Doing multiplex in one lane only

[mcrepeau]

We want to do a little multiplex sequencing using the Illumina kit and recipes, but we don't need to do a lot. We'd like to do a single lane on a flow cell with all the other lanes containing standard PE libraries. Problem is the multiplex recipes use a different read 2 sequencing primer. Illumina tech support tells me that some customers are just using an equimolar mixture of the multiplex and standard read 2 sequencing primer. Seems reasonable to me. Has anyone tried it, or something similar?

[kmcarr]

I don't think this could work. The primer for the index read is complementary to the same strand as the primer for read 1. The index read is supposed to be performed prior to cluster resynthesis (if you are doing a paired read). I have attached a picture from one of Illumina's publications showing the relationship of the read1, index read and read 2 primers and order of operations.

[mcrepeau]

Hmmm... Perhaps I wasn't clear. I didn't intend to mix the index sequencing primer with the PE read 2 sequencing primer. I meant to mix the multiplex read 2 sequencing primer with the PE read 2 sequencing primer (because the multiplex library uses a different read 2 sequencing primer, supplied with the kit). I don't really see any show stopper there so long as the two primers don't anneal efficiently to the ends from the other library type (i.e. the PE read 2 sequencing primer doesn't anneal to the multiplex kit library ends and the multiplex read 2 sequencing primer doesn't anneal to the PE kit library ends). Does that make more sense?

[kmcarr]

In the immortal words of Emily Litella...

Never mind.

You were making sense.

(Must remember not to post before my morning coffee.)

[ScottC]

Has anyone tried this yet? Or heard anything from Illumina about it?

[Aaron Cooper]

Is anyone able to comment on whether mixing standard PE read 2 and multiplex PE read 2 primers works for getting read 2 in a flow cell with mixed libraries?

I am also interested in this topic-- we are only going to do single lanes most of the time, so I am trying to make the libraries as flexible as possible so that I can jump into a SE, PE, or PE multiplex run. My libraries will be multiplex PE.

[ScottC]

Yes, you can mix the primers. Run it like a multiplex run... Just be sure to get the volume (hence concentration) of primers right according to the volume in the multiplex rd2 primer tube. Use multiplexing PhiX if you're using phi control lanes. We have done this many times. I think yhere may be another thread about this too...

[Aaron Cooper]

Thanks for the response. Are you saying to use the multiplexing PhiX control just as a control for the multiplex reads, or because the software has a multiplexing mode that will throw errors if it doesn't see indexes from the PhiX reads? (i.e. would the core have to do that to make the run work at all, or is it just a recommendation?)

[ScottC]

Hi,

Sorry, I should have been more clear... It's just a recommendation, not a requirement. I simply meant that, if you're using a PhiX control, don't forget that there's a special version of the PhiX library for multiplexing. The software doesn't strictly require it.

[Aaron Cooper]

Ah, that is great to hear-- thanks for the advice. Ultimately it will be up to the core here regarding running the two in the same flow cell, but at least it is feasible.

[csquared]

Mixing index and non-index lanes on a flowcell is fine. However, there are a few considerations to ensure success.

The multiplex and standard READ 2 primers can be mixed and in fact are mixed in the new TruSeq kits (HP7 is the mixed reagent). If you are using TruSeq, read 2 will sequence either the index or standard adaptors. If you are not using TruSeq, just mix the primers with the usual amount of each in the final primer mix.

The VERY important consideration is related to the focus of the instrument and this is a known problem on the HiSeq. When the index read is being performed in the lanes that do not contain any clusters since they are not indexed, it will throw the focus off for read2. This issue is not 100% consistent and can manifest itself in some odd ways but the quality of the run is definitely affected.

The easy fix is to include index PhiX at 1% in all lanes. That will allow all lanes to have some active clusters for every cycle (Read 1, Index read, and read 2). That will maintain the focus and prevent any surprises.

[Aaron Cooper]

Thanks csquared-- this is extremely valuable and helpful information. Our core spikes in 1% PhiX into all lanes, but I will need to be sure that they use index PhiX for my runs.

If I am in a SE run and want to read my index, I won't need to worry about the focusing issue, correct? I am mainly interested in using index adapters for convenience in the library protocol I am working up. Being able to jump into a PE run is just a nice side effect.

[csquared]

Correct. For a SE run, the focus issue is not a problem. Only affects read 2. In our experience, it has been quite variable. Everything from killing read 2 on a whole flowcell to no effect. Typically it results in a few lanes to drop out on read 2.

Including the index PhiX at 1% or more completely solves the issue. Really important to note that this issue is not like a control lane. You need something detectable for the index read in ALL lanes to avoid the focus issue.

[BTS]

Is this focus issue a problem with the GAIIx? I'm just completing a small indexing run and was unaware of the indexed PhiX. We had two lanes drop out for read two. The coincidence may be that these two lanes were prepped in a different way and may have some other issues...

[Aaron Cooper]

A related question-- is it possible to feed a custom sequencing primer to just one lane, or is the whole flow cell fed with the same mixes?