Hi

Thank you for confirming my suspicions. The distance between base peaks is off.

I'll keep on searching for mutations then.

Also in my opinion this is not an insertion; there is no room for an extra peak between G and A.

There is no insertion.

In the case of doubt, try to sequence the complementary strand.

useful link:

http://www.nucleics.com/DNA_sequencing_support/DNA-sequencing-troubleshooting.html

Hi,

I agree with David and Albino that it is unlikely to be an insertion based on your chromatogram. In our group, we always sequence the amplicon twice (once with forward primer and once with reverse primer) and if a variations appears in both forward and reverse sequence, we consider it a real variation. If it is only in one of the sequences, then it is most likely a sequencing error by the machine. Of course, one should correlate the finding with chromatogram as well, if it makes sense.

Hi,

I also agree it is not a insertion-delition. You want to see the shape of a peak.

Yes, I have ordered both forward and reverse sequencing. I was kind of let down that for a product that is only 150 nucleotides long, neither forward nor reverse sequencing reached the end of the product. I thought Sanger can sequence a couple hundred nucleotides with no problem.

As you can see in the attachment after about 97 nucleotides the chromatogram is unreadable, even though the product I've amplified is longer than that. There were no unspecific bands, no bad primer design, everything was amplified and cleaned-up perfectly. Is it normal?

Hi there,

The truth is the overall quality of the chromatogramm/sequence is very poor: there are multiple peaks overlapping that makes everything very confusing. Normally, such run should be considered as "does not pass quality test". At minimum, it should be repeated, though I expect there might be little help. What is the origin of the DNA and what kind of primers did you use (preset or you designed them yourself)?

Hi, the fact that you don't get 150nt is already telling you that something is wrong. Would think that you are sequencing more than one template, i.e. you have some contamination problem. Or you have multiple primer binding sites.

Such chromatogram depicts a bad sequencing results,peaks should be clear and distinct,. you should check concentration of templet required for sequencingbefore sending it. Also you want to do Digestion conformation before sending it.

It is possible that you have a mixture of two DNAs, which are identical to a certain point, until which you get a fine sequence (until position 97). From there on, as the sequences diverge from each other, you get overlaps of different sequences rendering the whole unreadable. Such DNA mixtures can occur as cloning artifacts.

An alternative is that the DNA has massive secondary structure, which can make sequencing very difficult. I am wondering whether that is the problem in your case, as you say the problem occurs with both the forward and the reverse primer.

I agree with earlier posts. You definitely have multiple DNA sequences. The fact that many peaks are clearly doubled and not just of bad quality is a sign of either contamination or multiple primer binding sites as suggested before. Did you have a negative control in your PCR? You could repeat the PCR, or since it looks like the two products may not be of the same size, you could run the PCR product through an electrophoresis and you will probably see two bands. Then you can cut the bands out of the gel and sequences each band independently.

As I stated earlier, the reaction was specific, optimized and there were no other bands on the gel. The reaction's products were cleaned up using Axygen's PCR Clean-Up Kit. The primers were designed thoroughly as well. I checked the PCR product concentration and it was very high (5–10 ng/uL). I did use a negative control and there were no bands so my reagents are pure.

I have sequenced 4 different products varying in length (100-300bp) using 4 different sets of primers which gave me 8 sequences altogether, both forward and reverse. All of them drop the quality as shown on the picture above about 30-50 nucleotides before the actual end of the amplified product.

The sequence before the signal drops is sequenced correctly.

I don't see any error on my side. I have provided them with primers of proper concentration (5uM = 5 pmol/ul) and purified the products to sequence. I knew that the initial 20-40 nucleotides of each read are lost due to poor quality, but now the final nucleotides appear to be unreadable as well?!

When I have these kinds of problems, I just clone the PCR products and choose 5 clones to sequence the insert. There are many kits for cloning the PCR products for example from Qiagen

good luck

Sanger sequencing works for 700bp sequences in length so the technique used is ok. From my experience even at the begining of the sequencing you can find overlaping peaks but since 80bp of your primer the chromas should be ok. Anyway, in your chromatography it does looks that there is something else and as it has been suggested earlier, I would sequence it again from diferent clones, in forward and reverse directions (just to point out: remember that For and Rev reactions are runned separately!).

Good kuck!

