In a previous post I asked Why do NEB adapters have non-complementary sequence?
Since then, I realized that there is some other sequence in the p5 indexing primer, as well as in the p7 indexing primer.
Here is a diagram of the NEBNext protocol. The parts that I am confused about are in step 5.
- My first question is about the p5 index. Why is there a sequence
5'GATCTACAC 3'between the p5 flowcell oligo and the index?
- My second question is about both indices. Why is there no gap between the p7 index and the flowcell annealing oligo?
For reference, I fetched these indexing primer sequences from NEB on page 18 and 19 of NEBNext Multiplex Oligos for Illumina instruction manual.