Some question about pad sequence

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genovia
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Joined: Wed Dec 13, 2017 9:46 pm

Some question about pad sequence

Post by genovia » Fri Dec 15, 2017 4:40 am

Hello

I'm trying to design a couple of sets of primers for 16S V1-V2(forward AGAGTTTGATCMTGGCTCAG and reverse ACTCCTACGGGAGGCAGC), Could you give me some concrete methods about how to make a suitable pad sequence and link sequence ? or about the principle of pad sequence and link sequence?


I am really grateful if anyone can provide some suggestions!
Thanks very much in advance!



genovia

pschloss
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Re: Some question about pad sequence

Post by pschloss » Mon Dec 18, 2017 10:03 am

First, I would strongly discourage using anything other than the V4 region (see http://blog.mothur.org/2014/09/11/Why-s ... nce-matrix)

Second, the linker sequence is a 2 nt sequence that is anti-complimentary to any other sequence in the database and the pad is a ~6 nt sequence that increases the Tm of the sequencing primer to between 60 and 65C. You can see the details about primer design and sequencing on the wet lab SOP: https://github.com/SchlossLab/MiSeq_WetLab_SOP

Pat

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