Hi Filip,

I think most has been already told, all very correct. Just to sum up: I would never think at an insertion looking at the first attachment you show. Indeed you seem to be sequencing even 3 different fragments that diverge after A97 in your second attachment (but keep in mind your nt 1 in the chromatogram is for sure not the first nt after the primer... normally unincorporated dideoxys excess covers the signal of the first 20-30 nt). As already suggested I would throw your PCR product in a TA Vector and sequence a few clones with a standard T7 or M13 primer (which will help you sequence also your PCR primers). Finally, sequence quality does not seem good to me since you should not have after 100 nt the big signal of non incorporated dideoxys (the two large C signals and the large A signal...)

Good luck!

Thanks for the reply, what I think of those sequencings are: they diluted my DNA sample too much and the sequencing polymerase ran out of product after ~90 nucleotides. That's why my sequence is gone after that point, and instead it started sequencing some trash (which can always be present in minimal concentration after PCR). If those big peaks mean dideoxy excess, it supports my idea.

I've read about cloning and it overcomplicates everything. I want to use TA vector in stubborn and problematic cases, not in every single case I'm sequencing something.

Well Filip,

you would be right if the peak intensity dropped down suddenly after A97, but unfortunately it doesn't seem to be your case. You seem to have one main fragment (the higher peaks) and one or two minor fragments that are disturbing your sequencing.

Keep in mind that if your DNA template were too low, your primer for sequencing would be in strong excess and the reaction would produce many short sequencing fragments that use up your reagents, resulting in a very clear sequence at the beginning, with very intense peaks, and then suddenly the peaks go flat because you finish up the dNTPs.

I agree that it is in general simpler to just sequence your PCR fragment, but this case seems pretty unclear. I'm not sure what is your template but is seems you might have hit some kind of repetitive sequence. If you don't manage to come around it, cloning will surely help you sort it out, producing single clones (and sequences) out of your mixture, and in general proportionally to your fragment representation. TA cloning takes 5 min, since the topoisomerase on the TA vector avoids the use of the ligase. Unless you are using a Pfu for your PCR, all Taq polymerases add a final A overhang to your PCR fragment (although in different percentage), but it always allows you to straightaway clone in a TA vector quite efficiently.

Again... good luck!!

The C is characteristic reading artifact. You should always sequence the opposite strand. Then you will be able to detect artifacts

As Wolfang said, the best way to detect this kind of artifacts is to sequence the opposite strand.

What is your the ploïdy of your template ? As many contributors said before, and if it is not an artifact, it may just be the result of the sequencing of two alleles.

A heterocygosity wouold not show this type of trailing. It is a characteristic consequence of distinct base sequences which force the DNA polymerase to drop off.without terminator ddNTP

Maybe, just not to confuse the young people, I would remark that the fluorescent peaks derive from the presence of the ddNTPs, which are each labelled with one of four fluorophores with different excitation/emission wavelengths. Fragments without a ddNTP are not detected, but can interfere with the migration of the labelled ones of similar length. Moreover the chromatography that separates the fragments based on their size/length is not completely insensitive to their sequence, which in some cases produces secondary structures that slow down or accelerate the migration of the fragments producing a non equal spacing of the specifically affected fragments. The large gaussian distribution of the C in question might be a very unfortunate case of a secondary structure that is affecting the separation of the fragment or a disturbance of the run by some unlabelled DNA fragment of similar size. So, although I would rule out an insertion, I would still leave open the possibility of a different allelic sequence, if this is compatible with the template Filip is using for the PCR, and as everybody has already suggested I would sequence the other strand, where maybe the structure/migration artifact might be absent and a clearer heterozygosity peak might be obtained.

I wouldn't agree with your inference. It could be very likely an insertion.

Is this a diploid sample? If there was a heterozygous insertion present, then the sequence would become nonsense after this point, because the two different products would be producing sequence shifted from each other by one base.

Such irritating signals occur regularly and are sequence specific results. Impurities/bad DNA quality enhance these problems, which cannot be avoided. If you repeat the sequencing reaction with the same material these signals will turn up again. So, interpretation is a question of experience. Again: ALWAYS sequence the other strand (this is a rule for any careful sequencing anyway).

OK, I have sequenced a lot more exons and now I have gained confidence in analysing them. Such signals do occur from time to time and this one is clearly an artifact.

Dear Garbiez,

It is indeed an artefact. Some times you get the shoulder peaks. If the intensity of the shoulder is above a threshold, the machine makes a base call.