I am using CAGEr to analyze some of my CAGE data. However I run into some errors when I tried to cluster the TSSs using clusterCTSS() function.

I use CAGEr 1.18 and R 3.3.2.

My input is the CTSS file for human genome 38 chr1, e.g.:

chr1    10594   -       1

chr1    10595   -       3

chr1    10597   -       14

chr1    10599   -       4

chr1    10600   -       17

Here are the commands I used before clustering:

>myCAGEset <- new("CAGEset", genomeName = "BSgenome.Hsapiens.UCSC.hg38", inputFiles = ctss_file, inputFilesType = 'ctss', sampleLabels=c("test”))

>getCTSS(myCAGEset)

>normalizeTagCount(myCAGEset, method = "powerLaw",fitInRange = c(5, 1000), alpha = 1.0, T = 10^6)

>ctss <- CTSStagCount(myCAGEset)

> clusterCTSS(object = myCAGEset, method = "paraclu")

Filtering CTSSs below threshold...

Clustering...

    -> test

Error in subset.default(clusters, (max_d >= (minStability * min_d)) &  : 

  object 'max_d' not found

Thanks a lot!

Thanks for the report. On a virtual machine running R 3.2.3, I tried the following test, which worked;

load(system.file("data", "exampleCAGEset.RData", package="CAGEr"))
clusterCTSS(object = exampleCAGEset, method = "paraclu")

Then I tried after normalisation and it did not work anymore.

load(system.file("data", "exampleCAGEset.RData", package="CAGEr"))
normalizeTagCount(exampleCAGEset, method = "powerLaw",fitInRange = c(5, 1000), alpha = 1.0, T = 10^6)
clusterCTSS(object = exampleCAGEset, method = "paraclu")

The reason is that the normalizeTagCount() command turned all the expression values to zero:

> summary(CTSSnormalizedTpm(exampleCAGEset))
    chr            pos          strand      sample1     sample2     sample3
 chr13:5272   Min.   :3000059   +:2840   Min.   :0   Min.   :0   Min.   :0  
              1st Qu.:4318065   -:2432   1st Qu.:0   1st Qu.:0   1st Qu.:0  
              Median :5238722            Median :0   Median :0   Median :0  
              Mean   :5370033            Mean   :0   Mean   :0   Mean   :0  
              3rd Qu.:6266246            3rd Qu.:0   3rd Qu.:0   3rd Qu.:0  
              Max.   :7993858            Max.   :0   Max.   :0   Max.   :0  

The problem seems to be that you set alpha = 1.0.  I recommend you to try a different value (for instance, following the output of plotReverseCumulatives) or a different normalisation method.