Hi Su-Kyung Kang

For Mycoplasma WGS, it’s better to centrifuge and collect the cell pellet before extraction rather than working directly from culture. This step removes media components that interfere with DNA quality and helps concentrate biomass.

With the Promega Wizard® HMW kit, make sure to:

• Pellet at ~10,000–15,000 × g, wash once with PBS, then proceed with lysis.
• Use gentle handling (no vortexing, wide-bore tips) to avoid shearing HMW DNA.
• Include RNase/proteinase K steps and check purity (A260/280 ~1.8, A260/230 ≥1.8).
• Quantify with Qubit and check fragment size (gel or TapeStation) to confirm long DNA.

Direct extraction from culture usually yields lower purity and is not ideal for PacBio long-read sequencing.

Dear Su-Kyung Kang

Mycoplasmas have a low DNA content. So, centrifuging the culture and collecting the pellet would be a better option as it will concentrate these cells from a large volume of liquid culture into a small pellet, ensuring enough starting material to meet the input requirements for WGS.

Besides, the liquid culture media typically contains components like serum and other additives that can interfere with DNA extraction kits and downstream sequencing reactions. When you pellet, you are separating the cells from these potential inhibitors.

Moreover, by following this procedure, you will be including a purification step namely, centrifuging and pellet washing with PBS, that will help to remove residual culture media. This is required to produce high-quality DNA for obtaining robust WGS data.

The amount of DNA yielded from a mycoplasma culture depends on several factors, such as the mycoplasma species, the density of the culture, and the extraction method used. Mycoplasma cells contain 4–7% DNA as a percentage of their total dry weight.

A high-density mycoplasma culture can reach 10^6 to 10^8 colony-forming units (CFU) per milliliter. Even with this high cell count, the total DNA will be low due to the small genome size of each individual bacterium. For instance, a quantitative result in a PCR assay could detect 6.3 picograms of M. orale DNA, equivalent to approximately 8.21 x 10^3 genome copies.

Some tips that you may want to consider.

A. Aim to extract at least 1 microgram of DNA, with higher yields being preferable. A high concentration is essential for sufficient material to prepare libraries for long-read sequencing.

B. You should aim for a A260/A280 ratio between 1.8 and 2.0, which indicates good DNA purity. Target a A260/A230 ratio greater than 2.0, ensuring the absence of contaminants like phenol or carbohydrates.

C. The DNA should be largely intact, with reads that are thousands of bases long. Long-read technologies require very long, intact strands of DNA. Any damage to the DNA will result in shorter reads and incomplete genomic assemblies.

D. Perform electrophoresis that should show a peak of DNA length greater than 50 kb, which will yield you good results.

E. Try to avoid harsh mechanical or enzymatic treatments that can shear the DNA into smaller fragments, as this will hinder long-read generation.

F. Use methods that achieve optimal quality namely, use commercial kits specifically designed for high molecular weight DNA or methods that include enzymatic lysis to lyse mycoplasma cells without high shear forces. After extraction, handle the DNA carefully to prevent physical shearing.

Good Luck!

Malcolm Nobre and Lucian Miles. Thank you for the detailed explanations. They’re very beneficial and will greatly assist me with my experiments.