I am currently looking at some splice variants of human genes and I have obtained sequence data from a PCR product which I had cut out of a gel. I have blasted this sequence against the human genome via blast at NCBI.
The results I got back raised a question which I am not sure how to interpret. I have a five nucleotide sequence in the pcr product which matches to the alternate assembly but not the primary assembly and so I was wondering what is the cause of this discrepancy?
Is it that some people may have these five nucleotides and other people don't?
Or is there just a mistake in the primary assembly sequence? If it was just one or two nucleotides I could understand this but five missed nucleotides in a row seems like a bit big to be a "pure" sequencing mistake or would this be an incorrect assumption?
Did the primary and alternate assembly genetic data come from the same person/people or do we know this?
Hi Joel
I have preformed the RT-PCR on about 8-10 different people but only 2 people give me the amplicon which i have the query about. I cut out one of these amplicons and sent it for sequencing and then blasted the sequence read i got against the NCBI human genome.
1 - the coding sequence is within the ORF
2 - the gene im looking at belongs to a multigene family
3 - yes the gene itself is found in other species
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