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Old 01-25-2015, 01:38 PM   #1
Location: Israel

Join Date: Mar 2014
Posts: 26
Default miseq run troubleshoot and advice on nextera xt library normalization


I would like to ask for some advice on normalization of Nextera XT libraries that will be sequenced on Miseq (paired ends 2x150). We got low output (~400K) in one run with our samples (bacterial genomes, ~2.5 Mb), and we believe something may not have worked well during or after the normalization step. So this time we want to do the normalization without following Nextera XT's protocol, that is, by quantitation of libraries with Qubit after clean up and Tapestation check.

1. To what concentration should we bring our libraries?

2. In what buffer should we dilute them?

3. Should this be done before denaturation or after?

4. How long can libraries (after PCR clean up, before normalization) be stored frozen before normalization etc. (in case we want to use our previous libraries and only normalize etc. them now)?

5. If someone has a protocol I would be glad to take a look because Illumina has many pieces of protocols and sheets which are a bit confusing to follow...

Attached are the last libraries we tried to run. For some reason we do not get longer libraries than these. Concentration of most of them seem ok, although we only checked them with Tapestation and not with Qubit last time. We are aware that some libraries are of low concentration. Is it possible that the addition of these low concentration libraries to beads normalization dropped the overall concentration even of the good libraries?

Since we want to sequence a lot of genomes, I wish we could use beads normalization. In your experience, would that work if we did not add the very low concentration libraries or dilutes the high concentration ones before this step?

Attached are some graphs from the last run. The only thing we thought about was the normalization and on steps.

Thanks a lot in advance!
Attached Images
File Type: png libraries.png (31.4 KB, 55 views)
File Type: png Fig 1.png (56.3 KB, 46 views)
File Type: png Fig 2.png (46.1 KB, 36 views)
File Type: png cluster dens.png (6.7 KB, 47 views)
File Type: png intensity..png (14.7 KB, 42 views)
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Old 02-02-2015, 06:00 AM   #2
Jessica D.
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Location: Washington, DC Metro

Join Date: Feb 2015
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I'll need to go back through some of my own notes for specifics on the Nextera preps, but my general MiSeq workflow is the same as my usual Illumina-- libraries all normalized to ~2nM and we quantify using qPCR and then size correct the results. You can use something like Qiagen EB buffer (from your PCR cleanup) to dilute your libraries if you need to.

Libraries should definitely be normalized before you denature, at least if you use qPCR for quantification. I haven't used either Tapestation or Qubit before, but qPCR doesn't give accurate quants for denatured DNA, or so I've been told, so normalization has to be done first.

Libraries should be stable for several months. I've never had to go back and repeat a library a year after making it, but based on things I've heard in user groups, libraries should be stable for at least that long. Having said that, your 2nM dilution and denatured libraries are going to degrade more rapidly, especially if you freeze/thaw them. I've also seen issues with (presumably?) contaminated buffers causing faster-than-normal library degradation, so if your cluster counts are drastically different than what you expected based on your quantification results, you might want to re-quantify and verify that your library hasn't degraded.

The short version is that a library stock should be stable for months. 2nM dilutions should be good for at least a few weeks, but I wouldn't re-use a denatured library unless it was going on MiSeq one day and then on a HiSeq (or being repeated on MiSeq) within a day or two. We tend to err on the side of caution as far as using diluted library-- when in doubt we usually just remake and requantify. It's cheaper than missing the target cluster density on a run and having to repeat the sequencing.

I hope that helps.
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Old 02-08-2015, 11:53 AM   #3
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The attached protocol of illumina may help.
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File Type: pdf Library Loading & Phix.pdf (269.8 KB, 95 views)
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Old 08-19-2015, 06:44 AM   #4
Location: Chile

Join Date: Aug 2011
Posts: 33

Hi Jessica D.
Do you use 2nM libraries always to work? And what is the cluster density that you obtain routinely?
Because I've working using 4nM libraries previous to denaturation and I've got cluster densities between 800-900

But the problem is that it is difficult to obtain libraries at that final concentration...
So, I want to hear your experience using 2nM

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Old 08-19-2015, 11:26 AM   #5
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Location: Washington, D.C. metro area

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Hi soleulloa,

I found my old login information for this site, so I'm back to posting under my previous username. Same Jessica, different last initial. Apologies in advance for any confusion.

To your question:
Yes, with v2 MiSeq chemistry, I'm generally taking my stock libraries and diluting them to 2nM prior to denaturing. For most applications, I'm been loading ~12-14pM and my cluster densities are generally 1000-1100K/mm^2.

If you use the current Illumina recommendation of 2nM library with 0.2N NaOH, you can't get a library concentration above 10pM, which I generally found to not be suitable for most of the applications I've been running.

When I first started using a MiSeq a few years ago, my FAS had told me to use 10ul of the 2nM DNA and 10ul of 0.1N NaOH so that when you neutralize the hydroxide with HT1, you only add 980ul. The final hydroxide ion concentration in all reactions should still be ~0.001N NaOH and the library concentration will be 20pM, which you can then dilute as you need for loading.

I don't have my old "Preparing Libraries for Sequencing" document from when I first got a MiSeq back in 2013, so I don't remember if the 0.1N NaOH concentration was ever an official recommendation from Illumina or if it was a fix that my FAS came up with since a lot of people were having trouble with poor cluster amplificiation at the time. I want to say it was an unofficial fix, so if you should ever decide to do it yourself, your mileage may vary.

Last edited by Jessica_L; 08-19-2015 at 11:29 AM. Reason: u/n clarification
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Old 08-20-2015, 07:46 AM   #6
Location: Chile

Join Date: Aug 2011
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Thank you so much Jessica!
I'll take your advices
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library preparation, miseq, nextera xt, normalization

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