[pmiguel]

There is no magic in the PPC. Adapters are not a complete "library element"...which is the beauty behind the Y-adapter. You do not have complimentary ends...they are "sticky", and only contain half of what you need to cluster (only half of your library elements have what it takes to hybridize to the flowcell). They should however qPCR with more accuracy than an order of magnitude...as only the first cycle of qPCR should be affected...but maybe thats enough....I haven't done the math.

From Illumina's FAQ:

Is there a PCR-free TruSeq Sample Prep protocol available?

The new index adapter design enables PCR-free protocols. (A single cycle of synthesis is required to separate the forked adapter.)

Upon querying Illumina tech support, I got the following response:

We have found that if you simply take the annealed fragment that has the forked adapters on each end and add it to the clustering reaction that even with NaOH treatment before clustering, there is poor initial hybridization and the resultant clustering is poor. By allowing at least one PCR cycle this separates the strands and the forked adapters, allows a single amplification of the entire strands, resulting in a clean, double-stranded species that gives cleaner clustering results.

This makes no sense unless one or both of the following is true:

(1) The adapter oligos are attached to each other in some way that resists their melting apart during NaOH treatment.

(2) The PPC primers themselves have some modification that causes them to bind especially well to the flow cell.

Or, at least, that is my take on the situation.

--
Phillip

[SeqR&D]

Neither of those are true. I'm certain that if you run a denaturing gel on the adapters, that they will run as 2 bands. And, PPC primers are complimentary to the adapter ends...nothing special.

Where you might run into issues are with A-tailing efficiency, ligation efficiency, possibly ligating 2 adapters together, having free adapters present when clustering a flowcell (they will take up space, but will not extend or show clusters). Doing some amplification enriches for library elements that have proper adapter ligation on both ends.

[pmiguel]

Neither of those are true. I'm certain that if you run a denaturing gel on the adapters, that they will run as 2 bands.

What makes you certain?

And, PPC primers are complimentary to the adapter ends...nothing special.

Why do they run ~80 nt on a pico RNA chip if they are generic oligos?

Where you might run into issues are with A-tailing efficiency, ligation efficiency, possibly ligating 2 adapters together,

Okay, I'm with you so far...

having free adapters present when clustering a flowcell (they will take up space, but will not extend or show clusters).

And PCR will dilute out the free adapters, okay, I guess.

Doing some amplification enriches for library elements that have proper adapter ligation on both ends.

I see your point. But when we tried no amp libraries we used qPCR to determine the concentration of library elements that had proper adapter ligation on both ends. That is how we determined how much to add to the flow cell. qPCR (SYBR green) should not be confounded by any of the factors you list above.

Yet, when we clustered using the unamplified libraries, we saw much lower cluster yields from those libraries. (Like 1% of what was expected, if I recall correctly.) Our qPCR was not otherwise way off -- all the other libraries we quantified this way clustered at about the expected efficiency. So it seems like the simple explanation is that there is something odd about the TruSeq system that Illumina has not revealed.

--
Phillip

[SeqR&D]

If you qPCR a non-ampified sample that has varying amounts of free adapter (with a keenly designed SPRI purification you can greatly minimize this), library elements with one ligated adapter and those with both, you will be quantifying the 2 adapter libraries, but the other stuff is in there as well. I'm not exactly sure what ligation efficiency is, but from what I understand it's not great...60% to 70%? (correct me if I'm wrong). That could leave a lot of non-clusterable DNA fragments floating around that will "block" the flowcell surface from clusterable library. I'm not sure if this can account for a 99% reduction in cluster density from one to the other. Keep in mind, this is only my best guess.

[pmiguel]

If you qPCR a non-ampified sample that has varying amounts of free adapter (with a keenly designed SPRI purification you can greatly minimize this), library elements with one ligated adapter and those with both, you will be quantifying the 2 adapter libraries, but the other stuff is in there as well.
I'm not exactly sure what ligation efficiency is, but from what I understand it's not great...60% to 70%? (correct me if I'm wrong).

You are right, I think. If anything 60%-70% is on the high side. Although if you presume that 60% means "60% of ends get adapters ligated to them", one would expect only 36% to get both ends adapter ligated.

Actually on Friday we looked at the results from a similar ligation chemistry -- the Roche Rapid Library prep kit for 454 library construction -- and saw 12% of the molecules were amplifiable via qPCR. Not entirely clear this is a result of whether adapters were ligated on or not. The genomic DNA given us was in questionable state -- prepared from old refrigerated leaf tissue or even twigs -- it was degraded to 5kb average length prior to any sonication. So the DNA may just have had a moderately high frequency of damage not circumventable by the qPCR polymerase.

