Dear Bioconductor-team,

I am very happy to have your helpful tool to analyze Nanostring data. However, I am struggling with the processNanostringData-function. I managed to upload my RCC-files combined in a vector and also uploaded a Sample_data-file containing the metadata. Still, I always get an error message if I try to use the processNanostringData-function:

Reading in .RCC files...... Checking codeset contents...... Checked gene name consistency in .RCC files. Fehler in dat[[1]] : Indizierung außerhalb der Grenzen

I tried to use a zip file format with RCC files and I also tried to use my uploaded vector. Please find the r-code below.

Do you have an advice for me?

Best regards, Anna

loading_meta_data

CME_Sample_data <- read.table(file = 'Sample_data.csv', header = TRUE, sep = ";")
str(CME_Sample_data)

'data.frame':   18 obs. of  5 variables:
 $ Sample_Tilte: chr  "20240411_31021086138-StemCell-AK-R01-555_Nanostring-CME-siA-B-Kombi-I-1_04" "20240411_31021086138-StemCell-AK-R01-555_Nanostring-CME-siBRD7Pool-1_01" "20240411_31021086138-StemCell-AK-R01-555_Nanostring-CME-siBRD9Pool-1_02" "20240411_31021086138-StemCell-AK-R01-555_Nanostring-CME-siCTRL1-1_06" ...
 $ Variable    : chr  "CME-AB-1" "CME-BRD7-1" "CME-BRD9-1" "CME-CTRL-1" ...
 $ Replicate   : int  1 1 1 1 1 1 2 2 2 2 ...
 $ Treatment   : chr  "treated" "treated" "treated" "control" ...
 $ Target      : chr  "siA-B" "siBRD7" "siBRD9" "siCTRL" ...

#Nanostring-RCC


CME_RCC_files <- read_merge_rcc(c("20240411_31021086138-StemCell-AK-R01-555_Nanostring-CME-siA-B-Kombi-I-1_04.RCC", "20240411_31021086138-StemCell-AK-R01-555_Nanostring-CME-siBRD7Pool-1_01.RCC", "20240411_31021086138-StemCell-AK-R01-555_Nanostring-CME-siBRD9Pool-1_02.RCC", "20240411_31021086138-StemCell-AK-R01-555_Nanostring-CME-siCTRL1-1_06.RCC", "20240411_31021086138-StemCell-AK-R01-555_Nanostring-CME-siIshii-Tamaki-1_05.RCC", "20240411_31021086138-StemCell-AK-R01-555_Nanostring-CME-siPBRM1Pool-1_03.RCC", "20240628_31021091122-StemCell-AK-R03-555_Nanostring-CME-siA-B-Kombi-I-2_04.RCC", "20240628_31021091122-StemCell-AK-R03-555_Nanostring-CME-siA-B-Kombi-I-3_10.RCC", "20240628_31021091122-StemCell-AK-R03-555_Nanostring-CME-siBRD7Pool-2_01.RCC", "20240628_31021091122-StemCell-AK-R03-555_Nanostring-CME-siBRD7Pool-3_07.RCC", "20240628_31021091122-StemCell-AK-R03-555_Nanostring-CME-siBRD9Pool-2_02.RCC", "20240628_31021091122-StemCell-AK-R03-555_Nanostring-CME-siBRD9Pool-3_08.RCC", "20240628_31021091122-StemCell-AK-R03-555_Nanostring-CME-siCTRL1-2_06.RCC", "20240628_31021091122-StemCell-AK-R03-555_Nanostring-CME-siCTRL1-3_12.RCC", "20240628_31021091122-StemCell-AK-R03-555_Nanostring-CME-siIshii-Tamaki-2_05.RCC", "20240628_31021091122-StemCell-AK-R03-555_Nanostring-CME-siIshii-Tamaki-3_11.RCC", "20240628_31021091122-StemCell-AK-R03-555_Nanostring-CME-siPBRM1Pool-2_03.RCC", "20240628_31021091122-StemCell-AK-R03-555_Nanostring-CME-siPBRM1Pool-3_09.RCC"), includeQC = TRUE, logfile = "")
str(CME_RCC_files)

Checking codeset contents......
Checked gene name consistency in .RCC files.
12 genes labeled as 'housekeeping'.List of 3
 $ exprs: num [1:784, 1:18] 641 12 2519 1370 6215 ...
  ..- attr(*, "dimnames")=List of 2
  .. ..$ : chr [1:784] "NM_002751.6" "NM_005442.2" "NM_001789.2" "NM_181837.3" ...
  .. ..$ : chr [1:18] "20240411_31021086138-StemCell-AK-R01-555_Nanostring-CME-siA-B-Kombi-I-1_04.RCC" "20240411_31021086138-StemCell-AK-R01-555_Nanostring-CME-siBRD7Pool-1_01.RCC" "20240411_31021086138-StemCell-AK-R01-555_Nanostring-CME-siBRD9Pool-1_02.RCC" "20240411_31021086138-StemCell-AK-R01-555_Nanostring-CME-siCTRL1-1_06.RCC" ...
 $ dict :'data.frame':  784 obs. of  3 variables:
  ..$ CodeClass: chr [1:784] "Endogenous" "Endogenous" "Endogenous" "Endogenous" ...
  ..$ Name     : chr [1:784] "MAPK11" "EOMES" "CDC25A" "ORC3" ...
  ..$ Accession: chr [1:784] "NM_002751.6" "NM_005442.2" "NM_001789.2" "NM_181837.3" ...
 $ qc   : chr [1:18, 1:7] "555" "555" "555" "555" ...
  ..- attr(*, "dimnames")=List of 2
  .. ..$ : chr [1:18] "20240411_31021086138-StemCell-AK-R01-555_Nanostring-CME-siA-B-Kombi-I-1_04.RCC" "20240411_31021086138-StemCell-AK-R01-555_Nanostring-CME-siBRD7Pool-1_01.RCC" "20240411_31021086138-StemCell-AK-R01-555_Nanostring-CME-siBRD9Pool-1_02.RCC" "20240411_31021086138-StemCell-AK-R01-555_Nanostring-CME-siCTRL1-1_06.RCC" ...
  .. ..$ : chr [1:7] "FovCount" "FovCounted" "ScannerID" "StagePosition" ...

CME_RCC_folder <- system.file("extdata", "CME_Raw", package = "NanoTube")
CME_Sample_folder <- system.file("extdata", "Sample_data.csv", package = "NanoTube")

#processNanostringData_Function - does not work yet 


processNanostringData(
      nsFiles = "CME_Raw",
      sampleTab = NULL, 
      idCol = NULL,
      groupCol = "Target",
      normalization = "nSolver",
      bgType = "none", bgPVal = 0.01,
      skip.housekeeping = FALSE,
      output.format = "ExpressionSet")


Reading in .RCC files......
Checking codeset contents......
Checked gene name consistency in .RCC files.
Fehler in dat[[1]] : Indizierung außerhalb der Grenzen