Hello All,

I am trying to analyze sc-RNA data using both scater and scran. While trying to run normalization using scran via the computeSumFactors method, I get the following error. Could someone please explain me what this error means and if there a way to fix it? Appreciate all your help and suggestions, thanks!!!

Code snippet: 

ScaterObject <- newSCESet(

    countData = mycountFrame,

    phenoData = pheno_data

)

keep_feature <- rowSums(counts(ScaterObject ) > 0) > 0

ScaterObject  <-ScaterObject [keep_feature,]

ScaterObject<- computeSumFactors(ScaterObject, sizes=c(20, 40, 60, 80))

summary(sizeFactors(ScaterObject))

Error:

Error in .local(x, ...) : 

  not enough cells in each cluster for specified 'sizes'

Calls: computeSumFactors ... .local -> computeSumFactors -> computeSumFactors -> .local

Execution halted

The problem is pretty much what the error message says; you don't have enough cells to use the pool sizes (i.e., in sizes) that you've specified to computeSumFactors. One option is to turn down the number of cells in each pool, e.g., from 10 - 40. This is probably fine as long as your genes are moderately to highly expressed. Otherwise you'll just end up with lots of zeroes, which defeats the purpose of pooling in the first place.

On a related note, it seems that you're keeping genes that are expressed at any level in any cell. This will probably result in a large number of genes that are only expressed in a handful (i.e., 1-2) of cells, which in turn leads to lots of zeroes in the pooled expression profiles. You might end up nonsensical size factors of zero when the median ratio is computed - at the very least, the use of the median ratio as a robust approximation to the mean won't be accurate for very low counts. I would suggest performing some more aggressive filtering (e.g., using only genes with a mean count above 1), for the sake of accurately calculating the size factors.