qPCR for library quantification

[Tarie]

Hi, I use the protocol Illumina DNA Prep with Enrichment for the preration of the libraries. In order to quantify libraries using KAPA q PCR, since I'm seeing a small increase in the cluster, I thought the problem might be due to correct library dilution. The results I get from qPCR are in pM and to convert it to nM using an excel spreadsheet where I enter the Standard fragment size (nt) values of 200 because the length of the fragments I get is Average library fragment size (nt) of 300. Is it correct ? or are there other methods ?

Thanks for your help

[Ben3]

Hello Tarie, are you using a specific excel spreadsheet for this? The last time I did it, I remember using one from the KAPA website that did all the conversions. Also, can you explain the fragment size you're using in a little more detail? The length sounds right but I wanted to be sure that the value you're using is correct.

[Tarie]

Ben3 I use a specific spreadsheet. I don't know if it's the same as the one that can be downloaded from the KAPA website, could you tell me where to download it from? I use fragment size 200 nt as standard because from the reports that I obtain ,at the end of the sequencing, there is a table in which the average length of the fragments is indicated for each sample, which is always around 200 nt.At this point I'm not sure if it's correct because the clusters are always slightly higher if not by a lot.

thanks a lot for the help.

[Ben3]

Tarie I couldn't find it on the website, and that's where I'm pretty sure I got it before. I recommend reaching out to the email listed on this contact page (https://sequencing.roche.com/us/en/c...ontact-us.html). They've sent me materials I've needed in the past so they should be able to send it to you.

And yeah, I have found qPCR to be quite tricky when it comes to library quantification. I had to change out pipettes and make a lot of small changes to ensure I was making it as accurate as possible. Let me know if they can't get it to you and I'll see if I can dig up the file I used before.

[Tarie]

Hi Ben3 sorry for disturbing. I wrote an email to roche to ask if there was an excel spreadsheet but they didn't reply. Have you the possibility to recover what you used ? because my doubt is tthat it is correct to put the value 200nt in the Standard fragment size

thank a lot .