Question about pooling calculations (Nextera XT)

[mdalessio]

I just used a Qubit to quantify all of my 24 samples. I am getting concentrations between 0.2 and 7 ng/ul for each sample. The issue is that I do not have access to a bioanalyzer, so should I assume size distribution is what Nextera XT kit is supposed to spit out, or how should I go forward?

I could still do bead normalization instead, but I am skeptical.

[dfhdfh]

At which point did you perform your quantitation?

[mcnelson.phd]

0.2ng/ul would be too low to be a viable library assuming you quantified after doing the PCR cleanup. Based on an 800bp average fragment size (which is what we usually get) that would be 0.4nM, which is well below the 4nM starting point for following the standard denaturing procedure for non-XT libraries.

Ideally you should have at least 2ng/ul which will equate to ~4nM. Illumina does have a protocol for starting with a 2nM library pool, but we haven't tried that yet so I can't comment on it.

I'm currently sequencing a set of libraries that were between 5 and 15 ng/ul after PCR cleanup, which is the range we normally get, and they're looking perfect so far with really even coverage and metrics.

If most of your libraries are on the low end of the spectrum, then you should just use the bead based normalization. If, however, most of them are above say 4ng/ul, then you can do standard normalization procedure IF you can somehow determine the average peak size. If you can't, then you're really shooting in the dark and open yourself up to sever over/under-clustering which will kill your run.

[helico93561]

based on my limited (but seems working) experience, for sample of 0.2ug/ul, I would dilute 13.2 ul of it with 586 ul (HT1). I work out based on a formula I generated from my previous good runs.

I did not pay too much attention to the DNA concentration (maybe I should). But according to above info and 800 bp fragment, that will generate 16pM DNA.

According to the guideline (1), we should be aiming at 8pM.

Reference
1. MiSeq run preparation, # 15027617 Rev. B, page 51, step 1

[mcnelson.phd]

based on my limited (but seems working) experience, for sample of 0.2ug/ul, I would dilute 13.2 ul of it with 586 ul (HT1). I work out based on a formula I generated from my previous good runs.

I did not pay too much attention to the DNA concentration (maybe I should). But according to above info and 800 bp fragment, that will generate 16pM DNA.

According to the guideline (1), we should be aiming at 8pM.

I'm really not sure I follow what you're talking about. If you're saying that your post-PCR cleanup library is 0.2ng/ul with an avg. size of 800bp, then you DO NOT have a library that would meet the 4nM threshold for the standard denaturing procedure, or even the 2nM level that Illumina has put out a guide for.

You can't just add HT1 buffer to a library without denaturing it first, so whatever you're talking about in your previous post won't work. If you want to play fast and loose with the rules, then by all means do what you want, but I'm advising you to remake those low yield libraries if you don't want to use the bead normalization process of the XT kit.

[helico93561]

Sorry for the misleading comment.

We did not quantify the DNA at post PCR and the beads normalisation.

We use ssDNA Qubit kit to measure the DNA concentration after the mixture of NaOH.

So if we did get 0.2 ng/ul at this stage, we will dilute 13.2 ul of it with 586 ul (HT1). The final NaOH concentration should be 0.0011 N and according to the guideline, it is not over the 0.0025 N limit.

according to the standard formula, 800 bp will give 16 pM; 1000 bp will give 13.3 pM.

we had done a few MiSeq runs base on this calculation and it always gives us a cluster density of around 1000

[rajasereddy]

If u want little more power over the low quantity libraries why dint you run a gel with the libraries where you have enough quantity the you will know the size distribution.