Description
HMW DNA Yield, Purity and Size Analysis
Assessing the quality of HMW DNA is critical to both judging the success of extractions as well as to predicting success in sequencing runs. Traditional tools for measuring high-molecular-weight DNA purity and yield are often sufficient. However, additional steps to assess fragment size can better predict long-read sequencing performance.
Yield and Purity
DNA was extracted from several sample types using the Wizard® HMW DNA Extraction Kit protocol specific to each sample type. The extracted DNA was found to be highly pure, which is a basic indicator of success in downstream applications. Yields were measured using the fluorescent dye-based QuantiFluor® dsDNA System. The yields and purities obtained are more than sufficient to meet the needs of common long-read sequencing applications.
Gel Analysis
DNA extracted from several unique samples was analyzed by pulsed-field gel electrophoresis (PFGE) to approximate the size distribution of high-molecular-weight fragments. All sample types yielded HMW DNA, which is commonly defined as being 50kb or larger. Of note, there were fragments up to 500kb for some samples.
Quantitative Fragment Analysis
The size distribution of fragments in HMW DNA samples were quantitated using the Agilent Femto Pulse System. This system produces data produced that can be viewed as a traditional electropherogram, or reported using the GQN (genomic quality number) metric. The GQN can be reported at any given fragment size and indicates the percentage of fragments that are of a designated size or larger. Using this approach, samples extracted using the Wizard® HMW DNA kit were analyzed, and the results reported in the table above. The data indicate that for even challenging sample types, most purified DNA is over 50kb, and in many cases, the majority of extracted DNA is over 100kb.
