3
$\begingroup$

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.

NEBNext protocol

  1. My first question is about the p5 index. Why is there a sequence 5'GATCTACAC 3' between the p5 flowcell oligo and the index?

enter image description here

  1. My second question is about both indices. Why is there no gap between the p7 index and the flowcell annealing oligo?

enter image description here

For reference, I fetched these indexing primer sequences from NEB on page 18 and 19 of NEBNext Multiplex Oligos for Illumina instruction manual.

$\endgroup$
4
$\begingroup$

Question 1: The additional sequences is needed because that is complimentary to the P5 sequence anchored to the flow cell. It is also the site for priming the Index 2 read on a MiSeq. The additional sequence is also needed for the read 1 primer in the cartridge to anneal to the correct place for the molecules.

Question 2: The "GT" sequence you're referring to is necessary for the Index 1 and Read 2 primer to anneal to the correct place.

enter image description here

$\endgroup$

Your Answer

By clicking “Post Your Answer”, you agree to our terms of service, privacy policy and cookie policy

Not the answer you're looking for? Browse other questions tagged or ask your own question.