I've found what looks like a heterozygous mutation in my DNA sequencing. It's more convincing in some reads than others but it definitely looks like the mutation is there. However it only shows up in the forward sequence and the reverse has just the wild type allele. I repeated the sequencing and got the same result. I SNP checked the primers and checked they're unique in the genome - all fine. There is a pseudogene related to the gene I'm investigating but it doesn't seem to have the same sequence in the region that I've sequenced.
I'd be grateful for any suggestions. Thanks.
There are only two things I can think could be the case based on the information you are providing:
1. The reverse primer has an issue binding to the mutant allele. It sounds like you accounted for that possibility (I assume that is to what "SNP checked" refers), but perhaps there is something about the primer itself, such as the way it was manufactured, that is causing an issue.
2. What you are interpreting as a heterozygous mutation is a sequencing error, albeit a recurrent one.
My suggestion would be to design new primers to different areas around the mutation and do another round of sequencing to see if the phenomenon still occurs.
I've seen two cases where the secondary structure around a piece of DNA results in an odd read in one direction and not the other. I would suggest amplifying with different primers and sequencing again. You can try smaller fragments and sequencing primers closer to the SNP. Let us know what you find out.
As Raymond said, the reverse primer might not bind because of a polymorphism in the primer site where the rare allele is in LD with the mutation you are sequencing. I have heard of that, but it is probably rare. To me, the "more convincing in some reads than others" in your question suggests a sequencing artifact, where something is migrating in the capillary (I am assume Sanger/BigDye?), which can be consistent. So, in addition trying new primers, I would consider going old school and do a restriction digest if you are lucky and can find a reasonable enzyme that cuts or doesn't cut at this mutation. Digest the PCR product.
If you are sequencing with the same primers that you have used for amplification there are no problems of SNPs. I had a similar case, which turned out to be an artifact of the sequencing reaction. I confirmed this by repeating the amplification and sequencing of the same region with a different primer couple.
I'd try to amplify the region with different primers, as everybody says. At least with a different reverse primer. The most plausible explanation is that there is a mutation/SNP in the region of annealing for the reverse primer.
Using new primers and sequencing parents as well might solve the proplem
I think this situation is one of the reasons we do fwd and rev sequencing.
Richard, if you cannot see the mutation on one of the reads, it is likely that it is an artifact, even if you can see it with multiple samples/ iterations. Like Michael said, use a restriction enzyme to confirm or if you do not have a suitable one, try new primers that will amplify the same region.
As every one opined, use new pair of primers is a better way. You mentioned about the presence of pseudo gene for the gene of your interest. Now this is different thing, if the gene on which you are working belongs to multigene family and the region which you targeted is highly variable, then do one thing, design fresh primers and go for sequencing then compare the sequence generated by old pair of primers. Otherwise design primers in such a way that for both the samples design same forward primer and different reverse primers. So if it is SNP as your thinking one pair of primers target the SNP allele and other targets normal allele. But finally, considered the advice of artifact given by others strongly. Good Luck
Is the mutation within 50bp of the forward primer? (Low amount of) primer dimers present in your amplicon sample might disturb your sequence in the first 50 bp.
I suggest never accept a mutation as such unless you get the same results from both forward and reverse primers. Many times if you get it only with one primer, this is an artifact.
Yer I thought it might just be an artifact - but was hoping it wasn't...
Many thanks for all your responses. Sounds like the consensus is to design new primers, but I might try the digest approach as well.
I observe exactly the same situation but I think that the mutation is real because it was previously detected by exome sequencing with illumina. The only explanation I can think of is, as suggested, a secondary structure issue because I am sequencing with the same primers I used for amplification. However, I do not see any additional snp that could account for a different secondary structure in PCR products with the mutation. I will design new primers and see what happens...
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