off-target position

[chuddy-ibk]

Dear Developer,

i have one question, since it seems I was wrong with what "off-target" meant in this tool.

Initially I though of the site, where interesting things happen like substitutions, indels, etc. However, today I realized that the position column only shows me the most left side of the binding site of the sgRNA when mismatched. When looking with snapgene at those sites, it means that when i copy paste the DNA column sequence, I will find the mismatch target site and the the starting point of that interval is the "position" column's value, right? well, minus 1, since snapgene should be 1-based and offinder is 0-based.

I always thought the tool had some fixed distance from the PAM region programmed and that would be the position site.

For the minus strand it is still the most "left" side of the mismatch interval, if left means lower genomic base numbers, only this time the position number is right next the the PAM sequence.

Am I totally wrong here?

Anyways, if I am right, then I will need to adjust the position number for the plus strand such that I add 21 to get to the expected cutting site, right?: 1 for 1-based + 20 for protospacer + 3 for NGG - 3 for distance to cut site relative to NGG.

For the minus strand I add 7 to get to the predicted cutting site: 1 for 1-based + 3 for NGG + 3 for distance to cut site relative to NGG

For bulge = 1 the minus strand should be unaffected. For the plus strand, however, I would need differentiate between RNA or DNA bulging. RNA bulge type: I add 20. DNA bulge type: I add 22.

I would appreciate your input as I realized this very late and I saw at least one publication using the predicted positions for overlap with vcf file, which would not make sense anymore, if this is not the off-target cut site.

Have a great day!

Tom