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Old 05-31-2011, 01:24 AM   #1
niceday
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Default KAPA qPCR

Hi,
I have a question about multiplication factor for library quantification after KAPA qPCR. We have been advised to multiply the qPCR result by the factor of standard fragment size (452bp) divided by the library size (~230bp). For this case it's a factor of 2.

This means our qPCR results are twice as concentrated as our qubit and bioanalyser results. It doesn't sound right but qPCR has always ben accurate for us. Is the multiplication factor wrong? We haven't seen this before because our libraries are usually much closer to the standard fragment size of 452bp.

I'm looking on the KAPA website but thought I would also post here.
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Old 06-17-2011, 07:37 AM   #2
John12824
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Hi Niceday,

I had a similar issue (average library size of ~250 bp), followed the KAPA instructions for size adjustment, and ended up underclustering my samples (i.e. the concentrations likely overestimated).

How did your samples turn out?

Going to try loading samples without the size adjustment and see what kind of densities I get.


UPDATE: There may have been a miscommunication in the amount of sample to load on the flow cell lane. Will update again once we solve the problem.

Last edited by John12824; 06-17-2011 at 02:40 PM.
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Old 06-22-2011, 01:29 AM   #3
niceday
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I have run the samples on a 36bp SR. I used the qPCR results even though they gave a higher concentration of DNA than both Qubit and the bioanalyser.

The cluster density ran from 250K to 280K per tile.

I have other libraries made from a customer and the qPCR results put some of the samples as much as 3 times what the Qubit and bioanalyser. I will cluster according to qPCR. It seems wrong and Kapa are not sure what is happening but other users have seen it. Thankfully I only see this if we make some RNA library preps and if some customers libraries dont have a tight peak.
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Old 06-22-2011, 01:37 AM   #4
niceday
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I should add for clarification I am using Kapa's standard concentration factor for all my calculations.
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Old 06-23-2011, 07:13 AM   #5
John12824
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Hi Niceday,

I re-titrated our libraries and the cluster densities were very close to what I expected.

The following numbers are raw cluster densities on an Illumina v3 flowcell. Samples were loaded according to the size adjusted concentrations determined by KAPA qPCR.

12 pM - 743 K/mm2
15 pM - 858 K/mm2
18 pM - 912 K/mm2

According to Illumina, at a loading concentration of 12 pM you should get a cluster density between 750 - 850 K/mm2, so we are very close. At least for our small insert size libraries, it seems like the size adjustment was appropriate and accurate.
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Old 07-31-2012, 11:44 PM   #6
Genquest
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Hi John12824,
There are a couple of questions that come to my mind,would appreciate if I get some help on that.

1. Do you take into account the exact concentration of libraries that you get from
qPCR,even though it is 2-3 times more than bioanalyzer or you do some manipulations
checking the trend its following?
2. Do you take into account the source of the samples (e.g. plant,animal,insect,sediment
or soil)from which the library has been made,as to how it will behave with qPCR or its
the standard protocol for all to hit the target at around 12-18pM?
We use pico green as our benchmark for quantitation,also doing qPCR with PhiX as standards.This shows lot of variation in qPCR,sometimes we attain the expected cluster density and sometimes it doesn't work out!!
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