Can you attach a sequence file ( .abi file of the its 1 and 4 sequences both good and failed please?

An insertion/deletion near to one primer will allow mainly good sequence from the other primer but a mixed and messy sequence from the other primer.

What are you sending for sequencing? is it purified pcr band in one tube and only one primer in a separate tube. You can only run sequencing with one primer so if you want a complete sequence you need to send 2 pcr samples or 1 large sample that can be split for sequencing) and 2 primer samples for the sequencing reaction. Is the company purifying your pcr band or are you purifying the pcr band and sending them the purified product?

Sir, Paul Rutland

Thanks a lot for responding. I have attached the .abi file.

I have used both forward and reverse primer in 1 pcr tube for amplification and sent that one tube for sequencing (and not only one primer in a separate tube). And it is not purified, they are purifying and sequencing

Thank you Rakhe Tepin

Forget what I said about indels this is a sequencing problem.

ITS1 is very good sequencing for 650 bases at an average intensity of over 300. The clean sequence indicates a clean pcr band and the lack of an ununcorporated dye peak says that the sequencing primer has bound well and all of the sequencing mix has been well used.

ITS4 looks like 2 sequences but really it is one sequence of intensity less than 30 which is getting mixed up with chemical background and electronic noise making the sequence unreadable. The company purified the dna and ITS1 worked so the problem has to be with your ITS4 primer, The primer did produce a pcr product so it should have been capable of sequencing so 2 possibilities seem likely to me.

1 The sequencing reaction anneals at 50c so if your ITS4 primer anneals at lower than 50c we may have poor annealing so a weak sequence.

or 2 The primer is degrading due to being stored in water or just having been stored for too long. If some of the primer is degraded it will not have anu effect on the pcr because the primer is in huge excess so there will always be some primer that will bind. In the sequencing reaction there is not a huge excess of primer so we may be getting a very weak signal because not much of the sequencing mix is being used.

You could try sequencing from the other its4 primer or or if all of your ITS4 sequences look like this then ordering a new primer may work better

Thanks a lot sir,

So it could be due to low concentration of DNA because of primer degradation and/or low sequencing mix used for sequencing?

I think annealing temperature could not be the problem as its4 has also been successfully sequenced (I've added another .abi file where its4 is good but its1 is bad) but i might try it too.

I've contacted the company they told me to resend more pcr mixture and after you've mentioned about low sequencing mixture I think the problem could be possibly due to low sequencing mix (or low DNA concentration) as they are asking me to send once again.

Next time I'll try to send 2 pcr tubes for each sequence hopefully they might be able to complete sequence.

I agree that with other good results the answer is not due to poor primer annealing. It does seem more likely that the company had a problem with the purification ( or the quality of the big dye mix but this seems lesslikely) and that running the samples again will work. The purification process should produce enough material for more than one sequence so the company asking for more seems odd and hopefully they are not charging you for the re sequencing

I hope they wont charge.

Again

Sir, thanks a lot for responding. I'm new to PCR and sequencing and you have helped me a lot with my doubts.