[ScottC]

It's possible for single read sequencing, or the first read of a paired end run, but not the second read of a paired end run.

Cheers,

Scott.

[Aaron Cooper]

It's possible for single read sequencing, or the first read of a paired end run, but not the second read of a paired end run.

Thanks for the response-- this is encouraging. I have somewhat funky libraries, and if I can use a custom primer for the first read, I would get an extra 22bp.

If you don't mind, could you please expand a bit on your statement, such as how the primer is fed in during the different cycles and why this is possible for SE but not PE? I might request this from our core and I would like to be a bit more knowledgeable before I suggest something that could endanger other people's libraries.

Thanks again,
Aaron

[ScottC]

I should point out that the technology I'm describing here is the GAIIx and Cluster Station.

The reason is very simply - it's the fluidics setup of the hardware that is the reason/limitation regarding the use of one primer or multiple primers.

Some background:
The cluster station ("CS") is a separate (from the sequencer) piece of equipment that is used to attach your template DNA (library) to the inside surface of the flowcell. It is amplified there to produce clusters, the DNA in the clusters is linearised and the sequencing primer is annealed (the CS also does a few other things in there that aren't relevant). The flowcell is then removed from the CS to the Genome Analyzer (GA) where the sequencing proper takes place. At the end of the first read, the strands are essentially turned upside-down on the flowcell and the second read primer is annealed (again, that's a rather simplified description for brevity). The paired-end module enables the flipping and rehybridisation of the 2nd read primer. It is yet another piece of equipment, but attached to the sequencer by some fluidics lines. It really only facilitates the pumping of the correct reagents into the flowcell as it sits in the sequencer.

The hardware related to the primer hybridisation:

The cluster station has a separate inlet port for each of the 8 lanes, so that you can flow on a separate template for sequencing. You can see the inlet lines in this image. They're currently taking reagents from the 8-tube PCR strip in the blue tube holder. The flowcell is located under the white clamp just behind those lines. These tubes are where you put your 8 libraries before they're taken into the flowcell. You change the strip tubes for different reagents, and at primer hybridisation time, you can replace the tubes with 8 different tubes of primer if you wish (or 8 tubes with the same primer). This is why you can also provide separate sequencing primers at this stage.

The paired end module has a single line that flows to the flowcell inlet. In fact, the whole flowcell has only a single inlet line on the GAIIx. This means that it pumps the same reagent through all 8 lanes. This is why the second read primer must be the same for all lanes. You can see the single input line on this image. The line runs through the manifold (orange plastic block) and into the flowcell (where the pipette tip is pointing).

I hope that helps.

PS: Images are linked from a Pasteur institute web page. They're not mine.

Cheers,

Scott.

[Aaron Cooper]

This all makes perfect sense. Thanks very much for the detailed explanation-- this is immensely helpful.

[ashchin]

what happens if some of the lanes have unbalanced bases ans some lanes have balanced indexes?

[cnicolet]

single lane multiplexing

also, we found out that HP7 has the indexing and the regular read 2 primer in it (that is, HP7 will work on plain old non-truseq PE libraries). While HP7 is not available to order as stand-alone (of course) illumina has been good about supplying this as a favor

regarding the analysis issue, our FAS said if we did have focus issues related to blank indexing lanes following the first read 2 base, just re-start the software and resume the run. Re-calibrating that first base supposedly fixes the problem (no direct experience with that, however)

[megalodon]

Custom Sequencing Primers

@ Scott and Aaron:

What Scott describes for the cluster station and GA is also true for the cBOT and HiSeq. It is possible to use different read1 sequencing primers for different lanes on the cBOT (each lane has an independent inlet port). This is not possible for the read2 sequencing primer since the sequencing primer during read2 resynthesis is supplied through a single inlet port just like on the GA.

[dagmar]

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.

Have you ever tried to sequence indexed libraries and non-indexed on the same lane?

[NextGenSeq]

1% PhiX is not enough to maintain focus. You have to restart HCS after the index read. Attached is the Illumina protocol on how to do this.

[kshankar]

While this is an old thread, perhaps someone can answer a similar issue.
I have two different kinds of libraries on the same flow cell (single read, GAIIx); 1) multiplexed libraries requiring the standard Read 1 and Index read seq primers from Illumina, and 2) 16S rRNA libraries requiring custom read 1 and custom index sequencing primers. These will obviously be on separate lanes, hence I can hybridize the respective primers for the primary read during cluster generation. However, for the index read will it be OK to mix equimolar concentrations of the two 'separate' index sequencing primers? Anyone see any major red flags? I have checked and don't see any overlap between the two primers.

[cnicolet]

We have done several runs mixing different index primers into the existing illumina index read mix (HP8), sometimes with the nextera indexing primers and sometimes with different custom primers. The nexteras are provided at 200x, that's easy. For custom we are assuming 100 uM is also 200x, and that has worked well. For cluster station and cBot, you can also mix the custom primer into the solution already containing the illumina primers, in our hands there has been no cross reactivity and it's easier than running the special routines to do individual lane primer hybs (for cbot just peel back the foil and add to the appropriate tube).

[megalodon]

I agree with the previous post. While our lab usually doesn't do multiplexed sequencing, we have mixed different sequencing primers for read1 and read2 and never had a problem with that. It should be the same for the index primer. As long as there is no overlap between the different primers you are using, you should be good to go.

[csquared]

Our results agree with the previous two posts. We've mixed non-overlapping primers for indexing with no issues. As long as the primers for one lane won't interfere with the other lanes and you keep the relative concentrations correct (be sure the concentration of EACH primer is the standard concentration), there shouldn't be any issues.