double peaks from base 270, but only for one primer

[PHS]

Dear all,

recently I am running into trouble when sequencing small subunit rDNA from nematode samples using BigDye v3.1 from Exo/Sap or Kit cleaned PCR products. I am sequencing closely related species from one genus, doing two PCR reactions of approximately 900 to 1000 bp each. When sequencing with the forward primers I get high quality reads of good length (up to 680 bases). However, when using the reverse primers this is only true for about half of my PCR products (i.e. half of my species), for the other half the quality drops at around 270 bases (or 370 for the 2nd PCR product). Traces look as if out of phase, or as if there was a second fragment being sequenced along. Needles to say, that I checked the gel for such a second product, bu there isn’t any. Also the forward primer wouldn’t work fine in that case as well, wouldn’t it? Basically the same should be true for secondary structure formation, would affect both primers wouldn’t it?
Also I checked for a second priming site within the expected product (i.e. checked in the products that worked), but that doesn’t seem to be the case. To make extra sure I made new PCRs and sent them away for sequencing to Macrogen Europe – I get the same results, as when I do the sequencing reaction myself and get the reads from our local seq. center. Albeit the Macrogen reads are a bit better as the bases are called for the whole product (not ‘Ns’ from 270 onwards), but not reliable as well, as the extra peaks are still there. Well, I’ve been sequencing small subunit PCR products for the last few years (hundreds and hundreds), but never had this kind of problem. Any help/hints/suggestions would be highly appreciated and I might as well provide more details on my protocols and traces to look at.

Kind regards

PHS

[Daniel]

This sounds like you have a SNP in your sequence. Since you are sequencing from PCR product you actually have two different PCR products from each allele. If one has a small insertion or deletions in one of the alleles then the sequencing will go out of phase after the point of the SNP.

Daniel Tillett
Nucleics Support

[Author]

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