Purify High-Molecular-Weight DNA for Long-Read Sequencing
High-Quality High-Molecular-Weight DNA from a Variety of Sample Types
De novo genome sequencing, genome finishing and structural variant detection have become significantly more commonplace due to advances in sequencing technology. Long-read sequencing in particular provides researchers with data to address challenges in fields such as oncology, inherited disease research, microbiology and agriculture. However, long-read technologies require high-quality, high-molecular-weight DNA. Obtaining DNA of sufficient, size, quantity and quality is critical to the process that has historically required costly or laborious methods.
The Wizard® HMW DNA Extraction Kit is specifically designed to isolate high-molecular-weight (HMW) DNA that will provide strong performance in long-read sequencing. This kit helps researchers obtain DNA up to 500kb with high purity to get the most out of precious samples and can be used with a wide range of sample types, including whole blood, plant leaf, tissue culture cells and Gram-positive and Gram-negative bacteria. With a workflow that can be completed in approximately 1.5 hours without specialized equipment, the Wizard® HMW DNA Extraction Kit relieves many of the challenges regularly faced by researchers.
HMW DNA in Long-Read Sequencing
Samples of human blood and cultured K562 mammalian cellswere used to extract high-molecular-weight DNA using the Wizard® HMW DNA Extraction Kit. These samples were prepped for sequencing using the recommended ligation-based library preparation method, and sequenced using an Oxford Nanopore Technologies® MinION® instrument.
|K562 Cultured Cells||Whole Human Blood|
|Percent Reads >100kb||1.3%||2.4%|
Sequencing run summary data comparing the extraction methods is shown above. Numerous measures indicate that the Wizard® HMW chemistry extracts large DNA fragments with excellent performance in sequencing. The N50 metric quantitates the read length above which half of the data is derived and the number of reads larger than 100kb benefits researchers who are tackling large structural variants or large gaps in genome assemblies.
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
|Sample||Input||Average Yield (µg)||Average Purity (A260/A230)||Average Purity (A260/A280)|
|E. coli||300µl overnight culture||27.3||2.1||1.9|
|L. innocua||300µl overnight culture||13.5||1.9||2.0|
|K562||1 × 106 cells||11.9||2.1||1.9|
|Holly||40mg leaf tissue||3.4||2.1||2.2|
|Spinach||40mg leaf tissue||1.7||2.2||2.1|
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
|Sample||Percent DNA >50kb (GQN 50kb)||Percent DNA >100kb (GQN 100kb)||Percent DNA >150kb (GQN 150kb)|
|E. coli (Gram negative)||91%||83%||61%|
|L. innocua (Gram positive)||65%||52%||28%|
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.
What’s in the box?
|Item/ Catalog number selected: A2920||Part #||Size||Available Separately|
|HMW Blood Lysis Buffer||A293A||1 × 50ml|
|HMW Lysis Buffer A||A294A||1 × 25ml|
|Protein Precipitation Solution||A795A||1 × 25ml||View Product|
|DNA Rehydration Solution||A796A||1 × 50ml||View Product|
|RNase A Solution||A797A||1 × 250μl||View Product|
|Proteinase K (PK) Solution||MC500C||1 × 1ml||View Product|
For Research Use Only. Not for Use in Diagnostic Procedures.