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Old 02-01-2016, 10:27 AM   #1
smitra
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Default Primer sequence search

Hi I want to pop this post up. Though this is not exactly my question but related. Actually I have few primer sequences, and I want to blast them to specific species strains.

My primer sequences looks like
Quote:
27Fmod
V1+V2+V3
AGRGTTTGATCMTGGCTCAG
ill519Rmod
V1+V2+V3
GTNTTACNGCGGCKGCTG
And I want to check them against B.bifidum,

http://www.ncbi.nlm.nih.gov/nuccore/U25952.1
First as a newbie, I tried direct blastn agains blast formatted B.bifidum strain. But then realise in the primers there are many other letters as M,R,G besides A,T,C,G;
So no wonder I don’t get a direct match with blastn. Can anybody please suggest me how should I proceed.
Thanks a lot, Mitra

Last edited by GenoMax; 02-01-2016 at 10:33 AM. Reason: Moved the question to a new thread
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Old 02-01-2016, 01:45 PM   #2
dgscofield
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If you have BioPerl available, you could use DISPR (Degenerate In-Silico PcR), which handles degenerate primers just as you have and searches directly within your sequence set of interest.

https://github.com/douglasgscofield/dispr
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Old 02-01-2016, 02:14 PM   #3
smitra
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Thanks dgscofield. I will take a close look to explore this. As a newbie seems will take a bit of time. Thanks
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Old 02-02-2016, 07:54 AM   #4
m_two
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Primer-blast

Link: http://www.ncbi.nlm.nih.gov/tools/primer-blast/
http://blast.ncbi.nlm.nih.gov/Blast....E=BlastNews#13
Contact: blast-help@ncbi.nlm.nih.gov

Primer-BLAST was developed at NCBI to help users make primers that are specific to the input PCR template. It uses Primer3 to design PCR primers and then submits them to BLAST search against user-selected database. The blast results are then automatically analyzed to avoid primer pairs that can cause amplification of targets other than the input template.
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Old 02-02-2016, 08:45 AM   #5
GenoMax
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@smitra: Bifidobacterium bifidum (taxid:1681) is available as an option for primer-blast @NCBI (choose "Genome: chromosomes from all organisms" database). That would be the easiest option as pointed out by @m_two.

Note: Bifidobacterium bifidum KCTC 3202 does not appear to be in the options list but perhaps there is an alternate ID (there are multiple bifidum strains) you could use.

Last edited by GenoMax; 02-02-2016 at 08:49 AM.
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Old 02-05-2016, 10:53 AM   #6
smitra
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Hello GenoMax and m_two,
I have been exploring primer blast. But from the tutorials and info, I could only find the ways to design primer using primer blast. But didn't manage to find how can I check or blast my existing primers against the Ref.
If I paste my primers in the Primer Parameters section and select the Bifidobacterium bifidum (taxid:1681) as Ref. Then how can I do that blast? Clicking Get primers is not the option that I want.
Any help will be really great. Thanks,smitra
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Old 02-05-2016, 11:27 AM   #7
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It appears the primer-blast @NCBI does not accept IUPAC codes so that is not going to work. I don't think blat accepts IUPAC codes either. So we will need some other option here.

@smitra: Is that a real example of a primer pair that is posted in #1? If not can you post a real pair?

Last edited by GenoMax; 02-05-2016 at 11:30 AM.
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Old 02-05-2016, 11:36 AM   #8
smitra
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Dear Genomax,
Thanks for your reply. Yes this is the point I am struggling. Seems like I need to write a code to get all possible combination of IUPAC codes then to local blast all against desired species. But I was hoping there are already something out there, if people need to check such primers.

And yes...they are real pair of primers. Actually I need to check three pairs. As pasted bellow

Quote:
Primer code 16S rRNA gene hypervariable region targeted Primer sequence
27Fmod V1+V2+V3 AGRGTTTGATCMTGGCTCAG
ill519Rmod V1+V2+V3 GTNTTACNGCGGCKGCTG

530R V4+V5 GTGCCAGCMGCNGCGG
bac926R V4+V5 CCGTCAATTYYTTTRAGTTT

926F V6+V7+V8 AAACTYAAAKGAATTGACGG
bac1394R V6+V7+V8 ACGGGCGGTGTGTRC
Thanks for helping, smitra

Last edited by smitra; 02-05-2016 at 11:39 AM.
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Old 02-05-2016, 11:41 AM   #9
GenoMax
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Found this thread which can atleast generate the primer combinations you need: https://www.biostars.org/p/6219/
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Old 02-05-2016, 11:45 AM   #10
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Looks good. I will try this. Thanks GenoMax
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Old 02-05-2016, 12:09 PM   #11
smitra
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The perl code works fine. Only needed to add M. Thanks

Now that I got my combination sequences, I am wondering if I do a local blast directly against the ref species , should I use the complement of these sequences..as these are primers after all. so complement should match right?

Last edited by smitra; 02-05-2016 at 12:21 PM.
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