Correcting allele-specific counts by library size factor before input to SCALE?

[angm...@gmail.com]

Hi Yuchao,

I was wondering if for input to SCALE, we are required to adjust by library size factor before running the package, or does SCALE do it automatically internally? Your publication writes that "For endogenous RNAs, SCALE takes as input the observed allele-specific read counts at heterozygous loci Qa and Qb, with adjustment by library size factor".

However, your demo code shows you correcting total read counts when examining heterogeneity in QC (see lines 64-66), but no adjustments were made to alleleA and alleleB before running analysis with SCALE. Should I divide alleleA and alleleB by library.size.factor matrix before running SCALE?



On a separate note, I have found a minor bug in your allelic_plot code. On line 41:

text(x=0.5,y=0.65,paste('Number of cells:',paste(names(results.list[[1]]),results.list[[1]],collapse = '; ')))

The results.list[[1]] should be updated to results.list[[i]]:

text(x=0.5,y=0.65,paste('Number of cells:',paste(names(results.list[[i]]),results.list[[i]],collapse = '; ')))

Regards,
Roy

[Yuchao Jiang]

HI Roy,

Thanks for finding the bug. I’ve already corrected it and pushed the update. 

For library size, in the QC procedure it needs to be calculated and adjusted. For estimating kinetics, library size is calculated and adjusted within the function allelic_kinetics() — see line 10 and 25 here https://github.com/yuchaojiang/SCALE/blob/master/package/R/allelic_kinetics.R .

Hope that this helps and please feel free to let me know if you have more questions or encounter any other problems.

Best,

Yuchao

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