Hmmm, actually the Roche Y adapters have FAM modification. So I guess we could actually assess the number of molecule ends with adapters ligated to them.

That could leave a lot of non-clusterable DNA fragments floating around that will "block" the flowcell surface from clusterable library. I'm not sure if this can account for a 99% reduction in cluster density from one to the other. Keep in mind, this is only my best guess.

Yeah, I like the way you think. The possibility of a blocking issue had not occurred to me prior to your previous post.

As it turns out the attempts we made with no-amp libraries were made from bacterial genomic DNA. So we were only shooting for 10% of a lane for each. (Even that would have been overkill...) So if there were material in the library blocking access of the flow cell oligos to library molecules we would see an effect on the other libraries sharing the lane commensurate with what we saw with with the no-amp libraries. But the other libraries seemed unaffected.

Also, we had previously ran amplified versions of these libraries and they seemed to produce plenty of clusters -- but they were terribly biased. All of this could be the result of the DNA of the organism (Deinococcus radiodurans) -- who know what modifications it hosts. But since Illumina tech support suggested that very poor clustering efficiency was to be expected for TruSeq libraries that had not undergone at least 1 cycle of amplification, I tend to draw other conclusions.

Obviously, I could have this all wrong. Just a couple of hypotheses...

--
Phillip

[Genquest]

Thank you Phillip and SeqR&D,

The discussion was very insightful!! It has given me novel aspects to think about...
I would like to add on quick questions where your suggestions would be needed...

1. Is there a way to detect presence of EtBr in our sample library before we proceed to purification?
2. I have observed variation in concentration of PhiX V2 & V3 libraries,the fragment size explains this.In this context do you suggest to quantitate phiX library with pico green as well to have a better comparison with sample library?
3. Do you use Truseq PPC for qPCR as well?As I have been using the in-house primers which was synthesized by the sequences provided by Illumina qPCR protocol.I have run melt curve and we see significant primer dimer peak which we have worked a lot on to remove but have been unsuccessful!!

At one point of time,i had used Illumina PPC for qPCR and the additional dimer peak disappeared and Efficiency improved but the PhIX conc. was even higher than previous ( i.e. when run by in-house primers).Don't remember which version of PhiX I used,will have to put a check on this as well!!
Suggestions and advises are welcomed!!
thank you once again

[pmiguel]

Thank you Phillip and SeqR&D,

The discussion was very insightful!! It has given me novel aspects to think about...
I would like to add on quick questions where your suggestions would be needed...

1. Is there a way to detect presence of EtBr in our sample library before we proceed to purification?

It would be possible fluorimetrically. But I don't have details. If you did some sort of clean-up after a step that used EtBr, you could at least hope the concentration of EtBr would drop below a threshold where it might be a problem.

2. I have observed variation in concentration of PhiX V2 & V3 libraries,the fragment size explains this.In this context do you suggest to quantitate phiX library with pico green as well to have a better comparison with sample library?

We now run a high sensitivity bioanalyzer chip on all new tubes of PhiX. That will give the length of the library and clue you into to abnormalities (like adapter dimer peaks...)

3. Do you use Truseq PPC for qPCR as well?As I have been using the in-house primers which was synthesized by the sequences provided by Illumina qPCR protocol.I have run melt curve and we see significant primer dimer peak which we have worked a lot on to remove but have been unsuccessful!!

A couple of things here:
(1) I don't think a bimodal melt curve is an indication of a primer dimer. Call me a heretic if you will, but why would we expect library melt curves to be monomodal? Any reason? Will not a not the complexity of a library, and the input sample from which it derives, cause one to see various melt curve modalities?

Anyway, I guess the TruSeq adapters themselves will be one of the peaks -- they don't have to be in an adapter or primer dimer to have their own melt temp.

A melt curve, I would hazard to guess, is for standard qPCR, where there will be a single gene being amplified. There your melt curve should be monomodal because it is a single sequence that is being amplified. But a library is a complex mixture of molecules all flanked by the same adapters.

(2) We just use cheap oligos we had synthesized for qPCR. I think of the PPC as being a limited resource, so it never occurred to me to use them.

--
Phillip

[RezaFalsafi]

deblocking FC

Hello I am new to this forum and sorry if the posts are old I just came across the all the above posts and the one about deblocking the FC and re-using it caught my eye. can it be please explained more as how to deblock? and is the FC used for the same run we tested with 6 cycles or just it is used for 6 cycle testings?
Thanks a lot