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？
First, I would strongly discourage using anything other than the V4 region (see http://blog.mothur.org/2014/09/11/Why-such-a-large-distance-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