Can you attach an original .abi sequence file for one control and one sample in case the sequencing parameters hold a hint of where the problem is arising please? Also have you run an agarose gel to check that 1 there is no primer dimer and

2 there are no non specific bands in the amplimers that you sent for sequencing

Could be missing something here, but isn't the fact that crispr editing introduces random indels going to innately hinder good sequencing data?

Like, your PCR primers will span the target site, so will amplify all edited (and unedited) targets, but there will be a ton of different mutations within that target site, all of which will be sequenced, leading to a whole mess of overlapping peaks.

Deconvoluting this mess of peaks is exactly what TIDE analysis is, after all.

Alternatively, assuming I am missing something, what's the purity of your PCR product like? You say you measure the concentration, so I assume that means nanodrop or similar: what's the 260/230 ratio like?

Most PCR cleanup/gel extraction kits use guanidium salts, and those absorb at 230: low 260/230 ratios are bad news for downstream applications.

Having said that, I find most PCR cleanup kits are pretty good in this respect (unlike gel extractions, which are almost always terrible).

Have you run the gel for gel purification?

Hi everyone, thanks a lot for your helpful comments!!

I always run a gel (using 10 ul of my total 50 ul reaction) to confirm that there is just one band, and then I purify the remaining 40 ul with the PCR cleaning kit. My bands on the gel are unique and very clear.

About the sequencing file, I am not talking about samples that underwent CRISPR cutting: those I expect them to be messy for the reason mentioned by John Hildyard. The point is that the control file itself is rejected by TIDE/ICE analysis as not being of enough quality to use it to align and thus deconvolute the messy/post CRISPR editing traces. Paul Rutland I attach you the control ab1 files I am talking about.

I will check when I am in the lab the 260/230 ratio again: in the case that this is effectively the problem, do you have any suggestions on how to improve this? do an additional ethanol wash before eluting the DNA from the column?

Eventually if I repeat all the PCR/purification/sequencing multiple times I think I will get to a decent sequencing, but repeating everything just to try by chance to get a better trace without changing anything in the protocol doesn't really adress the problem.

Thanks a lot!!

Thank you for the files Sofia Visconti . I will get back to you later today but meanwhile are you purifying away the pcr primers from the pcr product before sequencing? Are you using exo-sap degradation of the primers or is the sequencing company removing the primers for you please?

Thank you Paul

Hi

I agree with the suggestions above by Paul and John. In my opinion, the profiles looks like mix sequences which may occur due to the reasons indicated by Paul. Most kits will provide good quality products suitable for sequencing and such problems may be due to primer related issues.

1) The issue may be resolved by using exo-sap digestion or

2) You may also use minimum possible amount of primers for PCR reactions which will go for sequencing. This will ensure almost complete utilization of the primers and will nullify the associated problems in sequencing

3) One more concern is quality of primers, if the primers are old or subjected to several freeze-thaw cycles. Mixture of truncated primers may yield similar PCR product in the PCR, but when used for sequencing, the reaction will begin at different locations on the templates and even 1 based difference in primer length may give overlapping peaks at almost every position. Better to keep in mind this aspect also

all the best

Sofia Visconti

I have just re read your question and see that the pcr cleanup is nucleospin so do not worry about exosap.

These 2 sequences look completely different so I will treat them separately.

1 controllo dna.abi ( 661 bases)

This is a good sequence of a single template, well spaced and well cleaned up after the sequencing reaction. The intensity of the peaks (approximate) at base 30,60,90,120,150 and 180 are 657,459,234,219,19`,79,and 56 and get weaker with increasing bases.

This is a problem because expected intensities should be more than 800 for at least the first 500 bases and is important because you are looking for the secondary sequence but it will be a problem to identify because the background noise in your sequence is intensity 5 so when the true signal strength is 56 then the random background noise is 10% of the signal strength.

The problem here is how to get the signal strength up at least 10 fold so that the background becomes less than 1% of the main signal. There is no sign of primer dimer in the sequence data so the main areas to look at are 1 That the amount of primer in the reaction is 3.2uM

2 That the amount of template is 5-20 ng ( 10ng if the pcr product is 700 bases long)

3 that the sequencing mix is not being diluted before use and is in good condition….as your sequencing is being done commercially you have no control over this.

2 controllo.abi

This is a badly spaced and poor electropherogram. This is because either there are 2 sequences present or that you have 2 primers in the sequencing mix.. the signal strengths at 30,60,90…..240 are 657,459,234,219,191,79,56,55 so again the signal drops too quickly and much too low so again the signal to noise ratio makes it impossible to accurately analyse this sequence.

Again you have to improve the signal strength but unlike the first sample you may also have to worry about the quality of the pcr and also the quality of the sequencing primer.

Since your mutated samples sound like they sequenced well it is hard to say that the sequencing primer is sub standard and one control sequenced well but weak I would blame sample or primer mix up/contamination but as the signal intensities drop too quickly and are too low then again I would pay attention to the relative amounts of template and primer .

It might be worthwhile raising the annealing temperature of the pcr to make the pcr product more specific and asking the company to treat the samples as high GC but primer and template seem worth a look and unless the mutated sample sequences are very clean re ordering the sequencing primers may help

Hi Sofia,

You should notice that when you manipulate your cells with CRISPR, both alleles may undergo sequence changes, which may disturb the sequencing chromatogram. The possible solution is to separate the PCR product strands, clone them into TA-vector, and then send the vector for sequencing.

Hello Sofia,

I'm also agreed with possible reasons and suggestions made by @ Paul.

In my opinion, if the amplification and sequencing primers are the same then product purification is not the major case. In other case you need to purify your products.

For the Sanger sequence the quantity and quality of your DNA is the major case. Contaminants greatly hinder the ability to obtain good sequence data. Make sure to clean up DNA to get rid of excess salts, contaminants and PCR primers.

The two most common causes for failure to get good or any sequence data for your samples are purity and concentration of your template DNA.