Hi, I am looking for a script or tool to blasta all of my primer sequences against the reference fasta sequence. When i tried to use megablast i did not get any output eventhough those primers are supposed to hit somewhere in the reference sequence. I guess the problem here is the input sequences which are quite short. Any help is appreciated. Thanks in advance.....
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upendrakumar.devisetty
There must be something wrong there. Primer sequences should match the reference sequence perfectly, so almost every tool should find them (even a simple grep (need to compensate for linebreaks in sequences)). But try for example primersearch from EMBOSS or BWA, both should find them with standard parameters. Note to provide the correct input format for primresearch (not fasta). If you want to use blast, use blast+ program blastn with task option blastn-short and not megablast, see the documentation.
Unfortunately, there are so many things that can go wrong, these are only few pitfall I can think of:
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Hi, Thank you for your detailed information. I have used megablast with lower wordsize and it worked well.
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Megablast has a large default word-size. I think it's more than 20. If your primers are less than the word-size, then you won't get any results. Try lowering the word size to the minimum allowed.
You are right, the default word_size is 28 for megablast. The documentation of parameters and defaults can be found here: http://www.ncbi.nlm.nih.gov/books/NBK1763/#ui-ncbiinpagenav-heading-16 Table C2 shows blastn parameters
Thank you both. I have changed the word_size and the megablast was able to work with my primers.