Prime Editing Plot

[rehman220]

I have attached a screenshot of one of the prime editing outputs, would someone be able to explain what the different columns refer to (reference unmodified, reference modified, prime-edited unmodified, prime-edit modified, scaffold-incorporated, and ambiguous).

[kclem]

Hi @rehman220,

Thanks for using CRISPResso.

CRISPResso follows a 2-step process for prime editing analysis.

1. Each read is aligned to the WT reference sequence and the prime-edited sequence. The read is assigned to whichever reference sequence aligns best. At this point, the read will be classified as Reference, Prime-edited, or Scaffold-incorporated. If the read aligns equally well to both sequences, it is classified as Ambiguous. This usually occurs when the prime-edited region is deleted. (Learn more about debugging ambiguous alignments here)

2. Next, the each read is examined for modifications (indels, substitutions) in the quantification window for the corresponding reference. For reads that aligned to the WT reference sequence in step 1, if there is no modification in the quantification window, they will be assigned 'Reference UMODIFIED'. If there are modifications, the read will be assigned 'Reference MODIFIED'. For reads aligned to the prime-edited reference in step 1, CRISPResso checks for scaffold-incorporation at the end of the provided extension sequence. If scaffold sequence is present, the read is assigned 'Scaffold-incorporated'. Otherwise, if there are no modifications in the quantification window, it will be assigned 'Prime-edited UNMODIFIED', and if modifications are present, it will be classified as 'Prime-edited MODIFIED'.

Briefly:

• Reference UNMODIFIED: Read aligns to the WT sequence best and has no modifications in the quantification window.
• Reference MODIFIED: Read aligns to the WT sequence best but has modifications in the quantification window.
• Prime-edited UNMODIFIED: Read aligns to the prime-edited sequence best and has no modifications in the quantification window.
• Prime-edited MODIFIED: Read aligns to the prime-edited sequence best but has modifications in the quantification window.
• Scaffold-incorporated: Read aligns to the prime-edited sequence best but has bases from the scaffold incorporated at the end of the extension sequence.
• AMBIGUOUS: Read aligns to WT sequence and prime-edited sequence equally well.

I hope that helps!