I'm seeing a second call inside most peaks of my DNA sequencing data. This second (smaller) peak is exactly the next base called. Has any one seen this before? Is this a problem with primers? I'm attaching a picture.
one way that you can get this effect is having the sequencing primer either annealing one base further away from the normal site as well as at its usual site ( unlikely) but more likely when primers are made there is a failure rate at the addition of each base. If the n-1 mer is not purified away then the n-1 and n-2 etc may anneal ( depending on the amount present and the annealing temperature ) and produce an n-1 sequence . If the primer was HPLC or PAG purified this will not happen but on a poor synthesis ( if the primer contains many G bases ) and the synthesis was just desalted not further purified then this is possible. You could also get this effect if sequencing mitochondrial dna where ,say, 3% of the mitochondria have a one base deletion compared with the normal sequence and the deletion is within about 30 bases of the primer which produces this effect, Are you getting this effect from both ends when sequencing or is it just one primer? and the other sequence is ok
one way that you can get this effect is having the sequencing primer either annealing one base further away from the normal site as well as at its usual site ( unlikely) but more likely when primers are made there is a failure rate at the addition of each base. If the n-1 mer is not purified away then the n-1 and n-2 etc may anneal ( depending on the amount present and the annealing temperature ) and produce an n-1 sequence . If the primer was HPLC or PAG purified this will not happen but on a poor synthesis ( if the primer contains many G bases ) and the synthesis was just desalted not further purified then this is possible. You could also get this effect if sequencing mitochondrial dna where ,say, 3% of the mitochondria have a one base deletion compared with the normal sequence and the deletion is within about 30 bases of the primer which produces this effect, Are you getting this effect from both ends when sequencing or is it just one primer? and the other sequence is ok
100% agreement with Pauls statements! One simple addition to his anwer: the reverse primer could possibly answer the deletion/insertion question.
A deletion/insertion near the forward priming site or the n-1/n-2 problem could be the answer. However, if you observe only very weak secondary peaks, this shouldn't be a problem.
Does the small secondary peak matter for your assay? If not, you can honestly just chalk it up to "weird stuff happens" and not worry about trying to fix it.
Hi Katie. We're interested in the sequence data because we have a lot of projects depending on this idea working. Our bioinformatician was a bit concerned about it, especially because we didn't know why it was occurring. I've ordered the primers HPLC purified, so fingers crossed it gets rid of this n-1. If it doesn't, would you say it's no big deal? If you take a look at my attachment, the secondary peak is not predominant, but I would have to explain to the bioinformatician and PI why our data is still usable.
it would be interesting to see the sequence of the primer that creates this effect and the first 30 bases of the amplimer just in case the full length primer can anneal in 2 positions differing by one base . This might just be possible as pcr annealing often works at high temperatures but many sequencing kits suggest primer annealing at 50c which may be much lower so allowing the primer to anneal at the wrong position with fewer bases annealing. Very unlikely but just possible particularly if the primer has a single base repeat in its sequence. In this case increasing the annealing temperature of the sequencing reaction would make the effect disappear and decreasing the Ta would make it worse
It would also be of interest whether the templates are PCR-products or cloned DNA.
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