We use the NEBNext Library Quant Kit for Illumina to determine our Illumina library concentrations. Before taking the library to qPCR, we usually run the library on Bioanalyzer to get an idea of the concentration. Based on Bioanalyzer, we will dilute the library down to 5,000pM for qPCR. Most of the time, qPCR results will show libraries around 5,000pM (maybe 4,000-6000pM). In some cases, the qPCR concentration can be double or even triple the Bioanalyzer concentration. When this happens we QC the library by Qubit. In most cases, the Qubit concentration will be similar to the Bioanalzyer concentration.
This makes it challenging for us to determine which concentration to use for loading the sequencer. If we use the qPCR concentration, the runs will be under-clustered. We are trying to understand why we would get higher concentrations from qPCR. It makes more sense if the qPCR concentrations were lower than Qubit/BA suggesting that the adapter ligation was not very successful.
Why do you think we would get such high qPCR concentrations? One thought is that there may be single stranded DNA in the library which is not detected by Qubit or BA, but qPCR is able to amplify. Curious to know your thoughts.
Thank you,
Karrie
I also recently had this happen where our qPCR is double/triple our Qubit.
https://support.illumina.com/bulletins/2019/10/bubble-products-in-sequencing-libraries--causes--identification-.html
Using a fluorometric-based method to quantify the library when bubble product is present will result in inaccurate quantification. Fluorometric-based methods only quantify double-stranded DNA, while bubble product consists of both double-stranded DNA and single-stranded DNA. Fluorometric quantification will underestimate the library concentrations, which can negatively impact run performance.
Curious as to what you all used to load your sequencer? It seems most people say to trust the qPCR, but curious if you are finding that to be best.
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