Thanks for the great questions!
Please see my response below your questions.
Best regards,
Julie
Question 1- For a guide RNA what is the difference between top10OfftargetTotalScore and top5OfftargetTotalScore ? How shall I interpret them ?
Do I need to look at how off target score is calculated ?
Where does the N in topNOffTargetScore is used to calculate this score ?
Answer: top10OfftargetTotalScore means the sum of the scores from the top 5
offtargets and top10OfftargetTotalScore means the sum of the scores from
top 10 offtargets. Please look at the OffTargetAnalysis.xls file for detailed information on
individual offtargets
Please see the reference section in the help menu of offTargetAnalysis by
typing help(offTargetAnalysis) in a R session.
Please also type help(offTargetAnalysis) in a R session for detailed parameter
settings and interpretation.
You can set the parameter topN.OfftargetTotalScore for calculating the
topNOffTargetScore.
It is the sum of the scores from the top N offtargets where N is set by
topN.OfftargetTotalScore which specifies the top N offtargets used to calculate the topN.OfftargetTotalScore, default 10
In general, you would want to use gRNAs with high efficacy, low
topNOfftargetTotalScore where N can be 5,10, or a number you feel
comfortable, and a lower number of offtargets in offtargetAnalysis.xls.
Question 2- What information do the columns like top1Hit.onTarget.MMdistance2PAM provide , for eg if it has value of NMM ?
What are the different values that can be expected here ?
Answer: When there is no mismatch found in the top 1 hit, then it outputs NMM. If there is at least one perfect match in the genome, then it will output NMM.
Otherwise, it outputs the distance from the mismatch base to the PAM sequence.
Question 3- Does the columns uniqREin200 and uniqREin100 tell about Name of unique restriction enzymes with cut sites 200bp and 100bp upstream or downstream around the guide RNA ?
Answer: Yes, correct. uniqREin200 and uniqREin100 columns contain REs unique to the cleavage site within 100 or 200 bases surrounding the gRNA sequence. You can turn off this by setting outputUniqueREs = FALSE.
Please type help(offTargetAnalysis) in a R session for detailed parameter settings and interpretation.
Question 4- What does the column score mean ? Does it mean that the gRNA with high score has more offTargets ? How is it related to gRNAefficacy column ?
Answer: It depends on which method you use to calculate the score.
You can set scoring.method to "Hsu-Zhang" or "CFDscore".
If you set scoring.method to "CFDscore", the value is from 0 to 1.
If you set scoring.method to "Hsu-Zhang", the value ranges from 0 to 100.
The higher the value, the higher the cutting frequency.
For on-targets, it should be 1 for CFDscore and 100 for Hsu-Zhang score, and
it is close to 0 for off-targets with minimum cut.
Question: Does it mean that the gRNA with high score has more offTargets ?
Answer: No. The score is a relative cutting frequency of an offtarget site relative to the target site.
Question: How is it related to gRNAefficacy column ?
Answer: gRNAefficacy specifies how effective the gRNA is while score is the relative cutting frequency to the ontarget.
gRNAefficacy ranges from 0 to 1. 1 means 100% cutting efficacy while 0 means 0% cutting frequency.
gRNAefficacy refers the efficacy of a gRNA while score indicates the relative cutting frequency on an offtarget relative to that of the ontarget.
Question: Is the gRNAefficacy in OffTargetAnalysis.xls same as in Summary.xls ?
Answer: The gRNAefficacy in OffTargetAnalysis.xls for the target site is the same as that in the Summary.xls.
Question 5- What is isCanonicalPAM ? Is it telling whether the corresponding OffTargetSequence has a canonical PAM ?
Answer: Yes, you were right. Canonical PAM is the PAM sequence you specified in offTargetAnalysis. By default, PAM = "NGG". Please type help(offTargetAnalysis) in a R session for detailed parameter settings and interpretation.
