I am trying to establish a knock-out line by genome editing. In the sequencing results, I am getting these overlapping peaks (please see the attached image) in all my samples in this specific region, which is also the selected gRNA region. What might be the reason for these peaks and how to interpret or solve them?
If you get this in your edited cells but not in control cells than it is likely the result of having a mixed population of edited and unedited chromosomes in your culture.
There are online tools that allow you to analyze these and get the fraction of edited sequences as well as the types of edits.
you will need a sequence of a control unedited cells to be used as reference.
https://tide.nki.nl/
https://www.synthego.com/products/bioinformatics/crispr-analysis
Might be an artifact from the Sanger sequencing. Try sequencing from a primer facing the other direction.
Hi Ummey Kulsum
I think mostly looks like a PCR artifact and I would suggest a- using a proofread polymerase for your PCR reaction, b- gel purifying your PCR band, and c- using a sequencing primer at least 100 bp away from the cut site. After that, you can analyze whether this edited/non-edited cell population.
I concur with Katie A S Burnette that the first step is to be sure it is a real problem and not just a sequencing artifact. Sequencing from the other direction is the best simple solution.
Katie A S Burnette Michael J. Benedik Thank you for your suggestions. I sequenced from the opposite direction and got the same kind of result. What should I do in this situation?
Amr Salem I am using 2X KAPA mastermix and the PCR band is gel purified for sequencing. I will try your option C.
Please let me know if there is any other solution to this.
Thank you.
Is it possible that your cells have two copies which vary at this position? Or that you had two populations of cells that had not adequately segregrated so you are getting two different PCR products together?
If you read the sequence in the two different ways does it make sense (in other words is one the wild type sequence and one the mutant?)
Update:
It seems the problem I was facing was dye blob. I used XTerminator instead of ethanol precipitation after the sequencing reaction and it solved the problem.
Thanks everyone.
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