Custom amplicon sequencing with dual indexing

[tickseq]

custom amplicon dual indexing

Thanks for this link! It is just what I was looking for and is very helpful.

The reverse v5 sequence has a lower melting temperature than the reverse v4 so I need to modify the pad to increase the Tm. Can I just change some of the A's and T's to G's and C's in a non complementary way to the target group? Does the pad sequence need to be anti complementary?

For a dual index approach, have a look at:
http://www.mothur.org/w/images/0/0c/..._MiSeq_SOP.pdf

[RCJK]

For a dual index approach, have a look at:

http://www.mothur.org/w/images/0/0c/Wet-lab_MiSeq_SOP.pdf

[tickseq]

I am trying to use the Caporaso protocol for 16S amplicon sequencing but want to modify it to have dual indexing. I am designing the PCR primers to have the following format:
Forward:
5' Adaptor-Index-Primer Pad-Linker-16S Primer Sequence-3'
Reverse:
5'-RC Adaptor (P7)-Index-Primer Pad-Linker-16S Reverse sequence-3'

I would like to just use the Nextera or Truseq barcode sequences but when I spoke to the Illumina tech they said that the index sequences posted are the sequences as read by the MiSeq. I believe this means that the Index 1 (p7 side) should be reverse-complemented on my custom primer. But I'm not sure is the second Index (P5 side) also needs to be reverse complemented. Does anyone have experience with custom primers and how to insert the barcode sequence?

Also, I am looking at a different region V4 through V5 so my reverse primer sequence is different. I am trying to increase the Tm to be as close to 65 or 70C as possible. I have it up to 63 with a new primer pad. Is this sufficient?

Thanks for any help on these matters