On Target Score Characterization in Validating Existing sgRNA Library Tutorial

[stefanusbernard]

Hi, in the validating existing sgRNA library tutorial, it was mentioned that after creating the guideset, we can characterize off-target sgRNA by simply using addOffTargetScores() command and it directly output both CFD and MIT scores. Furthermore, I noticed we can do the same for on-target scoring by using addOnTargetScores() and it will output on-target score of our choice.

However, the on-target scoring method like rule set 1 and rule set 3 will also require the 4 bp of flank5 and 3 bp of flank3 sequences which actually discarded from the very beginning (as we only take the 20-mer spacer sequence) and there is no such columns in the guideset that define the flank5 and flank3 sequences.

I would like to know whether using addOnTargetScores() to validate the sgRNA library is still reliable in this case or do I need to do it manually by first defining the flank5 and flank3 sequences?.

[xguo-nveloptx]

I think the issue I ran into with addOnTargetScores() is that python 2.7 is no longer supported and can't be found in current conda chanels. Any hope to move away from to python 2.7?

[Jfortin1]

Hi @stefanusbernard,

You're correct that flanking nucleotides are needed to compute most of the scores. Based on the genomic coordinates stored in the GuideSet object, the addOnTargetScores() will automatically extract the extra needed nucleotides from the reference genome for each on-target scoring method separately, so no need to do anything from your side.