Hi all,

I have been trying to use the read.flowSet() command to read in multiple .fcs files but I keep getting the following error:

all <- read.flowSet(files=NULL,path=".",pattern=".fcs")

Error in asMethod(object) : 
  The individual flowFrames do not contain identical stains.
In addition: Warning message:
In ucol == sort(colNames[, 1]) :
  longer object length is not a multiple of shorter object length

On the contrary, I am able to read in the files individually using the command below. The files all follow the same format, only differences being the data and number of cells. They have the same row and column names despite the error message.

unstained1 <- read.FCS("Unstained.fcs",transformation="linearize")
> unstained1
flowFrame object 'Unstained.fcs'
with 16462 cells and 14 observables:
            name desc     range minRange  maxRange
$P1         Time <NA> 536870912        0 536870911
$P2          FSC <NA> 536870912        0 536870911
$P3          FSC <NA> 536870912        0 536870911
$P4          SSC <NA> 536870912        0 536870911
$P5          SSC <NA> 536870912        0 536870911
$P6   FL1-Height <NA> 536870912        0 536870911
$P7   FL2-Height <NA> 536870912        0 536870911
$P8   FL4-Height <NA> 536870912        0 536870911
$P9   FL6-Height <NA> 536870912        0 536870911
$P10  FL7-Height <NA> 536870912        0 536870911
$P11 FL10-Height <NA> 536870912        0 536870911
$P12 FL11-Height <NA> 536870912        0 536870911
$P13 FL12-Height <NA> 536870912        0 536870911
$P14 FL14-Height <NA> 536870912        0 536870911
101 keywords are stored in the 'description' slot

I have also tried the following command to create a flowSet but still get an error:

> all_data<-lapply(list.files(path=".",full.names=TRUE),function(x)read.FCS(x,transforation="scale"))
Error in if (!version %in% c("FCS2.0", "FCS3.0", "FCS3.1")) stop("This does not seem to be a valid FCS2.0, FCS3.0 or FCS3.1 file") : 

  argument is of length zero

I would like to perform probability binning but it requires a flowSet to be created, yet I cannot surpass this step. Any suggestions would be a great help! Thanks in advance.

> sessionInfo()
R version 3.2.4 (2016-03-10)
Platform: x86_64-apple-darwin13.4.0 (64-bit)
Running under: OS X 10.11.3 (El Capitan)
locale:
[1] en_GB.UTF-8/en_GB.UTF-8/en_GB.UTF-8/C/en_GB.UTF-8/en_GB.UTF-8

attached base packages:
[1] splines   stats     graphics  grDevices utils     datasets  methods 
[8] base   
 
other attached packages:
 [1] BiocInstaller_1.20.1      flowStats_3.28.1        
 [3] flowWorkspace_3.16.16     gridExtra_2.2.1         
 [5] ncdfFlow_2.16.1           BH_1.60.0-1             
 [7] RcppArmadillo_0.6.700.3.0 cluster_2.0.4           
 [9] mvoutlier_2.0.6           sgeostat_1.0-27         
[11] fda_2.4.4                 Matrix_1.2-5            
[13] flowFP_1.28.0             flowViz_1.34.1          
[15] lattice_0.20-33           flowCore_1.36.9         

loaded via a namespace (and not attached):
 [1] Biobase_2.30.0        jsonlite_0.9.19       assertthat_0.1      
 [4] sp_1.2-3              stats4_3.2.4          latticeExtra_0.6-28 
 [7] RBGL_1.46.0           robustbase_0.92-5     VIM_4.4.1           
[10] quantreg_5.21         RUnit_0.4.31          chron_2.3-47        
[13] RColorBrewer_1.1-2    minqa_1.2.4           colorspace_1.2-6    
[16] plyr_1.8.3            multicool_0.1-9       pcaPP_1.9-60        
[19] XML_3.98-1.4          misc3d_0.8-4          SparseM_1.7         
[22] zlibbioc_1.16.0       corpcor_1.6.8         mvtnorm_1.0-5       
[25] scales_0.4.0          lme4_1.1-12           MatrixModels_0.4-1  
[28] mgcv_1.8-12           car_2.1-2             ggplot2_2.1.0       
[31] nnet_7.3-12           BiocGenerics_0.16.1   hexbin_1.27.1       
[34] pbkrtest_0.4-6        magrittr_1.5          IDPmisc_1.1.17      
[37] ks_1.10.3             GGally_1.0.1          nlme_3.1-127        
[40] MASS_7.3-45           class_7.3-14          graph_1.48.0        
[43] tools_3.2.4           data.table_1.9.6      matrixStats_0.50.2  
[46] stringr_1.0.0         munsell_0.4.3         flowUtils_1.34.0    
[49] pls_2.5-0             e1071_1.6-7           vcd_1.4-1         
[52] grid_3.2.4            nloptr_1.0.4          cvTools_0.3.2       
[55] codetools_0.2-14      gtable_0.2.0          DBI_0.3.1           
[58] reshape_0.8.5         rrcov_1.3-11          R6_2.1.2            
[61] robCompositions_2.0.0 zoo_1.7-12            dplyr_0.4.3         
[64] sROC_0.1-2            KernSmooth_2.23-15    Rgraphviz_2.14.0    
[67] stringi_1.0-1         parallel_3.2.4        Rcpp_0.12.4         
[70] rgl_0.95.1441         DEoptimR_1.0-4        lmtest_0.9-34       

Check your folder to see if all files are valid FCS files.