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Old 04-15-2012, 05:43 AM   #1
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Join Date: Apr 2012
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Default Nucleotide blast help please?

Dear all,

I'm looking for some help with Blastn/megablast.

I am trying to find out if my antisense oligonucleotide sequences bind non-specifically to targets other than the intended one.

I've selected "human genomic and transcript"

Then I think I should use megablast, as I am looking for similar sequences - is that correct? However, NCBI adjusts my search parameters because of using a short sequence to blastn. Can I stop it from doing that?

Now the problem starts - I want to only get HSPs where > 15 bp bind without gap and I want them all sorted according to the number of bases identity. How do I get BLAST to do this sorting and which parameters should I choose? I think I can do this by adjusting the word size to 15, but Blastn insists on adjusting my search parameters for "short sequences". I think I should ramp up the match/mismatch costs to 4/-5 to achieve only matches with complete homology - is that right? Again Blast adjusts this.

Also is this a reasonable way of deciding if I get off-target annealing, or are there other/better ways to do this?

Any and all help is much appreciated!

Last edited by LeserattePD; 04-15-2012 at 06:03 AM.
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