
wb<-createWorkbook()
for (neut_raw_file in neut_raw_files){
neut_file <- basename(neut_raw_file) #remove file path
neut_file = tools::file_path_sans_ext(neut_file) #remove extension
addWorksheet(wb,sheetName=neut_file)
writeData(wb, sheet = neut_file,
x = NormaliseForIC50::final_func(
neut_raw_file,
control_neg_column = control_neg_column,
control_pos_column = control_pos_column,
rotation_deg_needed = rotate_by))
print(paste0("normalised data written to:",neut_file))
}## processing input file: /home/runner/work/NormaliseForIC50/NormaliseForIC50/Validation/2022-09-04_reads//example1.xlsx## negative control = 83.75## positive control = 299325## Finished processing: /home/runner/work/NormaliseForIC50/NormaliseForIC50/Validation/2022-09-04_reads//example1.xlsx## [1] "normalised data written to:example1"## processing input file: /home/runner/work/NormaliseForIC50/NormaliseForIC50/Validation/2022-09-04_reads//example10.xlsx## negative control = 77.5## positive control = 361125## Finished processing: /home/runner/work/NormaliseForIC50/NormaliseForIC50/Validation/2022-09-04_reads//example10.xlsx## [1] "normalised data written to:example10"## processing input file: /home/runner/work/NormaliseForIC50/NormaliseForIC50/Validation/2022-09-04_reads//example2.xlsx## negative control = 96.25## positive control = 361825## Finished processing: /home/runner/work/NormaliseForIC50/NormaliseForIC50/Validation/2022-09-04_reads//example2.xlsx## [1] "normalised data written to:example2"## processing input file: /home/runner/work/NormaliseForIC50/NormaliseForIC50/Validation/2022-09-04_reads//example3.xlsx## negative control = 96.25## positive control = 354575## Finished processing: /home/runner/work/NormaliseForIC50/NormaliseForIC50/Validation/2022-09-04_reads//example3.xlsx## [1] "normalised data written to:example3"## processing input file: /home/runner/work/NormaliseForIC50/NormaliseForIC50/Validation/2022-09-04_reads//example4.xlsx## negative control = 93.75## positive control = 363150## Finished processing: /home/runner/work/NormaliseForIC50/NormaliseForIC50/Validation/2022-09-04_reads//example4.xlsx## [1] "normalised data written to:example4"## processing input file: /home/runner/work/NormaliseForIC50/NormaliseForIC50/Validation/2022-09-04_reads//example5.xlsx## negative control = 91.25## positive control = 370775## Finished processing: /home/runner/work/NormaliseForIC50/NormaliseForIC50/Validation/2022-09-04_reads//example5.xlsx## [1] "normalised data written to:example5"## processing input file: /home/runner/work/NormaliseForIC50/NormaliseForIC50/Validation/2022-09-04_reads//example6.xlsx## negative control = 87.5## positive control = 351112.5## Finished processing: /home/runner/work/NormaliseForIC50/NormaliseForIC50/Validation/2022-09-04_reads//example6.xlsx## [1] "normalised data written to:example6"## processing input file: /home/runner/work/NormaliseForIC50/NormaliseForIC50/Validation/2022-09-04_reads//example7.xlsx## negative control = 92.5## positive control = 393425## Finished processing: /home/runner/work/NormaliseForIC50/NormaliseForIC50/Validation/2022-09-04_reads//example7.xlsx## [1] "normalised data written to:example7"## processing input file: /home/runner/work/NormaliseForIC50/NormaliseForIC50/Validation/2022-09-04_reads//example8.xlsx## negative control = 101.25## positive control = 381600## Finished processing: /home/runner/work/NormaliseForIC50/NormaliseForIC50/Validation/2022-09-04_reads//example8.xlsx## [1] "normalised data written to:example8"## processing input file: /home/runner/work/NormaliseForIC50/NormaliseForIC50/Validation/2022-09-04_reads//example9.xlsx## negative control = 91.25## positive control = 385575## Finished processing: /home/runner/work/NormaliseForIC50/NormaliseForIC50/Validation/2022-09-04_reads//example9.xlsx## [1] "normalised data written to:example9"
saveWorkbook(wb,file=output_file,overwrite = TRUE)
print(paste0("saved collated sheets to excel file:",output_file))## [1] "saved collated sheets to excel file:/home/runner/work/NormaliseForIC50/NormaliseForIC50/Validation/test.xlsx"Example2: custom plating
