Quantify Illumina Libraries

[felvis56]

Please excuse this basic question but I have used the Kapa Quantification kit on an ABI 7500 to quantify my TruSeq libraries and I just wanted to check how I convert quantity to concentration in pMol? I have run the libraries at serial dilutions of 1:1000 to 1:8000. I know it's probably basic maths but I don't want to get it wrong as the libraries look quite good so far.

Thanks,

[pmiguel]

Please excuse this basic question but I have used the Kapa Quantification kit on an ABI 7500 to quantify my TruSeq libraries and I just wanted to check how I convert quantity to concentration in pMol? I have run the libraries at serial dilutions of 1:1000 to 1:8000. I know it's probably basic maths but I don't want to get it wrong as the libraries look quite good so far.

Thanks,

The way to think about it is to realize that the 1:1000 dilution you did maps nM concentrations into the pM range. The KAPA illumina standards are at 20, 2, 0.2, 0.02 and 0.002 pM. So for your 1:1000 samples you just read the pM values you get from the instrument (by comparison to the standard curve) as "nM".

For other dilutions you have to create a factor to multiply the instrument reading by dividing your dilution factor by 1000. So if you did a 1:2000 dilution of a sample you calculate your factor as 2000/1000=2. So, for example, if your sample read "0.5pM" by the instrument, then your undiluted sample would be 0.5x2 = 1nM.

This all presumes you instrument is not already doing these back-calculations for you somehow. (Like if you told it the sample you were measuring was a 1:2000 dilution of the original samples during set-up of the run.)

Also, don't forget that the KAPA standards are 452 bp and the signal produced is dependent on the number of bases of double stranded DNA created each cycle. So if your amplicons are really different in size from 452 bp, you definitely want to do a correction for that. That is, if your average amplicon sizes are 226 bp, then you need to calculate a factor for that (452/226 = 2). That is there are twice as many molecules of your amplicons as there would have been had your amplicons been 452 bp.

--
Phillip

[microgirl123]

Pmiguel is correct. Also the directions that came with your KAPA kit, if you still have them, explain exactly how to do the math.

[NewbieNGS]

I'm new to NGS and am going charge my first flowcell this week but I'm confused on how to proceed after qpcr quantification. I have followed the calculations given by Kapa and I have 112pM for my first library. Do I need to divide by 4 as I added 4ul of library to the qpcr reaction? Or do I have 112pM/ul? (ideally I want to charge 12pM on my flowcell)

Thanks for any advice....I don't want to make an expensive mistake!

[microgirl123]

If you have properly followed the KAPA math then you do not need to divide by 4. Your library is 112 pM, which is only 0.112 nM. If this is a MiSeq run, there is not nearly enough library to load on a flowcell. To load 12 pM, you need to start with at least a 2 nM library.

Also the unit of pM is not the same as pg/ul. So you cannot have 112 pM/ul.

[NewbieNGS]

Thank you microgirl123! I guess I will redo the bead normalization of my library. Can you please explain to me why my concentration cut-off should be 2nM. I'm really new at this and no one in our lab has experience with NGS either and I don't find this part explained well in the manual.

[NewbieNGS]

Nextera XT libraries with bead normalization

[microgirl123]

Ah...this is Nextera XT. We don't run many of those. I'll look into my last run and get back to you!

[microgirl123]

I've had a look at our last Nextera XT run - we just followed the protocol (no KAPA quantification) and went with the 1:25 dilution ratio called for.

Nextera XT is designed to be used without the KAPA quantification but we have very little experience with how well the bead-based normalization works. Maybe someone else who is running this a lot will have a better answer. Illumina tech support is also a good resource and responds fairly quickly to email inquiries.