Dear Sir/Madame,

I am using the EdgeR (https://bioconductor.org/packages/release/bioc/html/edgeR.html) - processAmplicons function just to raw count the number of sgRNAs I have in samples treated with custom-made sgRNA/CRISPR pooled libraries and I love the easy to use processAmplicons function as it also allows for easy detection of sgRNAs containing mismatches or shifts.

However, I was wondering if anyone has some experience to run the EdgeR processAmplicons function without barcodes or if it is possible to set the barcode start and end coordinates to 0/0 or 1/1. I am asking because if I just remove these variables or adjust them to 0 I am unable to run the function. Most of my reads do not contain barcodes anymore and some of them have shifted by 1-2 bp, hence the question. Although I might be able to perform similar tasks with other scripts/functions, I just love the basic and almost ready to use processAmplicons function and I would like to keep using it.

With kind regards,

Jasper

Here is the work around that I ended up using: 

Make a barcodefile with your sample ID, group, and replicate, and then a 2 base long barcode.  Can be anything.  Then when you call processAmplicons, set allowMismatch=TRUE and barcodeMismatchBase=2

This will make the barcode into any 2-mer, so every read will match a barcode.

If you've got multiple samples, read them each into separate DGEList objects this way then merge them with cbind.