SEQanswers

Go Back   SEQanswers > Sequencing Technologies/Companies > Illumina/Solexa



Similar Threads
Thread Thread Starter Forum Replies Last Post
REM e for more than two-lane plates flxlex 454 Pyrosequencing 2 01-04-2011 10:16 PM
16 lane run leakage TonyBrooks 454 Pyrosequencing 1 05-24-2010 08:05 AM
what's in solexa's control lane mingkunli Illumina/Solexa 5 01-27-2010 11:35 AM
When can I get away without a control lane? lparsons Illumina/Solexa 4 11-02-2009 12:37 AM
Control Lane - PhiX bioinfosm Illumina/Solexa 3 07-16-2008 03:18 PM

Reply
 
Thread Tools
Old 06-29-2009, 12:23 PM   #1
mcrepeau
Junior Member
 
Location: California

Join Date: Jun 2009
Posts: 2
Default Doing multiplex in one lane only

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?
mcrepeau is offline   Reply With Quote
Old 06-30-2009, 03:37 AM   #2
kmcarr
Senior Member
 
Location: USA, Midwest

Join Date: May 2008
Posts: 1,178
Default

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.
Attached Files
File Type: pdf IlmnIndexSeq.pdf (67.6 KB, 310 views)
kmcarr is offline   Reply With Quote
Old 06-30-2009, 10:19 AM   #3
mcrepeau
Junior Member
 
Location: California

Join Date: Jun 2009
Posts: 2
Default

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?
mcrepeau is offline   Reply With Quote
Old 06-30-2009, 10:33 AM   #4
kmcarr
Senior Member
 
Location: USA, Midwest

Join Date: May 2008
Posts: 1,178
Default

In the immortal words of Emily Litella...

Never mind.

You were making sense.

(Must remember not to post before my morning coffee.)
kmcarr is offline   Reply With Quote
Old 07-06-2009, 02:37 PM   #5
ScottC
Senior Member
 
Location: Monash University, Melbourne, Australia.

Join Date: Jan 2008
Posts: 246
Default

Has anyone tried this yet? Or heard anything from Illumina about it?
ScottC is offline   Reply With Quote
Old 02-26-2011, 11:39 AM   #6
Aaron Cooper
Member
 
Location: Los Angeles

Join Date: Feb 2011
Posts: 15
Default

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.
Aaron Cooper is offline   Reply With Quote
Old 02-26-2011, 01:56 PM   #7
ScottC
Senior Member
 
Location: Monash University, Melbourne, Australia.

Join Date: Jan 2008
Posts: 246
Default

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...
ScottC is offline   Reply With Quote
Old 02-26-2011, 02:02 PM   #8
Aaron Cooper
Member
 
Location: Los Angeles

Join Date: Feb 2011
Posts: 15
Default

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?)
Aaron Cooper is offline   Reply With Quote
Old 02-26-2011, 04:41 PM   #9
ScottC
Senior Member
 
Location: Monash University, Melbourne, Australia.

Join Date: Jan 2008
Posts: 246
Default

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.
ScottC is offline   Reply With Quote
Old 02-26-2011, 04:58 PM   #10
Aaron Cooper
Member
 
Location: Los Angeles

Join Date: Feb 2011
Posts: 15
Default

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.
Aaron Cooper is offline   Reply With Quote
Old 03-09-2011, 06:47 PM   #11
csquared
Member
 
Location: Huntsville, AL

Join Date: May 2008
Posts: 67
Default

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.
__________________
HudsonAlpha Institute for Biotechnology
http://www.hudsonalpha.org/gsl
csquared is offline   Reply With Quote
Old 03-09-2011, 08:39 PM   #12
Aaron Cooper
Member
 
Location: Los Angeles

Join Date: Feb 2011
Posts: 15
Default

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.
Aaron Cooper is offline   Reply With Quote
Old 03-10-2011, 06:29 AM   #13
csquared
Member
 
Location: Huntsville, AL

Join Date: May 2008
Posts: 67
Default

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.
__________________
HudsonAlpha Institute for Biotechnology
http://www.hudsonalpha.org/gsl
csquared is offline   Reply With Quote
Old 03-17-2011, 06:37 AM   #14
BTS
Member
 
Location: Wisconsin

Join Date: Sep 2010
Posts: 19
Default

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...
BTS is offline   Reply With Quote
Old 03-20-2011, 03:43 PM   #15
Aaron Cooper
Member
 
Location: Los Angeles

Join Date: Feb 2011
Posts: 15
Default

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?
Aaron Cooper is offline   Reply With Quote
Old 03-20-2011, 04:36 PM   #16
ScottC
Senior Member
 
Location: Monash University, Melbourne, Australia.

Join Date: Jan 2008
Posts: 246
Default

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.
ScottC is offline   Reply With Quote
Old 03-20-2011, 05:03 PM   #17
Aaron Cooper
Member
 
Location: Los Angeles

Join Date: Feb 2011
Posts: 15
Default

Quote:
Originally Posted by ScottC View Post
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
Aaron Cooper is offline   Reply With Quote
Old 03-20-2011, 06:20 PM   #18
ScottC
Senior Member
 
Location: Monash University, Melbourne, Australia.

Join Date: Jan 2008
Posts: 246
Default

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.

Last edited by ScottC; 03-20-2011 at 07:02 PM.
ScottC is offline   Reply With Quote
Old 03-20-2011, 07:01 PM   #19
Aaron Cooper
Member
 
Location: Los Angeles

Join Date: Feb 2011
Posts: 15
Default

This all makes perfect sense. Thanks very much for the detailed explanation-- this is immensely helpful.
Aaron Cooper is offline   Reply With Quote
Old 04-16-2011, 07:00 PM   #20
ashchin
Member
 
Location: USA

Join Date: Dec 2010
Posts: 19
Default

what happens if some of the lanes have unbalanced bases ans some lanes have balanced indexes?
ashchin is offline   Reply With Quote
Reply

Thread Tools

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off




All times are GMT -8. The time now is 08:25 AM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2020, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO