We performed sequencing of PCR product using a forward and the reverse primer. When the results came out and when we tried aligning the forward sequence with the reverse sequence, multiple gaps were present. How should we treat these gaps since they are supposed to be the same sequence? Can we fill in the missing gaps with reference to the forward or the reverse sequence?
I understand that you are using Sanger capillary sequencing. To obtain a reliable consensus sequence it is recommended to cover each position with two sequencing fragments in both directions (hence a total of 4). I do not know what software, if any, you were using. Useful to know is that there exists software that automatically aligns the sequencing fragments and allows to display the original sequencer output with peaks at locations where there are gaps/ambiguities, making it easy to use human judgment to resolve the problems. I myself are familiar with good old Staden software (http://staden.sourceforge.net/). Maybe it is worth the effort learning how to use it.
Simply check your sequence chromatograms for quality. find your SNPs/gaps in sequence and check for dye blobs/repeats problems and etc.
Also it depends on PCR product length. if the product is short, reaction with one primer may be performed better than with other. If product is long, then reverse primer will give better sequence quality from one end, F- from another. then take the best sequence for the reference.
If you sequence with seq company, you should clean your PCR product and/or try to upgrade the quality (perform amplification with pfu enzyme).
You might also have a sequence length polymorphism - particularly if you are amplifying a non-coding region. Does it look as if there are overlapping peaks in your chromatograms? If so then you could try cloning and sequencing individual clones or look at a program like "champuru" (http://jfflot.mnhn.fr/champuru/) to try and phase and disentangle the sequences.
Direct sequencing of PCR products is appealing, but can be problematic. Many sequencing companies, don't do it as well as one might hope. If you don't have very many products, I'd clone them and sequence the plasmids off both the M13 and T7 forward and reverse primers. Sequencing multiple clones will allow you to see any SNPs (they'll be non-polymorphic in the clone, right?) as well as any other variants, such as repeats or indels.
Good Luck!
G
Hi
Most of the time in a sequencing procedure, about 50 bp of start site, some errors could occure and furthermore about 600 bp would done without error. If you need the full sequence of PCR product, you may clone it in a T/A or etc. cloning vectors and use the vector primers to do sequencing. If the PCR product length is more than 1200 t0 1500 bps (with regard the company and the method used for sequencing), you may need the interior primer to cover all the sequence length.
God speed
I understand that you are using Sanger capillary sequencing. To obtain a reliable consensus sequence it is recommended to cover each position with two sequencing fragments in both directions (hence a total of 4). I do not know what software, if any, you were using. Useful to know is that there exists software that automatically aligns the sequencing fragments and allows to display the original sequencer output with peaks at locations where there are gaps/ambiguities, making it easy to use human judgment to resolve the problems. I myself are familiar with good old Staden software (http://staden.sourceforge.net/). Maybe it is worth the effort learning how to use it.
Thank you all for your suggestions. Since I was a beginner at sequence analysis, I was making a few mistakes. Now I have assembled my sequences properly. Thank you very much again
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