I have been using the invertebrate COI primers to amplify and sequence different set of samples for quite a while. But recently I noticed that my forward sequence are returning terribly poor sequences and my reverse sequences of the sample samples are of high quality.
I have use the primers in the past without any such problem. And I have used two different commercial sequencing facilities now and the pattern is the same.
Additionally, I didn't send my primers with the samples to the second sequencing facility as they have the primer pair on their universal list that can be used without additional cost to test if the problem is with my forward primer. But the problem persisted.
I don't know if anyone else has encountered this kind of problem before and how can I get passed this?
Hi Adebiyi,
Are you amplifying before sending to the sequencing lab? If so do you use the same primers to amplify that are used to sequence? I know the sequencing lab used their own primers for sequencing, but perhaps there is an issue (mismatch) in the primer that was used to generate the amplicon.
Good luck.
Frank
Hello Frank,
Yes, I amplifies before sending to the sequencing laboratory and use the same primers for sequencing.
It is confusing as I was not experiencing this before
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