<p>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.</p>

<p>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.</p>

<p><a href="https://i.sstatic.net/LHZV0.jpg" rel="noreferrer"><img src="https://i.sstatic.net/LHZV0.jpg" alt="enter image description here"></a></p>

CC BY-SA 4.0
2018-12-21T22:20:31.400Z
2018-12-21T22:20:31.400Z
Beth Nelson
<p><a href="https://bioinformatics.stackexchange.com/questions/4061/why-do-neb-adapters-have-non-complementary-sequence">In a previous post</a> I asked <strong>Why do NEB adapters have non-complementary sequence?</strong></p>

<p>Since then, I realized that there is some other sequence in the p5 indexing primer, as well as in the p7 indexing primer.</p>

<p>Here is a diagram of the NEBNext protocol. The parts that I am confused about are in step 5.</p>

<p><a href="https://i.sstatic.net/7Gc73.png" rel="noreferrer"><img src="https://i.sstatic.net/7Gc73.png" alt="NEBNext protocol"></a></p>

<ol>
<li>My first question is about the p5 index. Why is there a sequence <code>5'GATCTACAC 3'</code> between the p5 flowcell oligo and the index? </li>
</ol>

<p><a href="https://i.sstatic.net/1K2yM.png" rel="noreferrer"><img src="https://i.sstatic.net/1K2yM.png" alt="enter image description here"></a></p>

<ol start="2">
<li>My second question is about both indices. Why is there no gap between the p7 index and the flowcell annealing oligo?</li>
</ol>

<p><a href="https://i.sstatic.net/EhUwu.png" rel="noreferrer"><img src="https://i.sstatic.net/EhUwu.png" alt="enter image description here"></a></p>

<p>For reference, I fetched these indexing primer sequences from <a href="https://www.neb.com/-/media/catalog/datacards-or-manuals/manuale7780.pdf" rel="noreferrer">NEB on page 18 and 19 of NEBNext Multiplex Oligos for Illumina instruction manual</a>.</p>

<phylogenetics><illumina><adapter><pcr><library>
Why do NEBNext indexing primers have sequence between the p5 oligo and index?
# Why do NEBNext indexing primers have sequence between the p5 oligo and index?

(Asked by: username_0 on 2018-06-21)

[In a previous post](https://bioinformatics.stackexchange.com/questions/4061/why-do-neb-adapters-have-non-complementary-sequence) 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](https://i.sstatic.net/7Gc73.png)](https://i.sstatic.net/7Gc73.png)

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](https://i.sstatic.net/1K2yM.png)](https://i.sstatic.net/1K2yM.png)

2. 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](https://i.sstatic.net/EhUwu.png)](https://i.sstatic.net/EhUwu.png)

For reference, I fetched these indexing primer sequences from [NEB on page 18 and 19 of NEBNext Multiplex Oligos for Illumina instruction manual](https://www.neb.com/-/media/catalog/datacards-or-manuals/manuale7780.pdf).

**Tags:** phylogenetics, illumina, adapter, pcr, library

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## 1 answers

### Answer by username_1 on 2018-12-21. Score: 5 | Accepted answer

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](https://i.sstatic.net/LHZV0.jpg)](https://i.sstatic.net/LHZV0.jpg)


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