Dear All,
The forward and reverse sequences obtained from the PCR products are not identical at all.
Can I still make the contigs for doing BLASTn?
Individually both sequences are showing the same species in BLASTn.
Together, by placing one sequence after another they are showing a different species.
Kindly suggest a way to identify the organism by using both forward and reverse sequences.
Best regards
Arush
The sequences should be the reverse complement to each other, so you will need to convert one of your sequences to a reverse complement so you can combine them to form a consensus sequence.
If you did the above and it was still a problem, it is possible the sequence is long and there was a breakdown towards the end of sequencing, meaning there may be no clear sections of overlap. If this is the problem, you can rerun the PCR and sequencing using primers closer together to crease a shorter contig.
That being said, since both the forward and reverse sequences agree, you probably have identified the organism.
Matthew B Paddock is correct but I will add one more thing. If your amplimer has a small insert or deletion ( CA repeat or poly A or similar) near to one end then the sequence will look good from the other end but will be weak signalled and messy after the indel when sequenced from the indel end. You may have to trim off poorly sequenced bases at the ends of each sequence to get a good Blast result but if you are looking at gene coding sequences then genes are highly conserved through species and it may just be that your search finds many identical sequences and the one that you are interested in is way down a list of nearly identical sequences. You may have to increase the number of matches shown
The sequences of your PCR product, using forward and reverse primers, should be different but only because they are reverse complement of each other.
I agree with other answers. I would suggest you use SeqMan software by which you will be able to assemble your reverse and forward sequences and compare all nucleotides in terms of insertion/deletion or differences.
HERE YOU CAN NOT SAY COMPLETELY REVERSE COMPLEMENTARY TO EACH OTHER.
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