Concentration of DNA to be denatured, loaded onto flowcell

[ktabbada]

Hello everyone! I am helping to manage an Illumina/NGS Facility in the Cambridge, UK area. This is my first post, and I am hoping someone might help answer a question that I have.

Long story short: I would like to know what is the lowest concentration of library that can be denatured for loading onto the flowcell, given that you have to dilute out the NaOH used for denaturation. Please read details below.

I work at an NGS facility that performs sequencing as a service, and recently a DNA library with a very low concentration was submitted to me, and I am puzzled as to how to prepare this for sequencing.

This library will be a simple multiplex, for which three samples will be pooled in equimolar amounts. The starting concentrations of the 3 samples are different, with the lowest being less than 10 pM. Ten microlitres of each sample are available.

I have calculated that even if I use up all of the sample, the most concentrated pooled library (for the denaturation step) that I can produce will be have a concentration of approximately 66 pM of each sample, or 200pM in total. This, in turn, means that in order to produce 120µL of 10 pM denatured, diluted library for loading on the flowcell, I would dilute 6 µL of the 200pM denatured library to 120 µL with Hybridization Buffer, a dilution factor of only 1:20, whereas the normal dilution factor is 1:100 or greater. I am worried that having a large amount of the denatured library (and therefore NaOH) loaded on the flowcell will have an adverse effect on cluster generation.

I was wondering if anyone can say what is the lowest librray concentration that can be loaded on the flowcell (or alternatively, what is the largest volume of NaOH-denaturated DNA that can be diluted for hybridization to the flowcell).

I will be grateful for any information or advice.

[ScottC]

I don't recall off the top of my head, but I'm pretty sure the user manual for the cluster gen kits specify the maximum amount of NaOH that can be present in the sample before it inhibits cluster formation.

Cheers,

Scott.

[ktabbada]

Re: Concentration of DNA to be denatured, loaded onto flowcell

Thank you for your reply, Scott.

However, I was wondering if anyone has tried alternatives to NaOH, such as heat denaturation, or if it might be possible to neutralize the NaOH with HCl. The manual assumes that you have a sufficient amount of your library so that the DNA concentration can be adjusted and the NaOH diluted out, but if you have a very low amount/concentration of the library, then this approach isn't really possible.

[elaney_k]

There's a really great paper that came out from the sanger Institute (is that near you?), "A large genome center's improvements to the Illumina sequencing system" PMID 19034268 which addresses the problem of subnanomolar libraries by adding Tris-HCL to the hyb buffer. I'm not sure if this is still possible with the version 2 cluster gen kits but I would guess that they would be the best people to ask

[ktabbada]

Thank you very much for that reference; this is what I was looking for. I hope it works and thank you again!

[Susanne]

Did it work?

Hi,
would you mind sharing your experience with us, whether the described protocol workked for you, or any other experience you made? Generally, I would assume the material should be enough.

Thanks!

[ktabbada]

Hi Suzanne:

I tried the protocol described in the Sanger paper, although I had a somewhat more concentrated sample (a little less than 100 pM) to start with. I loaded the sample for hybridization at a final concentration of 10pM. The first base report and RTA indicated that we got somewhere between 125,000 and 145,000 clusters per tile, which looks like a reasonable yield.

However, I must add that when I inspected the tiles visually, I found that they had an odd morphology - there appeared to be some variation in the size of the clusters. However, as I don't know much about the sample in question, I can't tell if this is due to some property of the sample itself or an artifact of the sample preparation/denaturation.

I will post more information here when the run (Paired-end) is finished and some quality data is available.

Best wishes,

Kristina

[d17]

Update?

Hi Kristina,

Any chance you have an update on whether your subnanomolar libraries worked? I'd be interested to hear how this turned out!

I just stumbled on your thread today and am interested to see that you at least got pretty good cluster densities. We have tried the protocol described in the Sanger paper for subnanomolar libraries, and have had no success getting anywhere near a reasonable cluster density.

Thanks!

[ktabbada]

Hello d17,

I apologise for taking quite a while to reply to your inquiry. Unfortunately, although initially (that is, during the first read) it appeared that the very low concentration library that we loaded onto the flowcell yielded a reasonable number of clusters (126,000 clusters per tile), after the second read, the number of clusters abruptly dropped by about half. Furthermore, as I noted before the clusters had an unusual moprphology (variable sizes); this may have accounted for another problem with the sample, a very low number of clusters passing the filter.

I'm sorry I don't have better news to report. It would have been very useful if I had been able to validate use of the Sanger protocol for low concentration libraries.

[d17]

Thanks ktabbada! It's useful to hear about failures as well as successes.

I wonder what they're doing over at Sanger ! The protocol we're referring to [in the paper listed earlier in this thread, PMID 19034268] doesn't seem like it should be very difficult to get working ...
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