HMW DNA extraction
NGI now offers High Molecular Weight (HMW) DNA extraction as a service for sequencing projects at or in collaboration with NGI and SciLifeLab.
The success of any long-read sequencing project will depend mainly on the integrity and purity of the input DNA, which again can depend on storage and handling of both starting material and the extracted DNA. We have been working with High Molecular Weight (HMW) DNA extractions since 2016, and gained experience with extractions from many different organisms using commercially available HMW DNA extraction kits and traditional methods, as well as in-house developed workflows.
For de novo genome sequencing of higher plants and animals, HMW DNA in combination with long read sequencing technologies, such as PacBio or Nanopore, is highly beneficial for spanning genomic repeats and detecting large-scale rearrangements.
During planning of sequencing projects, we are happy to advice our users on how to best extract DNA depending on the sample and the sequencing requirements. We also now offer the possibility of HMW DNA extraction as a service to our users.
Bellow you can find a short description of extraction methods that have been extensively evaluated at NGI.
For a more comprehensive description of extraction methods, please consult extraction material guidelines.
Currently first choice extraction method unless circumstances dictate otherwise. In our experience, Nanobind kits provide superior recovery, length and purity of DNA compared to other kits. Circulomics Nanobind protocols for different sample types can be found at the Circulomics online store.
In situ agarose plug extraction is especially suited for Bionano optical mapping and samples requiring shipping as the agarose protects the DNA from fragmentation. It is not recommended for applications that do not require ultra HMW DNA as the viscosity and potential agarose remnants can interfere with library preparation and sequencing. Bionano’s protocols for agarose plug extractions can be found at Bionano’s website.
SDS/CTAB lysis and phenol-chloroform extraction
Recommended for samples displaying purity issues when using other extraction methods. Especially useful for samples high in polysaccharides, phenols, fats and/or pigments. For this type of extraction we tailors the workflow to each specific sample.
Only recommended for samples expected to be low in contaminants (such as vertebrate blood and tissue). This is a less expensive option and can provide 100 – 200 kb fragments from high quality material. The MagAttract protocol can be found in QIAGENS’s webshop.
High quality input material increases the chance of obtaining High Molecular Weight (HMW) DNA that meet the quality and quantity requirements for long read applications. The best option for obtaining quality HMW DNA is fresh samples. However, samples that have been flash frozen, stored at -80ºC and shipped on dry-ice perform just as well in most cases. If possible, we highly recommend dissecting your sample prior to freezing to avoid freeze/thaw cycles which promote cell disruption and DNA fragmentation.
Before submitting samples for extraction, please read carefully shipping instructions for extraction material.
HMW-DNA extractions can be very laborious. We are therefore unable to process a large number of samples simultaneously.