Hello everyone! I am helping to manage an Illumina/NGS Facility in the Cambridge, UK area. This is my first post, and I am hoping someone might help answer a question that I have.
Long story short: I would like to know what is the lowest concentration of library that can be denatured for loading onto the flowcell, given that you have to dilute out the NaOH used for denaturation. Please read details below.
I work at an NGS facility that performs sequencing as a service, and recently a DNA library with a very low concentration was submitted to me, and I am puzzled as to how to prepare this for sequencing.
This library will be a simple multiplex, for which three samples will be pooled in equimolar amounts. The starting concentrations of the 3 samples are different, with the lowest being less than 10 pM. Ten microlitres of each sample are available.
I have calculated that even if I use up all of the sample, the most concentrated pooled library (for the denaturation step) that I can produce will be have a concentration of approximately 66 pM of each sample, or 200pM in total. This, in turn, means that in order to produce 120µL of 10 pM denatured, diluted library for loading on the flowcell, I would dilute 6 µL of the 200pM denatured library to 120 µL with Hybridization Buffer, a dilution factor of only 1:20, whereas the normal dilution factor is 1:100 or greater. I am worried that having a large amount of the denatured library (and therefore NaOH) loaded on the flowcell will have an adverse effect on cluster generation.
I was wondering if anyone can say what is the lowest librray concentration that can be loaded on the flowcell (or alternatively, what is the largest volume of NaOH-denaturated DNA that can be diluted for hybridization to the flowcell).
I will be grateful for any information or advice.
Long story short: I would like to know what is the lowest concentration of library that can be denatured for loading onto the flowcell, given that you have to dilute out the NaOH used for denaturation. Please read details below.
I work at an NGS facility that performs sequencing as a service, and recently a DNA library with a very low concentration was submitted to me, and I am puzzled as to how to prepare this for sequencing.
This library will be a simple multiplex, for which three samples will be pooled in equimolar amounts. The starting concentrations of the 3 samples are different, with the lowest being less than 10 pM. Ten microlitres of each sample are available.
I have calculated that even if I use up all of the sample, the most concentrated pooled library (for the denaturation step) that I can produce will be have a concentration of approximately 66 pM of each sample, or 200pM in total. This, in turn, means that in order to produce 120µL of 10 pM denatured, diluted library for loading on the flowcell, I would dilute 6 µL of the 200pM denatured library to 120 µL with Hybridization Buffer, a dilution factor of only 1:20, whereas the normal dilution factor is 1:100 or greater. I am worried that having a large amount of the denatured library (and therefore NaOH) loaded on the flowcell will have an adverse effect on cluster generation.
I was wondering if anyone can say what is the lowest librray concentration that can be loaded on the flowcell (or alternatively, what is the largest volume of NaOH-denaturated DNA that can be diluted for hybridization to the flowcell).
I will be grateful for any information or advice.
Comment