I have a ~700 PCR amplicon sequenced from both directions. However, forward sequencing and reverse sequencing have different results. The forward one has almost perfect matches with existing sequences in BLAST. But, the reverse one only has the first ~400 bp matching in BLAST. I think it should be caused by sequencing quality (misreading in reverse sequencing). Is it right? Other than this, what else could cause this?
Thank you!
What do the rest of the results look like for the sequencing from the reverse primer? Have you looked at the quality score for the chromatograph? That would probably answer your questions. Go back and actually look at your data.
Not all primers work well for sequencing. You typically won't get quality reads for the first 50-100 bp.
Reverse compliment the reverse primer data, align it with the forward data and BLAST the resulting contig. If you still have a gap and need more sequence data, then use an internal primer. Or clone your PCR product into a plasmid and sequence from that.
Honestly, you should easily be able to get all 700 bp by sequencing from both ends.
What do the rest of the results look like for the sequencing from the reverse primer? Have you looked at the quality score for the chromatograph? That would probably answer your questions. Go back and actually look at your data.
Not all primers work well for sequencing. You typically won't get quality reads for the first 50-100 bp.
Reverse compliment the reverse primer data, align it with the forward data and BLAST the resulting contig. If you still have a gap and need more sequence data, then use an internal primer. Or clone your PCR product into a plasmid and sequence from that.
Honestly, you should easily be able to get all 700 bp by sequencing from both ends.
sometimes also the template can form a secondary structure which becomes double stranded which terminates the sequencing, This is often the case when the electropherogram intensity dies off quickly rather than reagent depletion caused by too many of the same base in the sequence when the sequence dies off slowly. Sequencing in 1M betaine or 3% DMSO (with an initial denaturing of 95c for 5 mins and using double the usual amount of sequencing mix ( some is destroyed in the denaturation step) can often inprove sequencing when secondary structure is a problem. A look at the .abi sequencing file would help for an accurate answer to this problem
I agree with the previous answers. If your chromatogram is good, one reason for the mismatched reverse sequence can be from nonspecific reverse primer that can amplify unintended sequence. Check if your DNA target has any homology to other locations in your DNA template.
- Badges
- Science method