We have sequenced promoter calpastatin genes. Sequence results are very good, but our sequence is very different with reference sequence in Genbank. After, do BLAST, our sequence didn't show closeness in any gene. Any suggestion?
Can you be more significant in question??Using which technology did you sequence?
Is it using NGS methods?
How did you retrieve the sequence ? Is it using chromosome location?
Dear Snijesh,
We use Single Pass DNA Sequencing (BigDye Terminator Sequencing Kit). Yes, we design primers from chromosome sequence data (Ensembl and NCBI). Is it a problem?
Thank you for responding my question.
The biggest question here would be whether your PCR of the promoter worked appropriately or if you somehow just got a background PCR product. The first thing I would do would be to change your BLAST search to search the entire NR database. This includes sequences from all kinds of sources and usually if you have picked up vector, bacteria or some other random sequence you can find it here. Another question would be what do you mean by very good for your sequence. Are there no N's in the reads? Have you looked at the traces to be sure that the peaks have good amplitude and spacing?
if the amplified sequence has no match to your target gene, the most likely explanation is that your primers were not specific enough and you amplified another region in the genome of the organism you are working on. In this case, you should be able to tell which region was amplified based on the BLAST result and can then check why your primers amplified this sequence (my guess is that one of primers is in a repetitive DNA region). If, one the other hand, there is no match to your organism of interest (and it's complete genome is present in the BLAST database) then your PCR product is likely from a contaminating DNA species.
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