df <- NormaliseForIC50::read_promega_plate_excel(input_promega_excel_file_path = "../Validation/custom_control_plating/rbm 5.xlsx")
NormaliseForIC50::final_func("../Validation/custom_control_plating/rbm 5.xlsx",control_plating = "custom",
control_neg_wells=c("H1","H2","H3","H4","H5","H6"),
control_pos_wells=c("H7","H8","H9","H10","H11","H12"),
rotation_deg_needed = rotate_by)## processing input file: ../Validation/custom_control_plating/rbm 5.xlsx## Warning in check_max(df.values, filename = filename): rbm 5.xlsx Warning: The
## largest value is found outside of the last three rows of column 8## Warning in check_max(df.values, filename = filename): rbm 5.xlsx Warning: The
## largest value is found outside of the last three rows of column 11## Warning in check_max(df.values, filename = filename): rbm 5.xlsx Warning: The
## largest value is found outside of the last three rows of column 12## negative control = 278.333333333333## positive control = 2250166.66666667## Finished processing: ../Validation/custom_control_plating/rbm 5.xlsx## 1 2 3 4 5 6 7 8 9 10
## 1 96.563 96.464 95.946 97.052 99.868 99.915 33.076 27.475 99.518 99.506
## 2 94.025 93.621 86.687 84.936 87.452 90.523 -61.507 -52.440 87.141 84.296
## 3 89.914 88.176 67.900 62.726 51.121 50.721 -56.351 -64.751 57.508 58.375
## 4 68.073 63.233 38.143 23.609 21.031 18.142 -28.127 -27.105 32.142 27.342
## 5 35.565 38.187 12.230 -2.882 -8.393 0.674 -14.971 -15.504 7.741 14.364
## 6 30.231 25.031 5.474 -17.016 -4.926 -0.748 -4.659 1.563 7.030 14.497
## 7 10.897 10.897 14.319 7.786 -0.526 -10.749 -13.371 8.941 0.674 1.696
## 11 12
## 1 35.520 33.476
## 2 -50.751 -66.974
## 3 -79.908 -84.530
## 4 -48.173 -49.906
## 5 -28.883 -23.016
## 6 5.252 -9.015
## 7 0.185 -10.749
neut_raw_files <- Sys.glob(paste0(
paste0("../Validation/custom_control_plating","/*.xlsx")) # find all .xlsx file in input directory
)
neut_raw_files## [1] "../Validation/custom_control_plating/rbm 3.xlsx"
## [2] "../Validation/custom_control_plating/rbm 5.xlsx"
#create xlsx and populate df into separate sheets. CHANGE rotation_deg_needed if the plate was ROTATED when setting up.
library(openxlsx)
wb<-createWorkbook()
for (neut_raw_file in neut_raw_files){
neut_file <- basename(neut_raw_file) #remove file path
neut_file = tools::file_path_sans_ext(neut_file) #remove extension
addWorksheet(wb,sheetName=neut_file)
writeData(wb, sheet = neut_file,
x = NormaliseForIC50::final_func(
neut_raw_file,
control_plating = "custom",
control_neg_wells = c("H1","H2","H3","H4","H5","H6"),
control_pos_column =c("H7","H8","H9","H10","H11","H12"),
rotation_deg_needed = rotate_by))
print(paste0("normalised data written to:",neut_file))
}## processing input file: ../Validation/custom_control_plating/rbm 3.xlsx## negative control = 236.666666666667## positive control = 954992.5## Finished processing: ../Validation/custom_control_plating/rbm 3.xlsx## [1] "normalised data written to:rbm 3"## processing input file: ../Validation/custom_control_plating/rbm 5.xlsx## Warning in check_max(df.values, filename = filename): rbm 5.xlsx Warning: The
## largest value is found outside of the last three rows of column 8## Warning in check_max(df.values, filename = filename): rbm 5.xlsx Warning: The
## largest value is found outside of the last three rows of column 11## Warning in check_max(df.values, filename = filename): rbm 5.xlsx Warning: The
## largest value is found outside of the last three rows of column 12## negative control = 278.333333333333## positive control = 800088.75## Finished processing: ../Validation/custom_control_plating/rbm 5.xlsx## [1] "normalised data written to:rbm 5"
saveWorkbook(wb,file=here::here("Validation/test_CustomPlating.xlsx"),overwrite = TRUE)
print(paste0("saved collated sheets to excel file: ",here::here("Validation/test_CustomPlating.xlsx")))## [1] "saved collated sheets to excel file: /home/runner/work/NormaliseForIC50/NormaliseForIC50/Validation/test_CustomPlating.xlsx"